阅读说明：本技术 利用心肌肌钙蛋白i辅助诊断和评估人类受试者的轻度创伤性脑损伤的方法 (Method for auxiliary diagnosis and evaluation of mild traumatic brain injury of human subjects by using cardiac troponin I ) 是由 B·麦奎斯顿 F·科利 A·贝希里 J·马力诺 S·德特维勒 于 2018-05-30 设计创作，主要内容包括：本文公开了使用cTnI辅助诊断和评估已遭受或可能已遭受头部损伤例如轻度或中度、重度、或中度至重度创伤性脑损伤(TBI)的人类受试者的方法。还公开了通过检测cTnI水平来确定是否对受试者进行头部计算机断层摄影的方法。最后,还公开了患有轻度TBI的受试者的结局的方法。(Disclosed herein are methods of using cTnI to assist in the diagnosis and assessment of human subjects who have suffered or may have suffered a head injury, e.g., mild or moderate, severe, or moderate to severe Traumatic Brain Injury (TBI). Also disclosed are methods of determining whether to perform head computed tomography on a subject by detecting a level of cTnI. Finally, methods of outcome in subjects with mild TBI are also disclosed.)

1. A method for assessing mild traumatic brain injury in a human subject, the method comprising:

a) assaying a sample obtained from the subject within about 24 hours after actual or suspected head injury to measure or detect a level of cardiac troponin i (ctni); and

b) determining whether the subject has suffered mild or moderate to severe Traumatic Brain Injury (TBI), wherein the subject is determined to have (1) moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI in the sample is above a cTnI reference level or (2) mild traumatic brain injury when the level of cTnI in the sample is below a cTnI reference level.

2. The method of claim 1, wherein the subject has received a glasgow coma scale score before or after performing the assay.

3. The method of claim 2, wherein the subject is suspected of having moderate, severe, or moderate to severe traumatic brain injury based on the glasgow coma scale score.

4. The method of claim 3, wherein the reference level is associated with a subject with moderate, severe, or moderate to severe traumatic brain injury.

5. The method of claim 4, wherein the reference level is associated with a glasgow coma scale score of 3-12.

6. The method of claim 2, wherein the subject is suspected of having mild traumatic brain injury based on the glasgow coma scale score.

7. The method of claim 6, wherein the reference level is associated with a subject with mild traumatic brain injury.

8. The method of claim 7, wherein the reference level is associated with a glasgow coma scale score of 13-15.

9. The method of any one of claims 1-8, wherein the reference level of cTnI is about 1.94pg/mL, about 2.54pg/mL, about 21.23pg/mL, or about 43.79 pg/mL.

10. The method of any one of claims 1 to 9, wherein the reference level (a) is determined by an assay having a sensitivity of between at least about 85% and 100% and a specificity of between at least about 30% and 100%; (b) as determined by an assay having a sensitivity of at least about 87.5% and a specificity of at least about 31%; or (c) between at least about 1pg/mL to about 50 pg/mL.

11. The method of any one of claims 1 to 10, wherein the sample is obtained within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of an actual or suspected head injury.

12. A method of treating mild or moderate, severe, or moderate to severe traumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample obtained from the subject within about 24 hours after an actual or suspected head injury to measure or detect the level of cTnI;

b) determining whether the subject suffers from mild or moderate to severe Traumatic Brain Injury (TBI), wherein the subject is determined to have (1) moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI in the sample is above a cTnI reference level or (2) mild traumatic brain injury when the level of cTnI in the sample is below a cTnI reference level; and

c) subjects assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury are treated with a traumatic brain injury treatment.

13. The method of claim 12, further comprising monitoring a subject assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury.

14. A method of determining whether to perform a head Computed Tomography (CT) scan on a human subject having actual or suspected head damage, the method comprising:

a) performing an assay on a sample obtained from the subject within about 24 hours after an actual or suspected head injury to measure or detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in the sample is above a cTnI reference level, and not performing a CT scan on the subject when the level of cTnI in the sample is below the cTnI reference level.

15. The method of claim 14, wherein the sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of actual or suspected head injury.

16. The method of claim 14 or 15, wherein the subject has received a CT scan before or after performing the assay.

17. The method of claim 16, wherein the subject is suspected of having traumatic brain injury based on a CT scan.

18. The method of any one of claims 14 to 17, wherein the reference level is associated with a head computed tomography positive.

19. The method of claim 14, wherein the reference level is associated with a control subject that does not suffer from head injury.

20. The method of any one of claims 14-19, wherein the reference level of cTnI is about 1.65pg/mL, about 2.16pg/mL, about 14.75pg/mL, or about 30.43 pg/mL.

21. The method of any one of claims 14 to 20, wherein the reference level (a) is determined by an assay having a sensitivity of between at least about 65% and 100% and a specificity of between at least about 30% and 100%; (b) as determined by an assay having a sensitivity of at least about 85% and a specificity of at least about 33%; or (c) between at least about 1.0pg/mL to about 50 pg/mL.

22. A method for assessing mild traumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after a head injury and the second sample is taken from the human subject about 3 hours to about 6 hours after the first sample, wherein the sample is a biological sample;

b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and

c) confirming the occurrence of moderate, severe, or moderate to severe traumatic brain injury if the detected level of cTnI has increased from the first sample to the second sample, and confirming the absence of mild traumatic brain injury if the detected level of cTnI remains unchanged or decreases from the first sample to the second sample.

23. A method for assessing mild traumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point and the second sample is taken from the human subject about 1 hour to about 4 hours after the first sample, wherein the sample is a biological sample;

b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and

c) confirming the occurrence of moderate to severe traumatic brain injury if the detected level of cTnI increases from the first sample to the second sample, and confirming the absence of mild traumatic brain injury if the detected level of cTnI remains unchanged or decreases from the first sample to the second sample.

24. The method of claim 22 or 23, wherein the first sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of an actual or suspected head injury.

25. The method of any one of claims 22-24, wherein the subject has abnormal CT in the head.

26. The method of any one of claims 22-25, wherein the amount of cTnI in the first sample is about 1.0 to about 50 pg/mL.

27. The method of any one of claims 22-26, wherein the amount of cTnI in the second sample is about 1.0 to about 50 pg/mL.

28. A method of treating mild or moderate, severe, or moderate to severe traumatic brain injury in a human subject, the method comprising:

a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point and the second sample is taken from the human subject about 3 hours to about 6 hours or about 1 hour to about 4 hours after the first sample, wherein the sample is a biological sample;

b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample;

c) confirming the occurrence of moderate to severe traumatic brain injury if the detected level of cTnI increases from the first sample to the second sample, and confirming the absence of mild traumatic brain injury if the detected level of cTnI remains unchanged or decreases from the first sample to the second sample; and

d) subjects assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury are treated with a traumatic brain injury treatment.

29. The method of claim 28, further comprising monitoring a subject assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury.

30. A method of aiding diagnosis and assessment of a human subject who has suffered or may have suffered a head injury, the method comprising:

a) performing an assay on a sample obtained from the subject within about 2 hours after an actual or suspected head injury to measure or detect the level of cTnI; and

b) determining whether the subject suffers from mild or moderate to severe Traumatic Brain Injury (TBI), wherein the subject is determined to have (1) moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI in the sample is above a cTnI reference level or (2) mild traumatic brain injury when the level of cTnI in the sample is below a cTnI reference level.

31. The method of claim 30, wherein the subject has received a glasgow coma scale score before or after performing the assay.

32. The method of claim 31, wherein the subject is suspected of having moderate, severe, or moderate to severe traumatic brain injury based on the glasgow coma scale score.

33. The method of claim 32, wherein the reference level is associated with a subject with moderate, severe, or moderate to severe traumatic brain injury.

34. The method of claim 33, wherein the reference level is associated with a glasgow coma scale score of 3-12.

35. The method of claim 31, wherein the subject is suspected of having mild traumatic brain injury based on the glasgow coma scale score.

36. The method of claim 35, wherein the reference level is associated with a subject with mild traumatic brain injury.

37. The method of claim 36, wherein the reference level is associated with a glasgow coma scale score of 13-15.

38. The method of any one of claims 30-37, wherein the reference level of cTnI is about 1.15pg/mL or about 1.29 pg/mL.

39. The method of any one of claims 30 to 38, wherein the reference level (a) is determined by an assay having a sensitivity of between at least about 85% and 100% and a specificity of between at least about 30% and 100%; (b) as determined by an assay having a sensitivity of at least about 87.5% and a specificity of at least about 31%; or (c) between at least about 0.5pg/mL to about 30 pg/mL.

40. The method of any one of claims 30 to 39, wherein the sample is taken within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, or within about 90 minutes after an actual or suspected head injury.

41. A method of treating mild, moderate, severe, or moderate to severe TBI, the method comprising:

a) performing an assay on a sample obtained from the subject within about 2 hours after an actual or suspected head injury to measure or detect the level of cTnI;

b) determining whether the subject suffers from mild or moderate to severe Traumatic Brain Injury (TBI), wherein the subject is determined to have (1) moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI in the sample is above a cTnI reference level or (2) mild traumatic brain injury when the level of cTnI in the sample is below a cTnI reference level; and

c) subjects assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury are treated with a traumatic brain injury treatment.

42. The method of claim 41, further comprising monitoring a subject assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury.

43. A method of assisting in determining whether to perform a head Computed Tomography (CT) scan on a human subject having an actual or suspected head injury, the method comprising:

a) performing an assay on a sample obtained from the subject within about 2 hours after an actual or suspected head injury to measure or detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in the sample is above a cTnI reference level, and not performing a CT scan on the subject when the level of cTnI in the sample is below the cTnI reference level.

44. The method of claim 43, wherein the subject has received a CT scan before or after performing the assay.

45. The method of claim 44, wherein the subject is suspected of having traumatic brain injury based on a CT scan.

46. The method of any of claims 43-45, wherein the reference level is associated with a head computed tomography positive.

47. The method of claim 43, wherein the reference level is associated with a control subject that does not suffer from head injury.

48. The method of any one of claims 43-47, wherein the cTnI reference level is about 5.8pg/mL or about 4.7 pg/mL.

49. The method of any one of claims 43 to 48, wherein the reference level (a) is determined by an assay having a sensitivity of between at least about 65% and 100% and a specificity of between at least about 30% and 100%; (b) as determined by an assay having a sensitivity of at least about 85% and a specificity of at least about 33%; or (c) between at least about 0.5pg/mL to about 25 pg/mL.

50. The method of any one of claims 43 to 49, wherein the sample is taken within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, or within about 90 minutes after an actual or suspected head injury.

51. A method for predicting outcome in a human subject with mild traumatic brain injury, the method comprising:

a) performing an assay on a sample from the human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after a head injury and the second sample is taken from the human subject about 0 hours to about 4 hours after the first sample, wherein the sample is a biological sample;

b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and

c) an adverse outcome is predicted if the detected level of cTnI increases by at least about 20% from the first sample to the second sample, and a favorable outcome is predicted if the detected level of cTnI remains unchanged, decreases, or increases by less than about 20% from the first sample to the second sample.

52. The method of claim 51, wherein the first sample is taken from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of a head injury.

53. The method of claim 51 or 52, wherein the method predicts outcome at about 1 month, 3 months, or 6 months after actual or suspected head injury.

54. The method of claim 53, wherein the subject is predicted to have an expanded Grossog Outcome Scale (GOSE) score of 5 or less.

55. The method of any one of claims 51-54, wherein the subject's head CT scan is normal.

56. The method of any one of claims 51-55, wherein the subject has received a Glasgow coma scale score before or after performing the assay.

57. The method of any one of claims 51-56, wherein the subject receives a Glassy coma Scale score of 13-15 and is suspected of having mild traumatic brain injury based on the Glassy coma Scale score.

58. The method of any one of claims 51-57, wherein the subject has received an extended Glasgow coma Scale (GOSE) score before or after performing the assay.

59. A method for predicting outcome in a human subject with mild traumatic brain injury, the method comprising:

a) determining a sample obtained from the subject within about 28 hours after injury to measure or detect a level of cTnI, wherein the sample is a biological sample; and

b) predicting an adverse outcome for the human subject if the level of cTnI in the sample is above a cTnI reference level, and predicting a favorable outcome for the human subject if the level of cTnI in the sample is below a cTnI reference level.

60. The method of claim 59, wherein the sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of a head injury.

61. The method of claim 59 or 60, wherein the method predicts outcome at about 1 month, 3 months, or 6 months after head injury.

62. The method of claim 61, wherein the subject is predicted to have an expanded Grossog Outcome Scale (GOSE) score of 5 or less.

63. The method of any one of claims 59 to 62, wherein the reference level (a) is determined by an assay having a sensitivity between at least about 80% and 100% and a specificity between at least about 45% and 100%; (b) as determined by an assay having a sensitivity of at least about 83.3% and a specificity of at least about 54.9%; (c) as determined by an assay having a sensitivity of at least about 100% and a specificity of at least about 49.2%; or (d) between at least about 1pg/mL to about 50 pg/mL.

64. The method of any one of claims 59-63, wherein the cTnI reference level is about 5.6pg/mL or about 5.7 pg/mL.

65. A method for predicting outcome in a human subject with mild traumatic brain injury, the method comprising:

a) performing an assay on a sample from the human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after a head injury and the second sample is taken from the human subject about 4 hours after the first sample, wherein the sample is a biological sample;

b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and

c) predicting an adverse outcome for the human subject if the level of cTnI in the sample increases from the first sample to the second sample, and predicting an adverse outcome for the human subject if the level of cTnI in the sample remains unchanged or decreases from the first sample to the second sample.

66. The method of claim 65, wherein the first sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours.

67. The method of claim 65 or 66, wherein the method predicts outcome at about 1 month, 3 months, or 6 months after head injury.

68. The method of claim 67, wherein the subject is predicted to have an extended Grossog Outcome Scale (GOSE) score of 5 or less.

69. The method of any one of claims 65-68, wherein the amount of cTnI in the first sample is about 1.0 to about 50 pg/mL.

70. The method of any one of claims 65-69, wherein the amount of cTnI in the second sample is about 1.0 to about 50 pg/mL.

71. The method of any one of claims 65-70, wherein the level of cTnI is decreased or increased by at least one absolute amount from the first sample to the second sample.

72. The method of claim 71, wherein the absolute amounts (a) are determined by an assay having a sensitivity of between at least about 80% and 100% and a specificity of between at least about 45% and 100%; (b) as determined by an assay having a sensitivity of at least about 82.4% and a specificity of at least about 69.5%; or (c) between at least about 1pg/mL to about 50 pg/mL.

73. The method of claim 72, wherein the absolute amount is about 5.6 pg/mL.

74. The method of any one of claims 51-73, further comprising treating a subject assessed as having an adverse outcome with a traumatic brain injury treatment.

75. The method of any one of claims 51-74, further comprising monitoring a subject assessed as having an adverse outcome with a traumatic brain injury treatment.

76. A method for predicting outcome in a human subject with mild traumatic brain injury, the method comprising:

a) performing an assay on a sample from the human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after a head injury and the second sample is taken from the human subject about 0 to about 4 hours after the first sample, wherein the sample is a biological sample;

b) determining the age of the subject; and

c) predicting an adverse outcome for the human subject if the level of cTnI in the first and/or second sample is above the cTnI reference level and the age of the subject is above a reference age, and predicting an adverse outcome for the human subject if the level of cTnI in the first and second sample is below the cTnI reference level and/or the age of the subject is below the reference age.

77. The method of any one of claims 1-76, further comprising treating the subject with at least one cardioprotective therapy.

78. The method of claim 77, wherein the at least one cardioprotective therapy comprises a beta-blocker, a diuretic, an Angiotensin Converting Enzyme (ACE) inhibitor, a calcium channel blocker, a lipid lowering therapy, a statin, a nitrate, an antiplatelet agent, an anticoagulant, or a combination thereof.

79. The method of any one of claims 1-78, wherein the level of cTnI is measured by an immunoassay or a clinical chemistry assay.

80. The method of any one of claims 1-79, wherein measuring the level of cTnI comprises:

C. contacting the sample with the following antibodies, simultaneously or sequentially in any order:

(1) a cTnI capture antibody that binds to an epitope on cTnI or a cTnI fragment to form a cTnI capture antibody-cTnI antigen complex, and

(2) a cTnI detection antibody comprising a detectable label and binding to an epitope on cTnI that is not bound by the cTnI capture antibody to form a cTnI antigen-cTnI detection antibody complex,

thereby forming a cTnI capture antibody-cTnI antigen-cTnI detection antibody complex, an

D. Measuring the amount or concentration of cTnI in the sample based on the signal generated by the detectable label in the cTnI capture antibody-cTnI antigen-cTnI detection antibody complex.

81. The method of any one of claims 1 to 80, wherein the sample is selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample and a plasma sample.

82. The method of any one of claims 1 to 81, wherein the sample is obtained after the subject has suffered a head injury caused by physical shock, blunt impact of external mechanical or other forces resulting in closed or open head trauma, one or more drops, explosions or explosions, or other types of blunt force trauma.

83. The method of any one of claims 1-81, wherein the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin.

84. The method of claim 83, wherein the chemical or toxin is fire, mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a gas, an organometallic, a drug of abuse, or one or more combinations thereof.

85. The method of any one of claims 1 to 84, wherein the sample is obtained from a subject having an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

86. A method according to one of claims 1-85, wherein the method can be performed on any subject without consideration of factors selected from the group consisting of: a clinical condition of the subject, a laboratory value of the subject, a grade of mild, moderate, severe, or moderate to severe traumatic brain injury of the subject, a low or high cTnI level of the subject, and a timing of any event that the subject may suffer from head injury.

87. The method of any one of claims 1 to 86, wherein the sample is a whole blood sample.

88. The method of any one of claims 1 to 86, wherein the sample is a serum sample.

89. The method of any one of claims 1 to 86, wherein the sample is a plasma sample.

90. The method of any one of claims 87-89, wherein the assay is an immunoassay.

91. The method of any one of claims 87-89, wherein the assay is a clinical chemistry assay.

92. The method of any one of claims 87-89, wherein the assay is a single molecule detection assay.

Technical Field

The present disclosure relates to methods of aiding diagnosis and assessment in a human subject suffering from or likely to suffer from (or having an actual or suspected) head injury, e.g., mild Traumatic Brain Injury (TBI), by detecting the level of cardiac troponin i (ctni) in a sample (or biological sample) taken from the human subject at a point in time of injury after the subject suffered from or likely to suffer from (or having an actual or suspected) head injury. The present disclosure also provides methods of determining whether to perform head computed tomography of a subject based on detecting various levels of cTnI. The disclosure also provides methods of predicting outcome in a subject having TBI.

Background

In the united states alone, over 500 million mild Traumatic Brain Injuries (TBI) occur each year. Currently, no simple, objective, accurate metric is available to assist in patient assessment. In fact, many TBI assessments and diagnoses are based on subjective data. Unfortunately, objective measures such as head CT and Glasgow Coma Score (GCS) are not very comprehensive or sensitive in assessing mild TBI. In addition, head CT is not able to show mild TBI for most of the time, is expensive, and exposes the patient to unnecessary radiation. In addition, a head CT negative does not mean that the patient has excluded a concussion; rather it simply means that some intervention, such as surgery, is not required. Clinicians and patients need objective, reliable information to accurately assess such situations to facilitate proper treatment triage and rehabilitation. To date, there is limited data available to utilize cardiac troponin I in acute care settings or hyperacute care settings (acute time points very early after injury) to assist in patient assessment and management.

Mild TBI or concussion is difficult to detect objectively and presents a daily challenge to emergency care units worldwide. Concussions do not usually lead to gross pathologies such as bleeding, there are no abnormalities in conventional computed tomography, but rather rapid onset of neuronal dysfunction that can spontaneously resolve within days to weeks. Approximately 15% of patients with mild TBI suffer from persistent cognitive dysfunction. The needs of mild TBI victims have not been met in the field, in emergency rooms and clinics, sport arenas, and military activities (e.g., combat).

Current algorithms for assessing the severity of brain damage include glasgow coma scale scoring and other measures. These measures may sometimes be applicable to the relevant acute severity, but are not sufficiently sensitive to subtle pathologies, which may lead to persistent deficiencies. GCS and other measures also fail to distinguish between damage types and may not be applicable. Thus, patients grouped into clinical trials at a single GCS level may vary greatly in severity and lesion type. Improper classification undermines the sophistication of the clinical trial because outcomes can vary accordingly. Improved lesion classification will enable a more accurate delineation of disease severity and type in TBI patients in clinical trials.

Furthermore, current brain damage tests rely on outcome measures, such as the Extended glasgow outcome Scale Extended, which can capture the overall phenomenon but cannot assess subtle differences in outcome. Thus, 30 consecutive trials of the brain injury therapeutic failed. Sensitive outcome measures are needed to determine the extent to which a patient recovers from brain injury in order to test for therapeutic and prophylactic agents.

Patients with Traumatic Brain Injury (TBI) have at least three times higher probability of death from cardiovascular causes than the general population. Cardiac injury caused by TBI is also associated with neurogenic pulmonary edema. The phenomenon of cardiac injury in neurological conditions has been described in patients with spontaneous subarachnoid hemorrhage and is thought to be due to sudden increases in catecholamine levels. However, the underlying mechanisms of excessive cardiovascular death in TBI are poorly studied and are therefore not well understood. Therefore, it is not clear: whether the onset of cardiac injury occurs in the acute or chronic phase of TBI; whether a particular subtype of TBI is preferentially affected by cardiac injury; and what is the biological trigger for cardiac injury in TBI. Many retrospective studies have investigated myocardial injury in the acute phase of TBI. These studies reported that 30% of patients with severe TBI had suffered heart damage (as determined by elevated troponin levels) within 24 hours of injury using conventional cardiac troponin assays. Cardiac injury in TBI is related to the severity and age of the injury. The risk of hospitalization mortality is higher in TBI patients with cardiac injury than in TBI patients without cardiac injury. However, there is a spectrum bias in these findings since they are from retrospective studies and troponin measurements are made at the discretion of the clinician (who rarely does so in the routine care of TBI patients). Furthermore, the relationship between cardiac injury and neurological outcome in TBI has not been studied. Furthermore, the role of cardiac injury in mild and moderate TBI has not been studied.

Disclosure of Invention

In some embodiments, the present disclosure relates to a method of aiding diagnosis and assessment of mild traumatic brain injury in a human subject. The method may comprise the steps of:

a) assaying a sample obtained from the subject within about 24 hours after the actual or suspected head injury to measure or detect a level of cardiac troponin i (ctni); and

b) determining whether the subject suffers from mild or moderate, severe, or moderate to severe Traumatic Brain Injury (TBI), wherein the subject is determined to be (1) moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI in the sample is above the cTnI reference level or (2) mild traumatic brain injury when the level of cTnI in the sample is below the cTnI reference level.

In some embodiments of the above methods, the subject is diagnosed or determined to have suffered a mild traumatic brain injury. In other embodiments of the above methods, the subject is diagnosed or determined to have suffered a moderately traumatic brain injury. In still other embodiments of the above methods, the subject is diagnosed or determined to have suffered a severe traumatic brain injury. In still other embodiments, the subject is diagnosed or determined to suffer from moderate to severe traumatic brain injury.

In some embodiments of the above methods, the subject receives a glasgow coma scale score before or after performing the assay. In some embodiments, the subject may be suspected of having traumatic brain injury based on a previously conducted glasgow coma scale score. For example, depending on the medical condition of the subject, the glasgow coma scale score may be assessed shortly after the subject arrives at an emergency room, trauma center, or other location in order to assess and/or assess whether the subject has a TBI. Such a glasgow coma scale score may be provided prior to the assay to be performed to confirm and determine whether the subject has mild or moderate, severe, or moderate to severe TBI. After the determination is made, one or more subsequent glasgow coma scale scores may be made based on the determination results as part of the TBI management of the physician (or other medical personnel) (e.g., to determine whether surgical and/or pharmacological intervention may be required). In other embodiments, the subject may not receive a glasgow coma scale score prior to performing the assay.

In some embodiments of the above methods, the subject is suspected of having moderate, severe, or moderate to severe traumatic brain injury based on the glasgow coma scale score.

In some embodiments of the above methods, the reference level is associated with (corresponds to) a subject having moderate, severe, or moderate to severe traumatic brain injury. In some embodiments of the above methods, the reference level is associated with (corresponds to) a moderate traumatic brain injury. In other embodiments of the above methods, the reference level is associated with (corresponds to) a severe traumatic brain injury.

In some embodiments, the subject may be suspected of having mild TBI based on the glasgow coma scale score. In other aspects, the subject may be suspected of having moderate TBI based on the glasgow coma scale score. In other aspects, the subject may be suspected of having severe TBI based on the glasgow coma scale score. In other aspects, the subject may be suspected of having moderate to severe TBI based on the glasgow coma scale score. In other aspects, the reference level or levels of GFAP correlate or correspond to a glasgow coma scale score of 13-15 (mild TBI). In other aspects, the reference level is associated with or corresponds to a glasgow coma scale score of 3-8 (severe TBI). In other aspects, the reference level is associated with or corresponds to a glasgow coma scale score of 9-13 (moderate TBI). In other aspects, the reference level is associated with or corresponds to a glasgow coma scale score of 3-12 (moderate to severe TBI).

In some embodiments of the above methods, the reference level of cTnI is about 1.94pg/mL, about 2.54pg/mL, about 21.23pg/mL, or about 43.79 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 1.94 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 2.54 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 21.23 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 43.79 pg/mL.

In some embodiments of the above methods, the reference level (a) is determined by an assay having a sensitivity between at least about 85% and 100% and a specificity between at least about 30% and 100%; (b) as determined by an assay having a sensitivity of at least about 87.5% and a specificity of at least about 31%; or (c) between at least about 1pg/mL to about 50 pg/mL. In some embodiments of the above methods, the reference level is determined by an assay having a sensitivity between at least about 85% and 100% and a specificity between at least about 30% and 100%. In some embodiments of the above methods, the reference level is determined by an assay having a sensitivity of at least about 87.5% and a specificity of at least about 31%. In some embodiments of the above methods, the reference level is between at least about 1pg/mL to about 50 pg/mL.

In some embodiments of the above methods, the sample is obtained within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of the actual or suspected head injury. Specifically, in some embodiments of the above methods, the sample is taken within about 30 minutes of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 1 hour of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 2 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 3 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 4 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 5 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 6 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 7 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 8 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 9 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 10 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 11 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 12 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 13 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 14 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 15 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 16 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 17 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 18 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 19 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 20 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 21 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 22 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 23 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 24 hours of an actual or suspected head injury.

In some embodiments, the above methods further comprise treating the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the methods further comprise monitoring the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury prior to treatment with the traumatic brain injury therapy. In some embodiments, the methods further comprise monitoring the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury after treatment with the traumatic brain injury therapy.

In some embodiments, the method further comprises monitoring the subject assessed as having mild traumatic brain injury. In some embodiments, the above methods further comprise treating the subject assessed as having mild traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the method comprises monitoring a subject assessed as having mild traumatic brain injury prior to treatment with the traumatic brain injury therapy. In other embodiments, the method comprises monitoring a subject assessed as having mild traumatic brain injury after treatment with a traumatic brain injury treatment.

In another embodiment, the present disclosure relates to a method of assisting in determining whether to perform a head Computed Tomography (CT) scan on a human subject who has suffered or may have suffered (or has actual or suspected) a head injury. The method may comprise the steps of:

a) performing an assay on a sample obtained from the subject within about 24 hours after the actual or suspected head injury to measure or detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in the sample is above the cTnI reference level; when the level of cTnI in the sample is below the cTnI reference level, no CT scan is performed on the subject.

In some embodiments of the above methods, the subject is subjected to a CT scan. In other embodiments of the above methods, the subject is not subjected to a CT scan.

In some embodiments of the above methods, the sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of the actual or suspected head injury. Specifically, in some embodiments of the above methods, the sample is taken within about 30 minutes of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 1 hour of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 2 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 3 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 4 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 5 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 6 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 7 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 8 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 9 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 10 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 11 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 12 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 13 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 14 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 15 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 16 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 17 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 18 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 19 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 20 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 21 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 22 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 23 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 24 hours of an actual or suspected head injury.

In some embodiments of the above methods, the subject has received a CT scan before or after performing the assay, and wherein the subject is suspected of having TBI based on the CT scan results. In some embodiments, the subject may be suspected of having traumatic brain injury based on CT scans that have been performed. For example, depending on the medical condition of the subject (e.g., if the patient is unconscious), a CT scan may be performed shortly after the subject arrives at an emergency room, trauma center, or other location in order to assess and/or assess whether the subject has a TBI. Such CT scans can be provided prior to the assay to be performed to confirm and determine whether the subject has mild or moderate, severe, or moderate to severe TBI. After the determination is made, one or more subsequent CT scans may be performed based on the determination results as part of the TBI management of the physician (or other medical personnel) (e.g., to determine whether surgical and/or pharmacological intervention may be required). In other embodiments, the subject may not receive a CT scan prior to performing the assay.

In some embodiments of the above methods, the subject is suspected of having traumatic brain injury based on a CT scan. In some embodiments, the subject is diagnosed with traumatic brain injury based on a CT scan. In other embodiments, the subject is diagnosed as free of traumatic brain injury based on the CT scan.

In some embodiments of the above methods, the reference level is associated with (corresponds to) a head computed tomography positive.

In some embodiments of the above methods, the reference level is associated with (corresponds to) a control subject that has not suffered a head injury.

In some embodiments of the above methods, the reference level of cTnI is about 1.65pg/mL, about 2.16pg/mL, about 14.75pg/mL, or about 30.43 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 1.65 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 2.16 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 14.75 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 30.43 pg/mL.

In some embodiments of the above methods, the reference level (a) is determined by an assay having a sensitivity of between at least about 65% and 100% and a specificity of between at least about 30% and 100%; (b) as determined by an assay having a sensitivity of at least about 85% and a specificity of at least about 33%; or (c) between at least about 1.0pg/mL to about 50 pg/mL. In some embodiments of the above methods, the reference level is determined by an assay having a sensitivity between at least about 65% and 100% and a specificity between at least about 30% and 100%. In some embodiments of the above methods, the reference level is determined by an assay having a sensitivity of at least about 85% and a specificity of at least about 33%. In some embodiments of the above methods, the reference level is between at least about 1.0pg/mL to about 50 pg/mL.

In another embodiment, the present disclosure relates to a method of aiding in the diagnosis and assessment of mild traumatic brain injury in a human subject. The method may comprise the steps of:

a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after the head injury and the second sample is taken from the human subject about 3 hours to about 6 hours after the first sample, wherein the sample is a biological sample;

b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and

c) if the detected level of cTnI increases from the first sample to the second sample, moderate, severe, or moderate to severe traumatic brain injury is confirmed to have occurred, and if the detected level of cTnI remains unchanged or decreases from the first sample to the second sample, mild traumatic brain injury is confirmed to not have occurred.

In some embodiments of the above methods, the subject is identified as having mild traumatic brain injury. In other embodiments of the above methods, the subject is identified as having moderate traumatic brain injury. In still other embodiments of the above methods, the subject is confirmed to have a certain severe traumatic brain injury. In other embodiments, the subject is confirmed to have moderate to severe traumatic brain injury.

In some embodiments of the above methods, the first sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of the actual or suspected head injury. Specifically, in some embodiments of the above methods, the sample is taken within about 30 minutes of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 1 hour of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 2 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 3 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 4 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 5 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 6 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 7 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 8 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 9 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 10 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 11 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 12 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 13 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 14 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 15 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 16 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 17 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 18 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 19 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 20 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 21 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 22 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 23 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 24 hours of an actual or suspected head injury.

In some embodiments of the above methods, the CT of the head of the subject is abnormal.

In some embodiments of the above methods, the amount of cTnI in the first sample is about 1.0 to about 50 pg/mL.

In some embodiments of the above methods, the amount of cTnI in the second sample is about 1.0 to about 50 pg/mL.

In some embodiments of the above methods, the method further comprises treating the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the methods further comprise monitoring the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury prior to treatment with the traumatic brain injury therapy. In some embodiments, the methods further comprise monitoring the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury after treatment with the traumatic brain injury therapy.

In some embodiments, the method further comprises monitoring the subject assessed as having mild traumatic brain injury. In some embodiments, the above methods further comprise treating the subject assessed as having mild traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the method comprises monitoring a subject assessed as having mild traumatic brain injury prior to treatment with the traumatic brain injury therapy. In other embodiments, the method comprises monitoring a subject assessed as having mild traumatic brain injury after treatment with a traumatic brain injury treatment.

In another embodiment, the present disclosure relates to a method of aiding in the diagnosis and assessment of mild traumatic brain injury in a human subject. The method may comprise the steps of:

a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after the head injury and the second sample is taken from the human subject about 1 hour to about 4 hours after the first sample, wherein the sample is a biological sample;

b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and

c) if the detected level of cTnI increases from the first sample to the second sample, moderate, severe, or moderate to severe traumatic brain injury is confirmed to have occurred, and if the detected level of cTnI remains unchanged or decreases from the first sample to the second sample, mild traumatic brain injury is confirmed to not have occurred.

In some embodiments of the above methods, the subject is identified as having mild traumatic brain injury. In other embodiments of the above methods, the subject is identified as having moderate traumatic brain injury. In still other embodiments of the above methods, the subject is confirmed to have a certain severe traumatic brain injury. In other embodiments, the subject is confirmed to have moderate to severe traumatic brain injury.

In some embodiments of the above methods, the first sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of the actual or suspected head injury. Specifically, in some embodiments of the above methods, the sample is taken within about 30 minutes of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 1 hour of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 2 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 3 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 4 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 5 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 6 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 7 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 8 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 9 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 10 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 11 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 12 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 13 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 14 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 15 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 16 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 17 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 18 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 19 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 20 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 21 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 22 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 23 hours of an actual or suspected head injury. In other embodiments of the above methods, the sample is taken within about 24 hours of an actual or suspected head injury.

In some embodiments of the above methods, the CT of the head of the subject is abnormal.

In some embodiments of the above methods, the amount of cTnI in the first sample is about 1.0 to about 50 pg/mL.

In some embodiments of the above methods, the amount of cTnI in the second sample is about 1.0 to about 50 pg/mL.

In some embodiments of the above methods, the method further comprises treating the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the methods further comprise monitoring the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury prior to treatment with the traumatic brain injury therapy. In some embodiments, the methods further comprise monitoring the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury after treatment with the traumatic brain injury therapy.

In some embodiments, the method further comprises monitoring the subject assessed as having mild traumatic brain injury. In some embodiments, the above methods further comprise treating the subject assessed as having mild traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the method comprises monitoring a subject assessed as having mild traumatic brain injury prior to treatment with the traumatic brain injury therapy. In other embodiments, the method comprises monitoring a subject assessed as having mild traumatic brain injury after treatment with a traumatic brain injury treatment.

In another embodiment, the present disclosure relates to a method of aiding diagnosis and assessment of a human subject who has suffered or may have suffered (or has actual or suspected) a head injury. The method may comprise the steps of:

a) performing an assay on a sample obtained from the subject within about 2 hours after the head injury or suspected injury to measure or detect a level of cTnI in the sample; and

b) determining whether the subject suffers from mild or moderate, severe, or moderate to severe Traumatic Brain Injury (TBI), wherein the subject is determined to be (1) moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI in the sample is above the cTnI reference level or (2) mild traumatic brain injury when the level of cTnI in the sample is below the cTnI reference level.

In some embodiments of the above methods, the subject receives a glasgow coma scale score before or after performing the assay. In some embodiments, the subject may be suspected of having traumatic brain injury based on a previously conducted glasgow coma scale score. For example, depending on the medical condition of the subject, the glasgow coma scale score may be assessed shortly after the subject arrives at an emergency room, trauma center, or other location in order to assess and/or assess whether the subject has a TBI. Such a glasgow coma scale score may be provided prior to the assay to be performed to confirm and determine whether the subject has mild or moderate, severe, or moderate to severe TBI. After the determination is made, one or more subsequent glasgow coma scale scores may be made based on the determination results as part of the TBI management of the physician (or other medical personnel) (e.g., to determine whether surgical and/or pharmacological intervention may be required). In other embodiments, the subject may not receive a glasgow coma scale score prior to performing the assay.

In some embodiments, the subject may be suspected of having mild TBI based on the glasgow coma scale score. In other aspects, the subject may be suspected of having moderate TBI based on the glasgow coma scale score. In other aspects, the subject may be suspected of having severe TBI based on the glasgow coma scale score. In other aspects, the subject may be suspected of having moderate to severe TBI based on the glasgow coma scale score. In other aspects, the reference level or levels of GFAP correlate or correspond to a glasgow coma scale score of 13-15 (mild TBI). In other aspects, the reference level is associated with or corresponds to a glasgow coma scale score of 3-8 (severe TBI). In other aspects, the reference level is associated with or corresponds to a glasgow coma scale score of 9-13 (moderate TBI). In other aspects, the reference level is associated with or corresponds to a glasgow coma scale score of 3-12 (moderate to severe TBI).

In some embodiments of the above methods, the reference level of cTnI is about 1.15 pg/mL. In some embodiments of the above methods, the reference level of cTnI is about 1.29 pg/mL.

In some embodiments of the above methods, the reference level (a) is determined by an assay having a sensitivity between at least about 85% and 100% and a specificity between at least about 30% and 100%; (b) as determined by an assay having a sensitivity of at least about 87.5% and a specificity of at least about 31%; or (c) between at least about 0.5pg/mL to about 30 pg/mL. In some embodiments of the above methods, the reference level is determined by an assay having a sensitivity between at least about 85% and 100% and a specificity between at least about 30% and 100%. In some embodiments of the above methods, the reference level is determined by an assay having a sensitivity of at least about 87.5% and a specificity of at least about 31%. In some embodiments of the above methods, the reference level is between at least about 0.5pg/mL to about 50 pg/mL.

In some embodiments of the above methods, the sample is taken within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, or within about 90 minutes after the actual or suspected head injury. In some embodiments of the above methods, the sample is taken within about 5 minutes after the actual or suspected head injury. In other embodiments, the sample is taken within about 10 minutes of a suspected head injury. In still other embodiments, the sample is taken within about 12 minutes of a suspected head injury. In still other embodiments, the sample is taken within about 15 minutes of a suspected head injury. In still other embodiments, the sample is taken within about 20 minutes of a suspected head injury. In still other embodiments, the sample is taken within about 60 minutes of a suspected head injury. In still other embodiments, the sample is taken within about 90 minutes of a suspected head injury.

In some embodiments, the methods further comprise monitoring the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury prior to treatment with the traumatic brain injury therapy. In some embodiments, the methods further comprise monitoring the subject assessed as having moderate, severe, or moderate to severe traumatic brain injury after treatment with the traumatic brain injury therapy.

In some embodiments, the method further comprises monitoring the subject assessed as having mild traumatic brain injury. In some embodiments, the above methods further comprise treating the subject assessed as having mild traumatic brain injury with a traumatic brain injury treatment. In some embodiments, the method comprises monitoring a subject assessed as having mild traumatic brain injury prior to treatment with the traumatic brain injury therapy. In other embodiments, the method comprises monitoring a subject assessed as having mild traumatic brain injury after treatment with a traumatic brain injury treatment.

In another embodiment, the present disclosure relates to a method of assisting in determining whether to perform a head Computed Tomography (CT) scan on a human subject who has suffered or may have suffered (or has actual or suspected) a head injury. The method may comprise the steps of:

a) performing an assay on a sample obtained from the subject within about 2 hours after the actual or suspected head injury to measure or detect a level of cTnI in the sample; and

b) performing a CT scan on the subject when the level of cTnI in the sample is above the cTnI reference level; when the level of cTnI in the sample is below the cTnI reference level, no CT scan is performed on the subject.

In some embodiments of the above methods, the subject is subjected to a CT scan. In other embodiments of the above methods, the subject is not subjected to a CT scan.

In some embodiments of the above methods, the subject has received a CT scan before or after performing the assay, and wherein the subject is suspected of having TBI based on the CT scan results. In some embodiments, the subject may be suspected of having traumatic brain injury based on CT scans that have been performed. For example, depending on the medical condition of the subject (e.g., if the patient is unconscious), a CT scan may be performed shortly after the subject arrives at an emergency room, trauma center, or other location in order to assess and/or assess whether the subject has a TBI. Such CT scans can be performed prior to the assay to be performed to confirm and determine whether the subject has mild or moderate, severe, or moderate to severe TBI. After the determination is made, one or more subsequent CT scans may be performed based on the determination results as part of the TBI management of the physician (or other medical personnel) (e.g., to determine whether surgical and/or pharmacological intervention may be required). In other embodiments, the subject may not receive a CT scan prior to performing the assay.

In some embodiments of the above methods, the subject is suspected of having traumatic brain injury based on a CT scan. In some embodiments, the subject is diagnosed with traumatic brain injury based on a CT scan. In some embodiments, the subject is diagnosed as free of traumatic brain injury based on a CT scan.

In some embodiments of the above methods, the reference level is associated with (corresponds to) a head computed tomography positive.

In some embodiments of the above methods, the reference level is associated with (corresponds to) a control subject that has not suffered a head injury.

In some embodiments of the methods above, the reference level of cTnI is about 5.8pg/mL in some embodiments of the methods above, the reference level of cTnI is about 4.7pg/mL.

In some embodiments of the above methods, the reference level (a) is determined by an assay having a sensitivity of between at least about 65% and 100% and a specificity of between at least about 30% and 100%; (b) as determined by an assay having a sensitivity of at least about 85% and a specificity of at least about 33%; or (c) between at least about 0.5pg/mL to about 25 pg/mL. In some embodiments of the above methods, the reference level is determined by an assay having a sensitivity between at least about 65% and 100% and a specificity between at least about 30% and 100%. In some embodiments of the above methods, the reference level is determined by an assay having a sensitivity of at least about 85% and a specificity of at least about 33%. In some embodiments of the above methods, the reference level is between at least about 0.5pg/mL to about 25 pg/mL.

In some embodiments of the above methods, the sample is taken within about 5 minutes, within about 10 minutes, within about 12 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 60 minutes, or within about 90 minutes after the actual or suspected head injury. In some embodiments of the above methods, the sample is taken within about 5 minutes after the actual or suspected head injury. In other embodiments, the sample is taken within about 10 minutes of a suspected head injury. In still other embodiments, the sample is taken within about 12 minutes of a suspected head injury. In still other embodiments, the sample is taken within about 15 minutes of an actual or suspected head injury. In still other embodiments, the sample is taken within about 20 minutes of an actual or suspected head injury. In still other embodiments, the sample is taken within about 60 minutes of an actual or suspected head injury. In still other embodiments, the sample is taken within about 90 minutes of an actual or suspected head injury.

In yet another embodiment, the present disclosure relates to a method of treating mild or moderate, severe, or moderate to severe TBI, the method comprising: a) performing an assay on a sample obtained from the subject within about 24 hours after the head injury to measure or detect the level of cTnI; b) determining whether the subject suffers from mild or moderate to severe Traumatic Brain Injury (TBI), wherein the subject is determined to be (1) moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI in the sample is above the cTnI reference level or (2) mild traumatic brain injury when the level of cTnI in the sample is below the cTnI reference level; and c) treating the subject assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury with a traumatic brain injury treatment.

Embodiments of the above methods further comprise monitoring the subject assessed as having mild traumatic brain injury. Embodiments of the above methods further comprise monitoring a subject assessed as having moderate, severe, or moderate to severe traumatic brain injury.

In some embodiments of the above methods, the treatment of traumatic brain injury in a subject with mild TBI can comprise allowing the subject to rest for a period of time, avoiding physical activity for a period of time, administering one or more therapeutic agents (e.g., a drug that provides relief from headache or migraine headache, etc.), or a combination thereof. In other embodiments of the above methods, a subject with moderate, severe, or moderate to severe TBI is treated for traumatic brain injury comprising administering one or more therapeutic agents (e.g., a medication such as a diuretic, an antiepileptic drug, etc.), performing one or more surgical procedures (e.g., removing a hematoma, repairing a skull fracture, dehiscence of a flap, decompression, etc.), receiving or providing one or more therapies (e.g., rehabilitation, physical therapy, occupational therapy, cognitive behavior therapy, anger management, etc.), or any combination thereof. Optionally, such methods may also include providing one or more cardioprotective therapies. Such cardioprotective therapies can be administered, depending on the situation, in combination with treatment for TBI, or alone without any TBI treatment.

In yet another embodiment, the present disclosure relates to a method of treating mild or moderate, severe, or moderate to severe traumatic brain injury in a human subject, the method comprising: a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point and the second sample is taken from the human subject about 3 hours to about 6 hours after the first sample, wherein the sample is a biological sample; b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; c) confirming the occurrence of moderate to severe traumatic brain injury if the detected level of cTnI increases from the first sample to the second sample; confirming the absence of mild traumatic brain injury if the detected level of cTnI remains unchanged or decreases from the first sample to the second sample; and d) treating the subject assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury with a traumatic brain injury treatment.

Embodiments of the above methods further comprise monitoring the subject assessed as having mild traumatic brain injury. Embodiments of the above methods further comprise monitoring a subject assessed as having moderate, severe, or moderate to severe traumatic brain injury.

In some embodiments of the above methods, the treatment of traumatic brain injury in a subject with mild TBI can comprise allowing the subject to rest for a period of time, avoiding physical activity for a period of time, administering one or more therapeutic agents (e.g., a drug that provides relief from headache or migraine headache, etc.), or a combination thereof. In other embodiments of the above methods, the subject with moderate, severe, or moderate to severe TBI is treated for traumatic brain injury comprising administering one or more therapeutic agents (e.g., a medication such as a diuretic, an antiepileptic, etc.), performing one or more surgical procedures (e.g., removing a hematoma, repairing a skull fracture, dehiscence of a flap, etc.), receiving or providing one or more therapies (e.g., rehabilitation, physical therapy, occupational therapy, cognitive behavior therapy, anger management, etc.), or any combination thereof. Optionally, such methods may also include providing one or more cardioprotective therapies. Such cardioprotective therapies can be administered, depending on the situation, in combination with treatment for TBI, or alone without any TBI treatment.

In yet another embodiment, the present disclosure relates to a method of treating mild or moderate, severe, or moderate to severe traumatic brain injury in a human subject, the method comprising: a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point and the second sample is taken from the human subject about 1 hour to about 4 hours after the first sample, wherein the sample is a biological sample; b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; c) confirming the occurrence of moderate to severe traumatic brain injury if the detected level of cTnI increases from the first sample to the second sample; confirming the absence of mild traumatic brain injury if the detected level of cTnI remains unchanged or decreases from the first sample to the second sample; and d) treating the subject assessed as having mild, moderate, severe, or moderate to severe traumatic brain injury with a traumatic brain injury treatment.

Embodiments of the above methods further comprise monitoring the subject assessed as having mild traumatic brain injury. Embodiments of the above methods further comprise monitoring a subject assessed as having moderate, severe, or moderate to severe traumatic brain injury.

In some embodiments of the above methods, the treatment of traumatic brain injury in a subject with mild TBI can comprise allowing the subject to rest for a period of time, avoiding physical activity for a period of time, administering one or more therapeutic agents (e.g., a drug that provides relief from headache or migraine headache, etc.), or a combination thereof. In other embodiments of the above methods, the subject with moderate, severe, or moderate to severe TBI is treated for traumatic brain injury comprising administering one or more therapeutic agents (e.g., a medication such as a diuretic, an antiepileptic, etc.), performing one or more surgical procedures (e.g., removing a hematoma, repairing a skull fracture, dehiscence of a flap, etc.), receiving or providing one or more therapies (e.g., rehabilitation, physical therapy, occupational therapy, cognitive behavior therapy, anger management, etc.), or any combination thereof. Optionally, such methods may also include providing one or more cardioprotective therapies. Such cardioprotective therapies can be administered, depending on the situation, in combination with treatment for TBI, or alone without any TBI treatment.

In yet another embodiment, the present disclosure relates to a method for aiding in predicting or predicting the outcome of a human subject having mild traumatic brain injury, the method comprising: a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within 24 hours after the head injury and the second sample is taken from the human subject about 0 to about 4 hours after the first sample, wherein the sample is a biological sample; b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and c) predicting an adverse outcome for the human subject if the detected level of cTnI increases from the first sample to the second sample; a favorable outcome is predicted if the detected level of cTnI remains unchanged, decreases, or increases by less than about 20% from the first sample to the second sample.

In some embodiments of the above methods, the first sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours after the head injury. In another embodiment, the first sample is taken from the subject within about 30 minutes after the head injury. In another embodiment, the first sample is taken from the subject within about 1 hour after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 2 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 3 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 4 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 5 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 6 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 7 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 8 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 9 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 10 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 11 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 12 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 13 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 14 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 15 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 16 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 17 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 18 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 19 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 20 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 21 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 22 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 23 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 24 hours after the head injury.

In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 1 month post head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 1 month after the head injury. In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 3 months after the head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 3 months after the head injury. In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 6 months after the head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 6 months after the head injury.

In some embodiments of the above methods, the CT scan of the subject's head is normal.

In some embodiments of the above methods, the subject has an abnormal CT scan of the head.

In some embodiments of the above methods, the subject has received a glasgow coma scale score before or after performing the assay. In some embodiments, the subject may be suspected of having traumatic brain injury based on a previously conducted glasgow coma scale score. For example, depending on the medical condition of the subject, the glasgow coma scale score may be assessed shortly after the subject arrives at an emergency room, trauma center, or other location in order to assess and/or assess whether the subject has a TBI. Such a glasgow coma scale score may be provided prior to the assay being performed to confirm and determine whether the subject has mild or moderate, severe, or moderate to severe TBI. After the determination is made, one or more subsequent glasgow coma scale scores may be made based on the determination results as part of the TBI management of the physician (or other medical personnel) (e.g., to determine whether surgical and/or pharmacological intervention may be required). In other embodiments, the subject may not have received a glasgow coma scale score prior to performing the assay.

In some embodiments of the above methods, the subject has an expanded Glasgow Outcome (GOSE) score of 5 or less. In some embodiments of the above methods, the GOSE score is obtained before or after performing the assay.

In some embodiments of the above methods, a subject assessed as having adverse outcome (with mild TBI or moderate, severe, or moderate to severe TBI) is treated with a traumatic brain injury treatment. In some embodiments of the above methods, the treatment of traumatic brain injury in a subject with mild TBI can comprise allowing the subject to rest for a period of time, avoiding physical activity for a period of time, administering one or more therapeutic agents (e.g., a drug that provides relief from headache or migraine headache, etc.), or a combination thereof. In other embodiments of the above methods, a subject with moderate, severe, or moderate to severe TBI is treated for traumatic brain injury comprising administering one or more therapeutic agents (e.g., a medication such as a diuretic, an antiepileptic drug, etc.), performing one or more surgical procedures (e.g., removing a hematoma, repairing a skull fracture, dehiscence of a flap, decompression, etc.), receiving or providing one or more therapies (e.g., rehabilitation, physical therapy, occupational therapy, cognitive behavior therapy, anger management, etc.), or any combination thereof.

In some embodiments of the above methods, a subject assessed as having an adverse outcome may also be monitored. The subject may be monitored regardless of whether the subject is undergoing traumatic brain injury therapy or other therapy (e.g., one or more cardioprotective therapies).

The present disclosure relates to a method for aiding in predicting or predicting the outcome of a human subject with mild traumatic brain injury, the method comprising: a) performing an assay on a sample obtained from the subject within about 28 hours after the head injury to measure or detect the level of cTnI, wherein the sample is a biological sample; and b) predicting an adverse outcome in the human subject if the level of cTnI in the sample is above the cTnI reference level; predicting a favorable outcome for the human subject if the level of cTnI in the sample is below the cTnI reference level.

In some embodiments of the above methods, the first sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours after the head injury. In another embodiment, the first sample is taken from the subject within about 30 minutes after the head injury. In another embodiment, the first sample is taken from the subject within about 1 hour after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 2 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 3 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 4 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 5 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 6 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 7 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 8 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 9 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 10 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 11 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 12 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 13 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 14 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 15 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 16 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 17 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 18 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 19 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 20 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 21 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 22 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 23 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 24 hours after the head injury.

In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 1 month post head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 1 month after the head injury. In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 3 months after the head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 3 months after the head injury. In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 6 months after the head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 6 months after the head injury.

In some embodiments of the above methods, the CT scan of the subject's head is normal.

In some embodiments of the above methods, the subject has an abnormal CT scan of the head.

In some embodiments of the above methods, the subject has received a glasgow coma scale score before or after performing the assay. In some embodiments, the subject may be suspected of having traumatic brain injury based on a previously conducted glasgow coma scale score. For example, depending on the medical condition of the subject, the glasgow coma scale score may be assessed shortly after the subject arrives at an emergency room, trauma center, or other location in order to assess and/or assess whether the subject has a TBI. Such a glasgow coma scale score may be provided prior to the assay being performed to confirm and determine whether the subject has mild or moderate, severe, or moderate to severe TBI. After the determination is made, one or more subsequent glasgow coma scale scores may be made based on the determination results as part of the TBI management of the physician (or other medical personnel) (e.g., to determine whether surgical and/or pharmacological intervention may be required). In other embodiments, the subject may not have received a glasgow coma scale score prior to performing the assay.

In some embodiments of the above methods, the subject has an expanded Glasgow Outcome (GOSE) score of 5 or less. In some embodiments of the above methods, the GOSE score is obtained before or after performing the assay.

In some embodiments of the above methods, the reference level is a reference level determined by an assay having a sensitivity between at least about 80% and 100% and a specificity between at least about 45% and 100%. In some embodiments of the above methods, the reference level is a reference level determined by an assay having a sensitivity of at least about 83.3% and a specificity of at least about 54.9%. In some embodiments of the above methods, the reference level is a reference level determined by an assay having a sensitivity of at least about 100% and a specificity of at least about 49.2%. In some embodiments of the above methods, the reference level is at least between about 1pg/mL to about 50 pg/mL.

In other embodiments of the above methods, the reference level of cTnI is about 5.6pg/mL in other embodiments of the above methods, the reference level of cTnI is about 5.7pg/mL.

In some embodiments of the above methods, a subject assessed as having adverse outcome (with mild TBI or moderate, severe, or moderate to severe TBI) is treated with a traumatic brain injury treatment. In some embodiments of the above methods, the treatment of traumatic brain injury in a subject with mild TBI can comprise allowing the subject to rest for a period of time, avoiding physical activity for a period of time, administering one or more therapeutic agents (e.g., a drug that provides relief from headache or migraine headache, etc.), or a combination thereof. In other embodiments of the above methods, a subject with moderate, severe, or moderate to severe TBI is treated for traumatic brain injury comprising administering one or more therapeutic agents (e.g., a medication such as a diuretic, an antiepileptic drug, etc.), performing one or more surgical procedures (e.g., removing a hematoma, repairing a skull fracture, dehiscence of a flap, decompression, etc.), receiving or providing one or more therapies (e.g., rehabilitation, physical therapy, occupational therapy, cognitive behavior therapy, anger management, etc.), or any combination thereof.

In some embodiments of the above methods, a subject assessed as having an adverse outcome may also be monitored. The subject may be monitored regardless of whether the subject is undergoing traumatic brain injury therapy or other therapy (e.g., one or more cardioprotective therapies).

In yet another embodiment, the present disclosure relates to a method for aiding in predicting or predicting the outcome of a human subject with mild traumatic brain injury, the method comprising: a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after the head injury and the second sample is taken from the human subject about 4 hours after the first sample, wherein the sample is a biological sample; b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and c) predicting an adverse outcome for the human subject if the level of cTnI in the sample increases from the first sample to the second sample; predicting a favorable outcome for the human subject if the level of cTnI in the sample remains unchanged or decreases from the first sample to the second sample.

In some embodiments of the above methods, the first sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours after the head injury. In another embodiment, the first sample is taken from the subject within about 30 minutes after the head injury. In another embodiment, the first sample is taken from the subject within about 1 hour after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 2 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 3 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 4 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 5 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 6 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 7 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 8 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 9 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 10 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 11 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 12 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 13 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 14 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 15 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 16 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 17 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 18 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 19 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 20 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 21 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 22 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 23 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 24 hours after the head injury.

In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 1 month post head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 1 month after the head injury. In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 3 months after the head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 3 months after the head injury. In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 6 months after the head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 6 months after the head injury.

In some embodiments of the above methods, the CT scan of the subject's head is normal.

In some embodiments of the above methods, the subject has an abnormal CT scan of the head.

In some embodiments of the above methods, the subject has received a glasgow coma scale score before or after performing the assay. In some embodiments, the subject may be suspected of having traumatic brain injury based on a previously conducted glasgow coma scale score. For example, depending on the medical condition of the subject, the glasgow coma scale score may be assessed shortly after the subject arrives at an emergency room, trauma center, or other location in order to assess and/or assess whether the subject has a TBI. Such a glasgow coma scale score may be provided prior to the assay being performed to confirm and determine whether the subject has mild or moderate, severe, or moderate to severe TBI. After the determination is made, one or more subsequent glasgow coma scale scores may be made based on the determination results as part of the TBI management of the physician (or other medical personnel) (e.g., to determine whether surgical and/or pharmacological intervention may be required). In other embodiments, the subject may not have received a glasgow coma scale score prior to performing the assay.

In some embodiments of the above methods, the subject has an expanded Glasgow Outcome (GOSE) score of 5 or less. In some embodiments of the above methods, the GOSE score is obtained before or after performing the assay.

In some embodiments of the above methods, the amount of cTnI in the first sample is about 1.0 to about 50 pg/mL.

In some embodiments of the above methods, the amount of cTnI in the second sample is about 1.0 to about 50 pg/mL.

In some embodiments of the above methods, the level of cTnI decreases or increases by at least one absolute amount from the first sample to the second sample. More specifically, the absolute amount is determined by an assay having a sensitivity between at least about 80% and 100% and a specificity between at least about 45% and 100%. In some embodiments of the above methods, the absolute amount is determined by an assay having a sensitivity of at least about 82.4% and a specificity of at least about 69.5%. In some embodiments of the above method, the absolute amount is between at least about 1pg/mL to about 50 pg/mL.

In some embodiments of the above method, the absolute amount is about 5.6pg/mL.

In some embodiments of the above methods, a subject assessed as having adverse outcome (with mild TBI or moderate, severe, or moderate to severe TBI) is treated with a traumatic brain injury treatment. In some embodiments of the above methods, the treatment of traumatic brain injury in a subject with mild TBI can comprise allowing the subject to rest for a period of time, avoiding physical activity for a period of time, administering one or more therapeutic agents (e.g., a drug that provides relief from headache or migraine headache, etc.), or a combination thereof. In other embodiments of the above methods, a subject with moderate, severe, or moderate to severe TBI is treated for traumatic brain injury comprising administering one or more therapeutic agents (e.g., a medication such as a diuretic, an antiepileptic drug, etc.), performing one or more surgical procedures (e.g., removing a hematoma, repairing a skull fracture, dehiscence of a flap, decompression, etc.), receiving or providing one or more therapies (e.g., rehabilitation, physical therapy, occupational therapy, cognitive behavior therapy, anger management, etc.), or any combination thereof.

In some embodiments of the above methods, a subject assessed as having an adverse outcome may also be monitored. The subject may be monitored regardless of whether the subject is undergoing traumatic brain injury therapy or other therapy (e.g., one or more cardioprotective therapies).

The present disclosure relates to a method of aiding diagnosis or assessment of mild traumatic brain injury in a human subject, the method comprising: a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point and the second sample is taken from the human subject about 1 hour to about 4 hours after the first sample, wherein the sample is a biological sample; b) determining whether the amount of cTnI increases or decreases from the first sample to the second sample; and c) confirming the occurrence of moderate to severe traumatic brain injury if the detected level of cTnI increases from the first sample to the second sample; the absence of mild traumatic brain injury is confirmed if the detected level of cTnI remains unchanged or decreases from the first sample to the second sample.

The present disclosure relates to a method for aiding in predicting or predicting the outcome of a human subject with mild traumatic brain injury, the method comprising: a) performing an assay on a sample from a human subject to measure or detect a level of cTnI in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after the head injury and the second sample is taken from the human subject about 0 to about 4 hours after the first sample, wherein the sample is a biological sample; b) determining the age of the subject; and c) predicting an adverse outcome for the human subject if the cTnI level in the first sample and/or the second sample is above the cTnI reference level and the age of the subject is above the reference age, and predicting a favorable outcome for the human subject if the cTnI level in the first sample and the second sample is below the cTnI reference level and/or the age of the subject is below the reference age.

In some embodiments of the above methods, the first sample is obtained from the subject within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours after the head injury. In another embodiment, the first sample is taken from the subject within about 30 minutes after the head injury. In another embodiment, the first sample is taken from the subject within about 1 hour after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 2 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 3 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 4 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 5 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 6 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 7 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 8 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 9 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 10 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 11 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 12 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 13 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 14 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 15 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 16 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 17 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 18 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 19 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 20 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 21 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 22 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 23 hours after the head injury. In some embodiments of the above methods, the first sample is obtained from the subject within about 24 hours after the head injury.

In some embodiments of the above methods, the subject is 18 to 30 years of age. In other embodiments of the above methods, the subject is 31 to 50 years of age. In still other embodiments of the above methods, the subject is 51 to 70 years of age. In still other embodiments of the above methods, the subject is 70 to 100 years of age. In still other embodiments of the above methods, the age is 18 years or less. In still other embodiments of the above methods, the age is 19-50 years. In yet other embodiments, the age is 51 to 70 years. In still other embodiments of the above methods, the age is greater than 70 years. In still other embodiments of the above methods, the subject is 20 to 30 years of age. In still other embodiments of the above methods, the subject is 31 to 40 years of age. In still other embodiments of the above methods, the subject is from 41 to 50 years of age. In still other embodiments of the above methods, the subject is 51 to 60 years of age. In still other embodiments of the above methods, the subject is 61 to 70 years of age. In still other embodiments of the above methods, the subject is 71 to 80 years of age. In still other embodiments of the above methods, the subject is 81 to 90 years of age. In still other embodiments of the above methods, the subject is 91 to 100 years of age.

In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 1 month post head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 1 month after the head injury. In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 3 months after the head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 3 months after the head injury. In some embodiments of the above methods, the method predicts a favorable outcome for the subject at about 6 months after the head injury. In some embodiments of the above methods, the method predicts that the subject is an adverse outcome at about 6 months after the head injury.

In some embodiments of the above methods, the CT scan of the subject's head is normal.

In some embodiments of the above methods, the subject has an abnormal CT scan of the head.

In some embodiments of the above methods, the subject has received a glasgow coma scale score before or after performing the assay. In some embodiments, the subject may be suspected of having traumatic brain injury based on a previously conducted glasgow coma scale score. For example, depending on the medical condition of the subject, the glasgow coma scale score may be assessed shortly after the subject arrives at an emergency room, trauma center, or other location in order to assess and/or assess whether the subject has a TBI. Such a glasgow coma scale score may be provided prior to the assay being performed to confirm and determine whether the subject has mild or moderate, severe, or moderate to severe TBI. After the determination is made, one or more subsequent glasgow coma scale scores may be made based on the determination results as part of the TBI management of the physician (or other medical personnel) (e.g., to determine whether surgical and/or pharmacological intervention may be required). In other embodiments, the subject may not have received a glasgow coma scale score prior to performing the assay.

In some embodiments of the above methods, the subject has an expanded Glasgow Outcome (GOSE) score of 5 or less. In some embodiments of the above methods, the GOSE score is obtained before or after performing the assay.

In some embodiments of the above methods, a subject assessed as having adverse outcome (with mild TBI or moderate, severe, or moderate to severe TBI) is treated with a traumatic brain injury treatment. In some embodiments of the above methods, the treatment of traumatic brain injury in a subject with mild TBI can comprise allowing the subject to rest for a period of time, avoiding physical activity for a period of time, administering one or more therapeutic agents (e.g., a drug that provides relief from headache or migraine headache, etc.), or a combination thereof. In other embodiments of the above methods, a subject with moderate, severe, or moderate to severe TBI is treated for traumatic brain injury comprising administering one or more therapeutic agents (e.g., a medication such as a diuretic, an antiepileptic drug, etc.), performing one or more surgical procedures (e.g., removing a hematoma, repairing a skull fracture, dehiscence of a flap, decompression, etc.), receiving or providing one or more therapies (e.g., rehabilitation, physical therapy, occupational therapy, cognitive behavior therapy, anger management, etc.), or any combination thereof.

In some embodiments of the above methods, a subject assessed as having an adverse outcome may also be monitored. The subject may be monitored regardless of whether the subject is undergoing traumatic brain injury therapy or other therapy (e.g., one or more cardioprotective therapies).

In some embodiments, in any of the above methods, the method further comprises providing a cardioprotective therapy to the subject. Such cardioprotective therapy can be any cardioprotective therapy known in the art, optionally including, but not limited to, one or more of a beta-blocker, a diuretic, an Angiotensin Converting Enzyme (ACE) inhibitor, a calcium channel blocker, a lipid lowering therapy, a statin, a nitrate, an antiplatelet agent, an anticoagulant, or a combination thereof. Such one or more cardioprotective therapies may be administered alone or in combination with one or more traumatic brain injury treatments.

In some embodiments of any of the above methods, the level of cTnI is measured by an immunoassay or a clinical chemistry assay. In other embodiments, the level of cTnI in the above methods can be measured as follows:

A. the sample is contacted with the following antibodies, simultaneously or sequentially in any order:

(1) a cTnI capture antibody that binds to an epitope on cTnI or a cTnI fragment to form a cTnI capture antibody-cTnI antigen complex, and

(2) a cTnI detection antibody comprising a detectable label and binding to an epitope on cTnI that is not bound by the cTnI capture antibody to form a cTnI antigen-cTnI detection antibody complex,

thereby forming a cTnI capture antibody-cTnI antigen-cTnI detection antibody complex, an

B. Measuring the amount or concentration of cTnI in the sample based on the signal generated by the detectable label in the cTnI capture antibody-cTnI antigen-cTnI detection antibody complex.

In some embodiments of any of the above methods, the sample is selected from the group consisting of a whole blood sample, a serum sample, a cerebrospinal fluid sample, and a plasma sample. In some embodiments of the above methods, the sample is obtained after the subject has suffered a head injury from physical shock, blunt impact of external mechanical or other forces resulting in closed or open head trauma, one or more drops, explosions or explosions, or other types of blunt force trauma. In other embodiments of the above methods, the sample is obtained after the subject has ingested or been exposed to a chemical, a toxin, or a combination of a chemical and a toxin. In some embodiments of the above method, the chemical substance or toxin is fire, mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, a glue, a gas, an organometallic, a drug of abuse, or one or more combinations thereof. In some embodiments of the above methods, the sample is obtained from a subject having an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a virus, meningitis, hydrocephalus, or a combination thereof.

In some embodiments of any of the above methods, the method can be performed on any subject without regard to factors selected from the group consisting of: clinical condition of the subject, laboratory value of the subject, grade of the subject having mild, moderate, severe, or moderate to severe traumatic brain injury, low or high cTnI levels in the subject, and timing of any event in which the subject may suffer head injury.

In some embodiments, in any of the above methods, the sample is a whole blood sample.

In some embodiments, in any of the above methods, the sample is a plasma sample.

In some embodiments, in any of the above methods, the sample is a serum sample.

In some embodiments, in any of the above methods, the assay is an immunoassay.

In some embodiments, in any of the above methods, the assay is a clinical chemistry assay.

In some embodiments, in any of the above methods, the assay is a single molecule detection assay.

In some embodiments, in any of the above methods, the sample is a whole blood sample and the assay is an immunoassay. In other embodiments, the sample is a plasma sample and the assay is an immunoassay. In still other embodiments, the sample is a serum sample and the assay is an immunoassay. In other embodiments, in the above methods, the sample is a whole blood sample and the assay is a clinical chemistry assay. In other embodiments, the sample is a plasma sample and the assay is a clinical chemistry assay. In still other embodiments, the sample is a serum sample and the assay is a clinical chemistry assay. In other embodiments, in the above methods, the sample is a whole blood sample and the assay is a single molecule detection assay. In other embodiments, the sample is a plasma sample and the assay is a single molecule detection assay. In still other embodiments, the sample is a serum sample and the assay is a single molecule detection assay.

Drawings

FIG. 1 shows a boxplot of the results of hsTnI measurements at time points.

Fig. 2 shows a boxplot of the correlation of hsTnI assay results with positive versus negative CT scan results at time point 1 (taken within 0 to 6 hours after head injury) and time point 2 (taken 3 to 6 hours after the sample at time point 1).

Fig. 3 shows a box plot of the absolute magnitude ("absolute Δ") of the hsTnI results (i.e., the absolute difference between time point 2 (taken 3 to 6 hours after the sample at time point 1) and time point 1 (taken within 0 to 6 hours after head injury)) versus the positive versus negative CT scan results.

Fig. 4 shows a boxplot of the hsTnI assay results versus mild to moderate/severe TBI GCS scores at time point 1 (taken within 0 to 6 hours after head injury) and time point 2 (taken within 3 to 6 hours after the sample at time point 1).

Fig. 5 shows a box plot of the absolute amount ("absolute Δ") of the hsTnI assay results (i.e., the absolute difference between time point 1 (taken within 0 to 6 hours after head injury) and time point 2 (taken 3 to 6 hours after time point 1 sample)) versus light to moderate/severe TBI GCS scores.

Figure 6 shows biomarker hsTnI results versus time since injury was judged based on CT scan results.

Figure 7 shows biomarker hsTnI results versus time from injury judged based on Glasgow Coma Scale (GCS) score.

Figure 8 shows a Receiver Operating Characteristics (ROC) analysis of the correlation of hsTnI levels to CT status (positive versus negative CT scan results) in samples taken within about 2 hours of suspected injury. The sample at time point 1 was taken within 2 hours after the head injury, while the sample at time point 2 was taken from about 3 to about 6 hours after the sample at time point 1 was taken.

Figure 9 shows a Receiver Operating Characteristics (ROC) analysis of hsTnI levels in samples taken within about 2 hours of suspected injury correlating to mild to moderate/severe TBI GCS scores. The sample at time point 1 was taken within 2 hours after the head injury, while the sample at time point 2 was taken from about 3 to about 6 hours after the sample at time point 1 was taken.

Figure 10 shows ROC curves correlating the results of hsTnI assays with positive versus negative CT scans for all subjects at time point 1.

Figure 11 shows ROC curves correlating the hsTnI assay results with mild to moderate/severe TBI GCS scores for all subjects at time point 1.

Figure 12 shows ROC analysis of the correlation of absolute amounts ("absolute Δ") of hsTnI assay results (i.e., the absolute difference between hsTnI levels at time point 2 and hsTnI levels at time point 1) to CT status (positive versus negative CT scan results). The sample at time point 1 was taken within 2 hours after the head injury, while the sample at time point 2 was taken from about 3 to about 6 hours after the sample at time point 1 was taken.

Figure 13 shows ROC analysis of the correlation of the absolute amount of hsTnI assay results ("absolute Δ") (i.e., the absolute difference between the hsTnI level at time point 2 and the hsTnI level at time point 1) to the mild to moderate/severe TBI GCS scores. The sample at time point 1 was taken within 2 hours after the head injury, while the sample at time point 2 was taken from about 3 to about 6 hours after the sample at time point 1 was taken.

Figure 14 shows ROC curves relating absolute amounts of hsTnI assay results ("absolute Δ") to positive versus negative CT scan results for all subjects.

Figure 15 shows ROC curves relating absolute amounts of hsTnI assay results ("absolute Δ") to positive versus negative CT scan results for all subjects.

Figure 16 shows the distribution of hsTnI levels (p ═ 0.0001) in TBI patients and trauma control patients.

Fig. 17 shows the distribution of hsTnI levels (p ═ 0.005) in TBI patients with abnormal and normal head CT scans.

Figure 18 shows the distribution of hsTnI levels (p ═ 0.46) in TBI patients with GCS scores <13, 14 or 15.

Figure 19 shows the distribution of hsTnI levels at 1 month post-injury in TBI patients with a GOSE score of 1, 3, 4,5, 6, 7 or 8 (p ═ 0.0001).

Figure 20 shows the distribution of hsTnI levels at 3 months post-injury in TBI patients with a GOSE score of 1, 2, 3, 4,5, 6, 7 or 8 (p ═ 0.02).

Figure 21 shows the distribution of hsTnI levels at 6 months post-injury in TBI patients with a GOSE score of 1, 3, 4,5, 6, 7 or 8 (p ═ 0.11).

Fig. 22 shows the distribution of hsTnI levels in samples taken at 0 hours, 4 hours, 24 hours, and 1 month post-injury in TBI patients with abnormal and normal head CT scans.

Figure 23 shows the distribution of the 4 hour absolute change in hsTnI levels at 1 month post injury in TBI patients with a GOSE score of 1, 3, 4,5, 6, 7 or 8.

Fig. 24 shows the following age-based: distribution of hsTnI levels in TBI patients aged 18 or less, 19 to 50 years, 51 to 70 years, and greater than 70 years.

Figure 25 shows the distribution of hsTnI values in TBI participants and trauma control participants. hsTnI values were higher in TBI participants than in trauma control participants.

Figure 26 shows a graphical display of the correlation between cardiac troponin I values and TBI outcome.

Figure 27 shows an area Analysis Under Receiver Operating Characteristics (AUROC) curve 0.7135 for subjects with 1 month GOSE <5 and GOSE >5 using initial hsTnI levels.

FIG. 28 shows an AUROC analysis using 4 hour hsTnI levels to distinguish subjects with 1 month GOSE <5 from GOSE >5 (AUROC curve ═ 0.7715).

FIG. 29 shows an AUROC analysis (AUROC curve 0.7070) using the relative change between initial and 4 hour hsTnI levels to distinguish subjects with 1 month GOSE <5 and GOSE > 5.

FIG. 30 shows an AUROC analysis (AUROC curve 0.7868) using 24-hour hsTnI levels to distinguish subjects with 1-month GOSE <5 > from GOSE > 5.

Figure 31 shows AUROC analysis (AUROC 0.8408) for a predictive model including initial hsTnI levels, 4 hour hsTnI levels, and age.

Detailed Description

The present disclosure relates to several findings involving the use of cardiac troponin i (ctni) as a biomarker associated with Traumatic Brain Injury (TBI). Cardiac troponin I is well known in the art as a highly specific biomarker for cardiac muscle injury. Indeed, for this purpose, there are several commercial assays available for measuring cTnI in blood or plasma. However, it is also known in the art that cardiac dysfunction is often observed in subjects with severe TBI. However, the significance of such cardiac dysfunction in these subjects is traditionally poorly understood.

In contrast, the present disclosure provides a new and improved method of assessing, diagnosing and/or stratifying whether a subject suffering from or believed to suffer from head injury has mild TBI, moderate TBI, severe TBI, moderate to severe TBI or no TBI at all, using the level and/or change in level of cTnI (e.g., by performing an assay to determine the level of cTnI in one or more biological samples and then comparing those levels to one or more reference levels) as an aid. The methods described herein can be performed rapidly — within only 2 hours and up to about 24 hours after a head injury or suspected injury. Distinguishing mild, moderate, severe, moderate to severe, or no TBI with cTnI in this manner was previously unknown. For patients identified or determined to have TBI, performing such methods not only allows the physician to quickly determine and classify (or reclassify) or treat the class of patients with TBI or without TBI, but the methods described herein also allow the physician to determine the type of TBI (mild to moderate, severe, or moderate to severe). The ability to quickly determine whether TBI is classified as mild, moderate, severe, or moderate to severe enables a physician to prescribe an appropriate course of treatment (e.g., a treatment plan) for a subject. Such a treatment plan may include whether (1) one or more additional tests are required to obtain further clinical information about TBI (e.g., MRI, etc.); (2) start (continue) monitoring the subject; (3) initiating treatment of the subject with a traumatic brain injury therapy (what type of therapy should be initiated if the therapy is initiated (e.g., one or more therapeutic therapies, protecting the airway, one or more surgical therapies, requiring rest, etc.), (4) initiating any cardioprotective therapy to protect the heart of the subject (e.g., optionally, by administering one or more beta-blockers, diuretics, angiotensin converting inhibitors, calcium channel blockers, lipid lowering therapies, statins, nitrates, antiplatelet therapies, anticoagulants, or combinations thereof known in the art), or (5) conducting any combination of (1) - (4).

Additionally, the present disclosure provides methods of using the level or change in level of cTnI as an aid in determining whether head Computed Tomography (CT) should be performed on a subject suffering from or believed to suffer from TBI. The methods described herein can be performed rapidly — within only 2 hours and up to about 24 hours after a head injury or suspected injury. The use of cTnI as an aid to help physicians determine whether head CT is to be performed on subjects with or thought to have TBI is previously unknown.

In addition, the disclosure also provides methods of using the level or change in level of cTnI to determine outcome in subjects with mild TBI. In particular, the methods described herein can be used to determine whether a subject diagnosed with mild TBI is more likely to have (1) a favorable outcome (optionally, the favorable outcome may be complete recovery of the subject and not continue to experience one or more symptoms of mild TBI); or (2) an adverse outcome (optionally, the adverse outcome may be one or more symptoms that the subject has not fully recovered and continues to experience mild TBI).

Alternatively, and optionally, favorable outcome may refer to a subject more likely to suffer no more than one symptom of postconcussion syndrome due to mild TBI, for example: (a) physical difficulties (e.g., headache, dizziness, fatigue, sensitivity to light noise and light, etc.); (b) cognitive difficulties (e.g., inattention, memory problems, restlessness, etc.); (c) mood difficulties (e.g., personality changes, irritability, depression, apathy, etc.); or (d) difficulty in sleeping (e.g., insomnia, etc.). Alternatively, and optionally, subjects with adverse outcome are more likely to suffer from more than one postconcussion syndrome symptom, such as: (a) physical difficulties (e.g., headache, dizziness, fatigue, sensitivity to light noise and light, etc.); (b) cognitive difficulties (e.g., inattention, memory problems, restlessness, etc.); (c) mood difficulties (e.g., personality changes, irritability, depression, apathy, etc.); or (d) difficulty sleeping (e.g., insomnia, etc.); or (e) any combination of (a) - (d). Alternatively, and optionally, adverse outcome may also refer to a subject exhibiting one or more symptoms of mild TBI. Alternatively, and optionally, adverse outcome may also refer to an exacerbation of the subject from mild TBI to moderate, moderate to severe, or severe. Furthermore, subjects with favorable outcomes may have a GOSE score of 5 or higher, while subjects with unfavorable outcomes may have a GOSE score of less than 5.

A subject who is unlikely to recover completely from mild TBI will likely require one or more additional therapeutic treatments, physical therapies and/or occupational therapies thereof following diagnosis of mild TBI for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years or 50 years. The use of cTnI to predict outcome in subjects with mild TBI was previously unknown.

In addition, the present disclosure provides methods of treating traumatic brain injury. In particular, the methods comprise assessing, diagnosing and/or stratifying whether a subject suffering from head injury or believed to suffer from mild TBI, moderate TBI, severe TBI, moderate to severe TBI or no TBI, using the levels and/or changes in levels of cTnI described herein (e.g., by performing assays to determine the level of cTnI in one or more biological samples and then comparing these levels to one or more reference levels). Once a subject has been identified, determined, classified or stratified as having mild TBI or moderate, severe, or moderate to severe TBI, the subject is treated with an appropriate traumatic brain injury treatment depending on the type of TBI (mild to moderate, severe, or moderate to severe). For example, for mild TBI, traumatic brain injury treatment may include resting the subject for a period of time, avoiding physical activity for a period of time, administering one or more therapeutic agents (e.g., drugs that provide relief from headache or migraine, etc.), or a combination thereof. For moderate, severe, or moderate to severe TB, traumatic brain injury treatment may include administering one or more therapeutic agents (e.g., drugs such as diuretics, antiepileptics, etc.), performing one or more surgical procedures (e.g., removing hematoma, repairing skull fracture, dehiscent decompression surgery, etc.), receiving or providing one or more therapies (e.g., rehabilitation, physical therapy, occupational therapy, cognitive behavioral therapy, anger management, etc.), or any combination thereof. Optionally, such methods may also include providing one or more cardioprotective therapies. Such cardioprotective therapies can be administered, depending on the situation, in combination with treatment for TBI, or alone without any TBI treatment.

In addition to performing the above-described methods, one skilled in the art (e.g., a physician) will also know and understand how to perform additional tests to detect or assess other co-diseases (e.g., other diseases, disorders, or conditions besides TBI). Furthermore, to confirm that a change in the amount or level of cTnI in the methods described herein is attributable to head injury or suspected head injury in the subject, rather than the outcome of an acute cardiac syndrome (e.g., myocardial infarction, heart failure, etc.), a physician or other health care provider may perform or conduct one or more additional tests or procedures to confirm the absence of an acute cardiac syndrome. Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

The section headings as used in this section and throughout the disclosure herein are for organizational purposes only and are not meant to be limiting.

1. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

As used herein, the terms "comprising," "including," "having," "may," "containing," and variations thereof, are not intended to exclude the possibility of additional acts or structures. The singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also encompasses other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

For the recitation of numerical ranges herein, each intervening number there between with the same degree of accuracy is specifically contemplated. For example, for the range 6-9, the numbers 7 and 8 are encompassed in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly encompassed.

"affinity matured antibody" is used herein to refer to an antibody having one or more changes in one or more CDRs that result in the affinity of the antibody for a target antigen (i.e., K) as compared to a parent antibody that does not have the changes_D、k_dOr k_a) And (4) improving. Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Various procedures for generating affinity matured antibodies are known in the art, including screening of combinatorial antibody libraries made using biological display. For example, Marks et al, Biotechnology 10:779-783(1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: barbas et al, Proc.Nat.Acad.Sci.USA,91: 3809-; schier et al, Gene,169:147-155 (1995); yelton et al, J.Immunol.,155:1994-2004 (1995); jackson et al, J.3310-3319 (1995); and Hawkins et al, J.mol.biol.,226:889-896 (1992). Selective mutagenesis at a site of selective mutagenesis and at a contact or hypermutation site by an activity enhancing amino acid residue is described in U.S. Pat. No.6,914,128B 1.

"antibody" and "antibodies" as used herein refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, birds (e.g., ducks or geese), sharks, whales and mammals (including non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) or non-human primates (e.g., monkeys, chimpanzees, etc.)), recombinant antibodies, chimeric antibodies, single chain Fv ("scFv"), single chain antibodies, single domain antibodies, Fab fragments, F (ab')₂Fragments, disulfide-linked Fv ("sdFv") and anti-idiotypic ("anti-Id") antibodies, double domain antibodies, Double Variable Domain (DVD) or Triple Variable Domain (TVD) antibodies (double variable domain immunoglobulins and methods of making the same are described in Wu, C et al, nature biotechnology,25(11):1290-1297(2007) and PCT international application WO2001/058956, the contents of each incorporated herein by reference), and epitope-binding fragments of the functional activity of any of the foregoing antibodies. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity, an antibody to an analyte is generally referred to herein as an "anti-analyte antibody" or simply an "analyte antibody" (e.g., an anti-cardiac troponin I antibody, or a cardiac troponin I antibody).

As used herein, "antibody fragment" refers to a portion of an intact antibody that comprises an antigen binding site or variable region. The portion does not include the constant heavy chain domain of the entire antibody Fc region (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab '-SH fragment, F (ab')₂Fragments, Fd fragments, Fv fragments, diabodies, single chain Fv (scfv) molecules, single chain polypeptides comprising only one light chain variable domain, single chain polypeptides comprising three CDRs of a light chain variable domain, single chain polypeptides comprising only one heavy chain variable region, and single chain polypeptides comprising three CDRs of a heavy chain variable region.

"area under the curve" or "AUC" refers to the area under the ROC curve. AUC under the ROC curve is a measure of accuracy. An AUC of 1 indicates a perfect test, while an AUC of 0.5 indicates a meaningless test. Preferred AUCs may be at least about 0.700, at least about 0.750, at least about 0.800, at least about 0.850, at least about 0.900, at least about 0.910, at least about 0.920, at least about 0.930, at least about 0.940, at least about 0.950, at least about 0.960, at least about 0.970, at least about 0.980, at least about 0.990, or at least about 0.995.

"bead" and "particle" are used interchangeably herein and refer to a substantially spherical solid support. One example of a bead or particle is a microparticle. The microparticles useful herein may be of any type known in the art. For example, the beads or particles may be magnetic beads or magnetic particles. The magnetic beads/particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO₂MnAs, MnBi, EuO and NiO/Fe. Examples of ferrimagnetic materials include NiFe₂O₄、CoFe₂O₄、Fe₃O₄(or FeO' Fe)₂O₃). The beads may have a magnetic solid core portion and be surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer surrounding a non-magnetic core. The microparticles can have any size that is functional in the methods described herein, for example, from about 0.75 to about 5nm, or from about 1 to about 3 nm.

"binding protein" is used herein to refer to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as, for example, a polypeptide, antigen, chemical compound or other molecule, or any kind of substrate. The binding protein specifically binds to a binding partner. Binding proteins include antibodies, as well as antigen-binding fragments thereof and other various forms and derivatives thereof known in the art and described below, as well as other molecules comprising one or more antigen-binding domains that bind to an antigenic molecule or a specific site (epitope) on an antigenic molecule. Thus, binding proteins include, but are not limited to, antibodies, tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity matured antibodies, and fragments of any such antibodies that retain the ability to bind antigen.

"bispecific antibody" is used herein to refer to a full-length antibody produced by the following technique: tetragenic hybridoma technology (see Milstein et al, Nature,305(5934):537-540 (1983)); chemical conjugation by two different monoclonal antibodies (see Staerz et al, Nature,314(6012):628-631 (1985)); or by a button-in-hole method or similar method (see Holliger et al, Proc. Natl. Acad. Sci. USA,90(14):6444-6448(1993)) which introduces mutations in the Fc region, which results in a variety of different immunoglobulin substances, only one of which is a functional bispecific antibody. Bispecific antibodies bind one antigen (or epitope) on one of their two binding arms (one HC/LC pair) and a different antigen (or epitope) on their second arm (the other HC/LC pair). According to this definition, bispecific antibodies have two different antigen binding arms (in terms of both specificity and CDR sequences) and are monovalent for each antigen to which they bind.

As used herein, the terms "cardiac troponin I", "cTnI" or "troponin I" are used interchangeably herein to refer to one of two distinct forms of cardiac troponin (the other distinct form being cardiac troponin T (also referred to as "cTnT")) that is released from the heart muscle into the blood where several may be present. The term "cardiac troponin I" or "cTnI" includes not only the full-length form of this form but also: (1) various complexes of cTnI (i.e. with each other and/or with cardiac troponin c (ctnc)); (2) a cTnI fragment resulting from proteolytic degradation; (3) phosphorylated and oxidized forms of cTnI (see, e.g., U.S. patent No.6,991,907, the contents of which are incorporated herein by reference); (4) any isoform of cTnI.

In some embodiments, the methods of the present disclosure allow for the detection and/or determination of the concentration of one or more of multiple forms of cTnI as individual entities in a sample, such as complexed cTnI, free cTnI (e.g., full length, fragments, isoforms, etc.), turbid cTnI (e.g., oxidized or phosphorylated), and optionally provide for the concentration of cTnI in a biological sample.

More specifically, in some embodiments, the disclosures described herein use high sensitivity assays that allow for the detection and quantification of cTnI at levels 10-100 fold lower than levels determined by traditional troponin assays known in the art (e.g., immunoassays). More specifically, an assay is defined as being highly sensitive (e.g., a highly sensitive assay for troponin) if such an assay satisfies at least two of the following conditions: : 1) coefficient of variance less than 10% in reference to the 99 th percentile of healthy population and 2) concentrations above the limit of detection can be measured in more than 50% of healthy individuals (see Apple FS et al, Clin chem.,58:54-61(2012), the contents of which are incorporated herein by reference). Examples of assays known in the art that allow for highly sensitive detection of Troponin include assays available from Quanterix (Simoa Human Troponin-I immunoassay) for research purposes only, as well as those described in U.S. patent No. 9,182,405, the contents of which are incorporated herein by reference.

As used interchangeably herein, a "cardiac troponin I state" or "cTnI state" may refer to the level or amount of cardiac troponin I at a certain point in time (e.g., using a single measure of troponin I), monitoring the level or amount of cardiac troponin I associated (e.g., repeated testing of a subject to confirm an increase or decrease in the amount of cardiac troponin I), the level or amount of cardiac troponin I associated with treatment of traumatic brain injury (whether primary brain injury and/or secondary brain injury), or a combination thereof.

"CDR" is used herein to refer to "complementarity determining regions" within antibody variable sequences. There are three CDRs in each variable region of the heavy and light chains. For each variable region, starting from the N-terminus of the heavy or light chain, these regions are denoted as "CDR 1", "CDR 2" and "CDR 3", respectively. The term "set of CDRs" as used herein refers to a set of three CDRs present in a single variable region that bind antigen. Thus, the antigen binding site may comprise six CDRs comprising a set of CDRs from each of the heavy and light chain variable regions. Crystallography analysis of polypeptides comprising a single CDR (e.g., CDR1, CDR2, or CDR3), which may be referred to as "molecular recognition unit" antigen-antibody complexes, has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR 3. Thus, the molecular recognition unit may be primarily responsible for the specificity of the antigen binding site. In general, CDR residues are directly and most substantially involved in affecting antigen binding.

The precise boundaries of these CDRs have been defined differently for different systems. The system described by Kabat (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides the precise residue boundaries that define the three CDRs, which may be referred to as "Kabat CDRs" Chothia and colleagues (Chothia and Lesk, J.mol.biol.,196:901-917(1987) and Chothia et al, Nature 342:877-883(1989)) found that, despite the large diversity at the level of amino acid Sequences, certain sub-portions within Kabat CDRs employ nearly identical conformational peptide backbones that are designated as "L1", "L2" and "L26" or "H1" and "H2", where these sub-portions are designated as "L1" and "H.6782" light CDR regions, which may be referred to as "heavy chain CDR boundaries" and "other CDRs" H6757 "that overlap with the chain CDRs, FASEB J.,9: 133-. Other CDR boundary definitions may not strictly follow one of the systems herein, but will still overlap with the Kabat CDRs, but they may be shortened or lengthened given that a particular residue or group of residues, or even the entire CDR, does not significantly affect the prediction or experimental findings of antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, but certain embodiments use Kabat or Chothia defined CDRs.

"component," "components," or "at least one component" generally refers to a capture antibody, detector, or conjugate, calibrator, control, sensitivity group, container, buffer, diluent, salt, enzyme, cofactor for the enzyme, detection reagent, pretreatment reagent/solution, substrate (e.g., as a solution), stop solution, and the like, that can be included in a kit for assaying a test sample (such as a patient urine, whole blood, serum, or plasma sample) according to the methods described herein and other methods known in the art. Some components may be in solution or lyophilized for reconstitution for use in an assay.

As used herein, "associated with …" means compared to ….

"CT scan" as used herein refers to a Computed Tomography (CT) scan. CT scans combine a series of X-ray images taken from different angles and use computer processing to create cross-sectional images or slices of bones, vessels and soft tissue in your body. The CT scan may use X-ray CT, Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), computed axial tomography (CAT scan), or computer-assisted tomography. The CT scan may be a conventional CT scan or a helical/spiral CT scan. In a conventional CT scan, the scan is performed slice by slice, and after each slice the scan is stopped and moved down to the next slice, e.g., from the top of the abdomen down to the pelvis. Conventional CT scans require the patient to hold their breath to avoid motion artifacts. Helical/spiral CT scanning is a continuous scan, which is taken in a spiral fashion, and is a faster process in which the scanned images are continuous.

As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence compared to the authentic or parent antibody and exhibits a modified domain structure. Derivatives may still be able to adopt the typical domain configurations found in natural antibodies, as well as amino acid sequences capable of specifically binding to the target (antigen). Typical examples of antibody derivatives are antibodies conjugated to other polypeptides, rearranged antibody domains, or antibody fragments. The derivative may also comprise at least one other compound, such as a protein domain, which is linked by covalent or non-covalent bonds. The ligation may be based on genetic fusion according to methods known in the art. The further domains present in the fusion protein comprising the antibody may preferably be linked by a flexible linker, advantageously a peptide linker, wherein said peptide linker comprises a plurality of hydrophilic peptide-bonded amino acids of sufficient length to span the distance between the C-terminus of the further protein domain and the N-terminus of the antibody, or vice versa. The antibody may be linked to an effector molecule having a conformation suitable for bioactive or selective binding to, for example, a solid support, a bioactive substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug.

"determined by assay" is used herein to refer to the determination of a reference level by any suitable assay. In some embodiments, determining the reference level may be accomplished by the same type of assay as the assay to be administered to the sample from the subject (e.g., by immunoassay, clinical chemistry assay, single molecule detection assay, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE, and western blot analysis, or western immunostaining, electrophoretic analysis, protein assay, competitive binding assay, functional protein assay, or chromatography or spectroscopy, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS)). In some embodiments, determining the reference level may be achieved by the same type of assay and under the same assay conditions as the assay to be administered to the sample from the subject. As noted herein, the present disclosure provides exemplary reference levels (e.g., calculated by comparing reference levels at different time points). It is well within the ability of one of ordinary skill in the art to modify the disclosure herein with respect to other assays based on the description provided herein to obtain assay-specific reference levels for those other assays. For example, a set of training samples, including samples obtained from human subjects known to suffer damage to the head (and more particularly, samples obtained from human subjects known to suffer (i) mild TBI; and/or (ii) moderate, severe, or moderate to severe TBI) and samples obtained from human subjects known not to suffer damage to the head, can be used to obtain assay-specific reference levels Within the abilities of one of ordinary skill in the art.

Indeed, the skilled person will balance the effect of the cut-off on sensitivity and specificity when distinguishing between subjects suffering or not suffering from traumatic brain injury, or subjects with mild and moderate, severe or moderate to severe traumatic brain injury. Raising or lowering the cutoff will have a clear and predictable impact on sensitivity and specificity as well as other standard statistics. It is well known that increasing the cut-off will increase specificity but may decrease sensitivity (the proportion of disease patients who test positive). In contrast, decreasing the cut-off will increase sensitivity but decrease specificity (the proportion of disease-free patients who test negative). It will be apparent to those skilled in the art that the consequences of detecting traumatic brain injury or determining mild to moderate, severe or moderate to severe traumatic brain injury will be apparent. In distinguishing whether a subject has traumatic brain injury or mild versus moderate, severe, or moderate to severe traumatic brain injury, the higher the cutoff, the higher the specificity, the more true negatives (i.e., the subject has no traumatic brain injury, no mild traumatic brain injury, no moderate traumatic brain injury, no severe traumatic brain injury, or no moderate to severe traumatic brain injury), as distinguished from a person having brain injury, mild brain injury, moderate brain injury, severe brain injury, or moderate to severe brain injury. However, at the same time, increasing the cut-off value reduces the number of cases identified as positive overall, as well as the number of true positives, and therefore sensitivity is necessarily reduced. Conversely, the lower the cut-off, the higher the sensitivity, the more true positive (e.g., with craniocerebral trauma, with mild craniocerebral trauma, with moderate craniocerebral trauma, with severe craniocerebral trauma, or with moderate to severe brain trauma), as distinguished from those without brain trauma, mild brain trauma, moderate brain trauma, severe brain trauma, or moderate to severe brain trauma. However, at the same time, decreasing the cut-off value increases the number of cases identified as positive overall, as well as the number of false positives, and thus specificity necessarily decreases.

Generally, high sensitivity values help a technician rule out a disease or condition (e.g., traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate to severe traumatic brain injury), and high specificity values help a technician incorporate a disease or condition. The skilled artisan will appreciate that the desire to exclude or include the disease depends on the outcome of each type of error to the patient. Therefore, the exact balance used to derive the test cut-off cannot be known or predicted without fully disclosing the underlying information on how to select the value. The balance between sensitivity and specificity and other factors will be determined as appropriate. This is why it is sometimes desirable to provide an alternative cut-off value (e.g. a reference value) so that the physician or practitioner can choose.

"drug of abuse" is used herein to refer to one or more additive substances (such as a drug) that are ingested for non-medical reasons (such as, for example, recreational and/or mental effects). Excessive drowsiness, use or dependence of such abused drugs is often referred to as "drug abuse". Examples of drugs of abuse include alcohol, barbiturates, benzodiazepines, cannabis, ***e, hallucinogens (such as ketamine, moschatine (peyote)), PCP, nudaphantin, DMT, and/or LSD), methaqualone, opioids, amphetamines (including methamphetamine), anabolic steroids, inhalants (i.e., substances containing volatile substances with psychotropic properties, such as, for example, nitrites, spray paints, cleaning fluids, markers, glues, and the like), and combinations thereof.

"Dual specific antibody" is used herein to refer to a full length antibody that can bind two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see PCT publication WO 02/02773). Thus, a dual specific binding protein has two identical antigen binding arms with the same specificity and the same CDR sequences, and is bivalent for each antigen it binds to.

"Dual variable domain" is used herein to refer to two or more binding sites on a binding protein, which may be bivalent (two antigen binding sites), tetravalent (four antigen binding sites), or multivalent. DVDs can be monospecific, i.e., capable of binding one antigen (or one specific epitope), or multispecific, i.e., capable of binding two or more antigens (i.e., two or more epitopes of the same antigenic molecule or two or more epitopes of different target antigens), a preferred DVD binding protein comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides and is referred to as a "DVD immunoglobulin" or "DVD-Ig". Such DVD-Ig binding proteins are therefore tetrameric and similar to IgG molecules, but provide more antigen binding sites than IgG molecules. Thus, each half of a tetrameric DVD-Ig molecule is similar to one half of an IgG molecule and comprises a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike the pair of heavy and light chains of an IgG molecule which provide a single antigen binding domain, the pair of heavy and light chains of a DVD-Ig provide two or more antigen binding sites.

Each antigen binding site of the DVD-Ig binding protein may be derived from a donor ("parent") monoclonal antibody, and thus comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) having a total of six CDRs each of which is involved in antigen binding. Thus, a DVD-Ig binding protein that binds two different epitopes (i.e., two different epitopes of two different antigenic molecules or two different epitopes of the same antigenic molecule) comprises an antigen binding site derived from a first parent monoclonal antibody and an antigen binding site of a second parent monoclonal antibody.

A description of the design, expression and characterization of DVD-Ig binding molecules is provided in PCT publication WO 2007/024715, U.S. Pat. No. 7,612,181 and Wu et al, NatureBiotech.,25:1290-1297 (2007). Preferred examples of such DVD-Ig molecules comprise a heavy chain comprising the structural formula VD1- (X1) n-VD2-C- (X2) n, wherein VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker (provided that it is not CH1, X2 is an Fc region), and n is 0 or 1, but preferably 1; and a light chain comprising VD1- (X1) n-VD2-C- (X2) n, wherein VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker (provided that it is not CH1, and X2 does not comprise an Fc region); and n is 0 or 1, but preferably 1. Such a DVD-Ig may comprise two such heavy chains and two such light chains, wherein each chain comprises variable domains connected in tandem without intervening constant regions between the variable domains, wherein the heavy and light chains associate to form a tandem functional antigen binding site, and one pair of heavy and light chains may associate with the other pair of heavy and light chains to form a tetrameric binding protein having four functional antigen binding sites. In another example, a DVD-Ig molecule can comprise heavy and light chains each comprising three variable domains (VD1, VD2, VD3) connected in series, without intervening constant regions between the variable domains, wherein one pair of heavy and light chains can associate to form three antigen binding sites, and wherein one pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetramer binding protein having six antigen binding sites.

In preferred embodiments, the DVD-Ig binding protein not only binds to the same target molecule bound by its parent monoclonal antibody, but also has the desired properties of one or more of its parent monoclonal antibodies. Preferably, such additional property is an antibody parameter of one or more of the parent monoclonal antibodies. Antibody parameters that may contribute to a DVD-Ig binding protein from one or more of its parent monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and orthologous antigen binding.

The DVD-Ig binding protein binds to at least one epitope in cardiac troponin I. Non-limiting examples of DVD-Ig binding proteins include DVD-Ig binding proteins that bind one or more epitopes in cardiac troponin I, DVD-Ig binding proteins that bind an epitope of human cardiac troponin I and an epitope of cardiac troponin I of another species (e.g., mouse), and DVD-Ig binding proteins that bind an epitope of human cardiac troponin I and an epitope of another target molecule.

As used herein, "dynamic range" refers to the range of an assay reading that is proportional to the amount of a target molecule or analyte in a sample being analyzed.

An "epitope" or "epitopes" or "epitope of interest" refers to any site on a molecule that is recognized and can bind to a complementary site on its specific binding partner. The molecule and the specific binding partner are part of a specific binding pair. For example, the epitope may be in a polypeptide, protein, hapten, carbohydrate antigen (such as, but not limited to, glycolipid, glycoprotein, or lipopolysaccharide), or polysaccharide. Its specific binding partner may be, but is not limited to, an antibody.

As used herein, "fragment antigen-binding fragment" or "Fab fragment" refers to a fragment of an antibody that binds an antigen and contains one antigen-binding site, one complete light chain, and a portion of one heavy chain. Fab is a monovalent fragment consisting of the VL, VH, CL and CH1 domains. Fab consists of one constant domain and one variable domain for each of the heavy and light chains. The variable domain contains a paratope (antigen binding site) that comprises a set of complementarity determining regions at the amino terminus of the monomer. Each arm of Y thus binds an epitope on the antigen. The Fab fragment can be produced as already described in the art, for example, using the enzyme papain, which can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant methods.

"F (ab')₂Fragment "refers to an antibody produced by pepsin digestion of an entire IgG antibody to remove most of the Fc region, while retaining some of the hinge region. F (ab')₂Fragments have two antigen-binding f (ab) moieties linked together by disulfide bonds and are therefore bivalent, having a molecular weight of about 110 kDa. Bivalent antibody fragment (F (ab')₂Fragments) are smaller than whole IgG molecules and are better able to penetrate into tissues, thereby promoting better antigen recognition in immunohistochemistry. F (ab')₂The use of fragments also avoids non-specific binding to Fc receptors or protein a/G on living cells. F (ab')₂The fragments can bind to and precipitate the antigen.

As used herein, "framework" (FR) or "framework sequence" can mean the remaining sequence of the variable region minus the CDRs. Because the precise definition of a CDR sequence can be determined by different systems (e.g., see above), the meaning of a framework sequence is susceptible to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2 and CDR-L3 for the light chain and CDR-H1, CDR-H2 and CDR-H3 for the heavy chain) also divide the framework regions on the light and heavy chains into four subregions on each chain (FR1, FR2, FR3 and FR4), with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR 4. Without specifying a particular sub-region as FR1, FR2, FR3 or FR4, the framework regions as otherwise mentioned represent the combined FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR denotes one of the four sub-regions, and FR denotes two or more of the four sub-regions constituting the framework region.

Human heavy and light chain FR sequences are known in the art and can be used as heavy and light chain "recipient" framework sequences (or simply "recipient" sequences) to humanize non-human antibodies by using techniques known in the art. In one embodiment, the human heavy and light chain recipient sequences are selected from publicly available databases such as V-base (hyper transfer protocol:// vbase. mr-cpe. cam. ac. uk /) or International ImmunoGene

Framework sequences listed in the information System (hypertext transfer protocol:// imgt. circuits. fr/texts/IMGTreperotoreire/Locusgenes /).

As used herein, "functional antigen binding site" may mean a site on a binding protein (e.g., an antibody) that is capable of binding a target antigen. The antigen binding affinity of the antigen binding site may not be as strong as the parent binding protein, e.g., parent antibody, from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any of a variety of methods known for assessing binding of proteins, e.g., antibodies, to antigens. Furthermore, the antigen binding affinity of each antigen binding site of a multivalent protein, e.g., multivalent antibody, herein need not be quantitatively the same.

As used herein, "glasgow coma scale" or "GCS" refers to a 15-component table used to estimate and classify brain injury outcomes based on overall social competence or dependence on others. The test measures motor response, verbal response, and eye-open response using the following values: I. motor responses (6-full compliance command; 5-location upon noxious stimulation; 4-retraction from noxious stimulation; 3-abnormal flexion, i.e. a state of the cortical shell; 2-extension, i.e. a state of the cerebrum; and 1-no response); II. Verbal response (5-alert and directional; 4-speech confusion but coherent; 3-word inappropriate and word-composed phrase confusion; 2-unintelligible sound; and 1-no sound; and III, open eyes (4-spontaneous open eyes; 3-open eyes while talking; 2-open eyes while painful; 1-open eyes; final score is determined by adding values of I + II + III.) the final score can be divided into four possible levels of survival, with lower numbers indicating more severe impairment and poorer prognosis: mild (13-15); moderate disability (9-12) (loss of consciousness for more than 30 minutes; possible or possible withdrawal of physical or cognitive impairment: and benefit from rehabilitation), severe disability (3-8) (coma: unconsciousness state: no meaningful response, no voluntary activity); and vegetative state (less than 3) (wake cycle; wake but no interaction with the environment; no localized response to pain). Moderate brain damage is defined as brain damage that causes loss of consciousness for 20 minutes to 6 hours and a glasgow coma scale of 9 to 12 points. Severe brain damage is defined as brain damage that causes loss of consciousness for more than 6 hours and has a glasgow coma scale of 3 to 8.

As used herein, "glasgow outcome scale" refers to a global scale for functional outcomes that scores patient status as one of five categories: death, vegetative state, severe disability, moderate disability or good recovery.

The "extended glasgow outcome scale" or "GOSE", used interchangeably herein, provides more detailed eight classifications by subdividing the categories of severe disability, moderate disability, and good recovery into the low-level categories and high-level categories in table 1.

TABLE 1

The term "humanized antibody" is used herein to describe antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., mouse) but in which at least a portion of the VH and/or VL sequences have become more "human-like", i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody, or a variant, derivative, analog or fragment thereof, that immunospecifically binds to an antigen of interest and comprises a Framework (FR) region having substantially the amino acid sequence of a human antibody and a Complementarity Determining Region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR whose amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. Humanized antibodies comprise substantially all of at least one and usually two variable domains (Fab, Fab ', F (ab')₂FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodimentsIn which the humanized antibody comprises a light chain and at least the variable domain of the heavy chain. Antibodies may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only humanized heavy chains. In particular embodiments, the humanized antibody contains only the humanized variable domains of the light chain and/or the humanized heavy chain.

Humanized antibodies may be selected from any class of immunoglobulin including IgM, IgG, IgD, IgA, and IgE, as well as any isotype including, but not limited to, IgG1, IgG2, IgG3, and IgG 4. Humanized antibodies may comprise sequences from more than one class or isotype, and specific constant domains may be selected to optimize desired effector function using techniques well known in the art.

The framework regions and CDRs of the humanized antibody need not correspond exactly to the parental sequences, e.g., the donor antibody CDRs or the consensus framework can be mutagenized by substituting, inserting, or/and deleting at least one amino acid residue such that the CDRs or framework residues at that site do not correspond to the donor antibody or the consensus framework. However, in preferred embodiments, such mutations will not be extensive. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework regions in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed by the most commonly occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" may comprise a "consensus framework region" and/or a "consensus CDR. In the immunoglobulin family, each position in the consensus sequence is occupied by the amino acid most commonly found at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.

"hyperacute" as used herein refers to being very acute or within about 2 hours of a head injury or suspected injury. Hyperacute is in an early stage, e.g., hyperacute biomarkers that can be used are early biomarkers, such as cTnI, that can be used to assess injury or suspected injury within about 2 hours of the early stage of injury or suspected injury.

"identical" or "identity" as used herein in the context of two or more polypeptide or polynucleotide sequences may mean that the sequences have a specified percentage of identical residues over a specified region. The percentage may be calculated by: optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions of the identical residue in the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions within the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. Where two sequences are of different lengths or are aligned to produce one or more staggered ends and the designated regions of comparison contain only a single sequence, the residues of the single sequence are included in the denominator, rather than the numerator, of the calculation.

"injury to the head" or "head injury" as used interchangeably herein refers to any trauma to the scalp, skull, or brain. Such injuries may include only a mild impact on the skull or may be severe brain injuries. Such injuries include primary injuries to the brain and/or secondary injuries to the brain. Primary brain injury occurs during the initial insult and is caused by the displacement of the physical structure of the brain. More specifically, primary brain injury is physical damage to parenchyma (tissue, blood vessels) that occurs during a traumatic event, resulting in shear and compression of surrounding brain tissue. Secondary brain injury occurs after primary injury and may involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period of time (from hours to days) after a primary brain injury. It includes the entire cascade of cellular, chemical, tissue or vascular changes in the brain that contribute to further destruction of brain tissue.

The damage to the head may be either occlusive or open (penetrating). Closed head injury refers to trauma to the scalp, skull, or brain in which the skull is not penetrated by an impacting object. Open head injury refers to a trauma to the scalp, skull, or brain in which the skull is penetrated by an impacting object. Head injuries may be caused by physical agitation of a person, by blunt impacts from external mechanical or other forces (e.g., traffic accidents such as in the case of automobiles, airplanes, trains, etc.; such as head strikes with baseball bats or from firearms), cerebrovascular accidents (e.g., stroke), one or more falls (e.g., such as sports or other activities), explosions or shock waves (collectively, "shock wave injuries"), and by other types of blunt force injuries. Alternatively, damage to the head may result from ingestion and/or exposure to chemicals, toxins, or a combination of chemicals and toxins. Examples of such chemicals and/or toxins include fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometallics (such as methyl mercury, tetraethyl lead, and organotin), and/or one or more drugs of abuse. Alternatively, the damage to the head may be due to the subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, one or more viruses, meningitis, hydrocephalus, hypoxia, or any combination thereof. In some cases, it is not possible to determine whether any such event or injury has occurred. For example, the patient or subject may have no medical history, the subject may not be able to speak, the subject may be aware of the event to which they were exposed, and the like. Such a situation is described herein as a subject "may have suffered a head injury". In certain embodiments herein, closed head injury does not include, and specifically excludes, cerebrovascular accidents, such as stroke.

As used herein, "isolated polynucleotide" may mean a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin, or a combination thereof) that, depending on its origin, is not associated with all or a portion of the polynucleotide at which the "isolated polynucleotide" is found in nature; operably linked to a polynucleotide to which it is not linked in nature; or not as part of a larger sequence in nature.

As used herein, "label" and "detectable label" refer to moieties that are attached to an antibody or analyte such that the reaction between the antibody and analyte is detectable, and the antibody or analyte so labeled is referred to as "detectably labeled". The marker may produce a signal that is detectable by visual or instrumental means. Various labels include signal-generating substances such as chromophores, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include light-generating moieties, such as acridinium compounds, and fluorescent moieties, such as fluorescein. Other markers are described herein. In this regard, the moiety may be undetectable by itself, but may become detectable upon reaction with another moiety. The use of the term "detectably labeled" is intended to encompass such labels.

Any suitable detectable label as known in the art may be used. For example, the detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium ester, thioester, or sulfonamide; luminol, isoluminol, phenanthridinium ester, and the like), a fluorescent label (such as a fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3' 6-carboxyfluorescein, 5- (6) -carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), a rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide capped cadmium selenide), temperature tags, or immuno-polymerase chain reaction tags. Introduction to labeling, labeling procedures and label detection is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2 nd edition, Springer Verlag, n.y. (1997); and Haughland, Handbook of Fluorescent Probes and Research Chemicals (1996), a combination Handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon. Fluorescent labels can be used in FPIAs (see, e.g., U.S. patent nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are incorporated herein by reference in their entirety). Acridinium compounds can be used as detectable labels in homogeneous chemiluminescent assays (see, e.g., Adamczyk et al, bioorg. Med ghem. Lett.16:1324-1328 (2006); Adamczyk et al, bioorg. Med chem. Lett.4:2313-2317 (2004); Adamczyk et al, biorg. Med chem.Lett.14:3917-3921 (2004); and Adamczyk et al, org. Lett.5:3779-3782 (2003)).

In one aspect, the acridine compound is acridine-9-carboxamide. Methods for preparing acridine 9-carboxamide are described in Mattingly, J.Biolumin.Chemilumin.6:107-114 (1991); adamczyk et al, J.org.chem.63:5636-5639 (1998); adamczyk et al Tetrahedron 55:10899-10914 (1999); adamczyk et al, org.Lett.1:779-781 (1999); adamczyk et al, Bioconjugate chem.11:714-724 (2000); mattingly et al, In luminescences Biotechnology Instruments and Applications; dyke, k.v. editing; CRC Press Boca Raton, pages 77-105 (2002); adamczyk et al, org.Lett.5:3779-3782 (2003); and U.S. patent nos. 5,468,646, 5,543,524, and 5,783,699 (each of which is incorporated by reference herein in its entirety for its teachings in this regard).

Another example of an acridine compound is an aryl acridine-9-carboxylate. An example of an aryl acridine-9-carboxylate ester having the formula II is 10-methyl-9- (phenoxycarbonyl) acridine fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing aryl acridine-9-carboxylates are described in McCapra et al, photochem. photobiol.4:1111-21 (1965); razavi et al, luminescences 15: 245-; razavi et al, Luminescience 15: 239-; and U.S. patent No. 5,241,070 (each of which is incorporated by reference herein in its entirety for its teachings in this regard). Such aryl acridine-9-carboxylates are chemiluminescent indicators that are highly efficient both in signal intensity and/or signal rapidity for hydrogen peroxide generated in the oxidation of an analyte by at least one oxidase. The chemiluminescent emission process of the aryl acridine-9-carboxylate ester is completed rapidly, i.e., within 1 second, while the chemiluminescent emission of acridine-9-carboxamide lasts up to 2 seconds. However, the acridine-9-carboxylic acid aryl esters lose their chemiluminescent properties in the presence of the protein. Thus, its use requires that no protein be present during signal generation and detection. Methods for isolating or removing proteins from a Sample are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and/or digestion (see, e.g., Wells, High Throughput biological Sample preparation, methods and analysis stratages, Elsevier (2003)). The amount of protein removed or isolated from the test sample may be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. More details regarding the aryl acridine-9-carboxylates and their uses are set forth in U.S. patent No. 11/697,835, filed on 9.4.2007. The aryl acridine-9-carboxylate ester may be dissolved in any suitable solvent, such as degassed anhydrous N, N-Dimethylformamide (DMF) or aqueous sodium cholate.

"linker sequence" or "linker peptide sequence" refers to a natural or artificial polypeptide sequence that is linked to one or more polypeptide sequences of interest (e.g., full length, fragments, etc.). The term "linked" refers to the joining of a linking sequence to a polypeptide sequence of interest. Such polypeptide sequences are preferably joined by one or more peptide bonds. The linker sequence may be about 4 to about 50 amino acids in length. Preferably, the linker sequence is about 6 to about 30 amino acids in length. The native linker sequence may be modified by amino acid substitutions, additions or deletions to produce an artificial linker sequence. The linker sequence may be used for many purposes, including in recombinant fabs. Exemplary linking sequences include, but are not limited to: (i) a histidine (His) tag, such as a 6X His tag, having the amino acid sequence of HHHHHHHHHH (SEQ ID NO: 4), useful as a linker sequence to facilitate isolation and purification of polypeptides and antibodies of interest; (ii) enterokinase cleavage sites, such as His tags, are used to isolate and purify proteins and antibodies of interest. Often, enterokinase cleavage sites are used with His-tags for the isolation and purification of proteins and antibodies of interest. Various enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence of DDDDK (SEQ ID NO: 3) and derivatives thereof (e.g., ADDDDK (SEQ ID NO: 4), etc.); (iii) miscellaneous sequences may be used to link or join the light and/or heavy chain variable regions of the single chain variable region fragments. Examples of other linking sequences can be found in Bird et al, Science 242:423-426 (1988); huston et al, PNAS USA 85: 5879-; and McCafferty et al, Nature348:552-554 (1990). The linking sequence may also be modified for additional functions, such as attachment of a drug or attachment to a solid support. In the context of the present disclosure, a monoclonal antibody, for example, may contain a linking sequence, such as a His tag, an enterokinase cleavage site, or both.

"magnetic resonance imaging" or "MRI" as used interchangeably herein refers to medical imaging techniques used in radiology to form pictures of the anatomy and physiological processes of the human body in health and disease. MRI is a form of medical imaging that measures the response of nuclei of human tissue to high frequency radio waves when in a strong magnetic field and produces images of internal organs. MRI scanners based on Nuclear Magnetic Resonance (NMR) science use strong magnetic fields, radio waves and field gradients to generate images of the interior of the human body.

As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigen. Furthermore, unlike polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

The term "multivalent binding protein" is used herein to refer to a binding protein that comprises two or more antigen binding sites (also referred to herein as "antigen binding domains"). Multivalent binding proteins are preferably engineered to have three or more antigen binding sites, and are not typically naturally occurring antibodies. The term "multispecific binding protein" refers to a binding protein that can bind to two or more related or unrelated targets, including binding proteins that are capable of binding to two or more different epitopes of the same target molecule.

"negative predictive value" or "NPV" as used interchangeably herein refers to the probability that a subject has a negative outcome given that they have a negative test result.

As used herein, "reference level" refers to a measured cut-off value used to assess the diagnosis, prognosis, or efficacy of a treatment, which has been correlated or correlated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression of disease, lack of progression or improvement, etc.). As used herein, "absolute amount" refers to the absolute value of the change or difference between at least two determinations taken or sampled at different time points and, similar to a reference level, has been correlated or correlated with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression of disease, lack of progression or improvement, etc.). As used herein, "absolute value" refers to the magnitude of the difference between real numbers, such as, for example, two comparison levels (e.g., a level taken at a first point in time and a level taken at a second point in time), regardless of its sign, i.e., whether it is positive or negative.

The present disclosure provides exemplary reference levels and absolute quantities (e.g., calculated by comparing reference levels at different time points). However, it is well known that reference levels and absolute amounts may vary depending on the nature of the immunoassay (e.g., antibody used, reaction conditions, sample purity, etc.), and that assays may be compared and standardized. It is further well within the ability of one of ordinary skill in the art to modify the disclosure herein with respect to other immunoassays based on the description provided herein to obtain immunoassay-specific reference levels and absolute amounts for those other immunoassays. Although the precise values of the reference levels and absolute amounts may vary between assays, the findings described herein should be generally applicable and capable of being extrapolated to other assays.

"point-of-care device" refers to a device for providing medical diagnostic tests at or near point-of-care (i.e., outside a laboratory), at the time and place of patient care, such as in a hospital, physician's office, emergency or other medical care facility, patient's home, nursing home, and/or long-term care and/or end-of-care facility. Examples of point-of-care devices include devices produced by Abbott laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT availability, ubiquitous biosensors (Rowville, Australia) (see US2006/0134713), Axis-Shield PoC AS (Oslo, Norway), and clinical laboratory products (Los Angeles, USA).

"positive predictive value" or "PPV" as used interchangeably herein refers to the probability that a subject has a positive outcome given that they have a positive test result.

"quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity groups. A calibration (standard) curve is typically established using a "calibrator" or "standard" (e.g., one or more, such as a plurality) to interpolate the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator may be used that is close to a reference or control level (e.g., "low," "medium," or "high"). Multiple calibrators (i.e., more than one calibrator or different amounts of calibrators) may be used in combination to form a "sensitivity group".

The "receiver operating characteristic" curve or "ROC" curve refers to a graphical plot that illustrates the performance of a binary classification system when its discrimination thresholds are varied. For example, the ROC curve is a plot of true positive rate versus false positive rate for different possible cut-off points of a diagnostic test. It was generated by plotting the true positive score in positives (TPR ═ true positive rate) versus the false positive score in negatives (FPR ═ false positive rate) at various threshold settings. TPR is also referred to as sensitivity, and FPR is the one minus the specificity or true negative rate. The ROC curve demonstrates a tradeoff between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the tighter the curve is along the left boundary of the ROC space, then the top boundary, the more accurate the test is; the closer the curve is to the 45-degree diagonal of the ROC space, the more inaccurate the test is; the slope of the tangent at the cut-off point gives the Likelihood (LR) of the test value; and the area under the curve is a measure of the accuracy of the test.

"recombinant antibody" and "recombinant antibodies" refer to an antibody prepared by one or more steps, including cloning, by recombinant techniques, a nucleic acid sequence encoding all or part of one or more monoclonal antibodies into an appropriate expression vector, and then expressing the antibody in an appropriate host cell. The term includes, but is not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugated abs, monoclonal antibodies, monoclonal,

And other antibodies described in (i) herein. (Dual variable domain immunoglobulins and methods of making them are described in Wu, C et al, Nature Biotechnology,25:1290-1297 (2007)). The term "bifunctional antibody" as used herein refers to an antibody comprising a first arm with specificity for one antigenic site and a second arm with specificity for a different antigenic site, i.e. a bifunctional antibody having dual specificity.

As used herein, "risk assessment," "risk classification," "risk identification," or "risk stratification" of a subject (e.g., patient) refers to evaluating factors, including biomarkers, to predict the risk of occurrence of a future event, including onset or progression of a disease, so that treatment decisions about the subject can be made on a more informed basis.

"sample", "test sample", "biological sample", "sample from a subject" and "patient sample" may be used interchangeably herein and may be a blood sample (such as whole blood), tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes or monocytes. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a serum sample. In some embodiments, the sample is a plasma sample. The sample may be used directly as obtained from the patient, or may be pre-treated, such as by filtration, dilution, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the characteristics of the sample in some manner discussed herein or otherwise known in the art.

Samples can be obtained using a variety of cell types, tissues, or body fluids. Such cell types, tissues, and fluids may include tissue sections, such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood (e.g., whole blood), plasma, serum, red blood cells, platelets, interstitial fluid, cerebrospinal fluid, and the like. Cell types and tissues may also include lymph, cerebrospinal fluid, fluids collected from tissues or cell types may be provided by removing cell samples from human and non-human animals, but may also be accomplished by using previously isolated cells (e.g., isolated by other humans, at other times, and/or for other purposes). Archival organizations, such as those with a history of treatment or outcome, may also be used. Protein or nucleotide isolation and/or purification may not be required.

As used herein, "sensitivity" of an assay refers to the proportion of subjects with positive outcome that are correctly identified as positive (e.g., correctly identifying those subjects with the disease or medical condition they are being tested for). For example, this may include correctly identifying those with TBI from subjects without TBI, correctly identifying those with moderate, severe, or moderate to severe TBI from subjects with mild TBI, correctly identifying those with mild TBI from subjects with moderate, severe, or moderate to severe TBI, correctly identifying those with moderate, severe, or moderate to severe TBI from subjects without TBI, or correctly identifying those with mild TBI from subjects without TBI, correctly identifying those with image or head CT scan or MRI from subjects who are unlikely to benefit from head imaging or CT scan or MRI).

As used herein, "specificity" of an assay refers to the proportion of subjects with negative outcomes that are correctly identified as negative (e.g., correctly identifying those subjects that do not have the disease or medical condition they are being tested for). For example, this may include correctly identifying those with TBI from subjects without TBI, correctly identifying those without moderate, severe, or moderate to severe TBI from subjects with mild TBI, correctly identifying those without mild TBI from subjects with moderate, severe, or moderate to severe TBI, or correctly identifying subjects without any TBI, or correctly identifying those with mild TBI from subjects without TBI).

"solid phase" or "solid support" as used interchangeably herein refers to any material that can be used to attach and/or attract and immobilize either (1) one or more capture agents or capture specific binding partners, or (2) one or more detection agents or detection specific binding partners. The solid phase may be selected for its inherent ability to attract and immobilize a capture agent. Alternatively, the solid phase may have a linker attached thereto, the linker having an attracting and immobilizing (1) a capture agent or capture specific binding partner, or (2) a detection agent or detection specific binding partner. For example, the linking agent can include a charged species that is oppositely charged relative to the capture agent (e.g., capture specific binding partner) or detection agent (e.g., detection specific binding partner) itself or relative to a charged species conjugated to (1) the capture agent or capture specific binding partner, or (2) the detection agent or detection specific binding partner. In general, the linking agent can be any binding partner (preferably heterogeneous) that is immobilized on (attached to) a solid phase and has the ability to immobilize by a binding reaction (1) a capture agent or capture specific binding partner, or (2) a detection agent or detect specific binding partner. The linking agent allows the capture agent to bind indirectly to the solid phase material prior to or during the performance assay. For example, the solid phase may be plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon, including, for example, test tubes, microtiter wells, flakes, beads, microparticles, chips and other configurations known to those of ordinary skill in the art.

As used herein, "specifically binds" or "specifically binds" may refer to the interaction of an antibody, protein or peptide with a second chemical, where the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical; for example, antibodies recognize and bind to specific protein structures, rather than to proteins broadly. If the antibody is specific for epitope "A", the presence of the epitope A-containing molecule (or free unlabeled A) will reduce the amount of labeled A bound to the antibody in the reaction between labeled "A" and the antibody.

A "specific binding partner" is a member of a specific binding pair. A specific binding pair comprises two different molecules that specifically bind to each other chemically or physically. Thus, in addition to antigen-antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin (or streptavidin); carbohydrates and lectins; a complementary nucleotide sequence; effector and receptor molecules; co-factors and enzymes; enzymes and enzyme inhibitors, and the like. In addition, a specific binding pair may include a member that is an analog of the original specific binding member, e.g., an analyte-analog. Immunoreactive specific binding members include isolated or recombinantly produced antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies and complexes and fragments thereof.

As used herein, "statistically significant" refers to the likelihood that a relationship between two or more variables is caused by other factors than random chance. Statistical hypothesis testing is used to determine whether the results of the data set are statistically significant. In statistical hypothesis testing, statistical significance results were obtained as long as the observed p-value of the test statistic was less than the study-defined significance level. The p-value is the probability of obtaining a result that is at least as extreme as the observed result, assuming the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon signed rank test, t test, chi-square or fisher's exact test. "significant" as used herein refers to a change that has not been determined to be statistically significant (e.g., it may not have undergone statistical hypothesis testing).

As used herein, "subject" and "patient" are used interchangeably to refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject may be human or non-human. In one embodiment, the subject is a human. The subject or patient may undergo other forms of treatment. In some embodiments, when the subject is a human, the subject does not include any person who has suffered a cerebrovascular accident (e.g., stroke).

"treating" or "treatment" is each used interchangeably herein to describe reversing, alleviating or inhibiting the progression of a disease and/or injury to which such terms apply, or one or more symptoms of such a disease. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease or preventing symptoms associated with a disease. Treatment may be performed in an acute or chronic manner. The term also refers to reducing the severity of a disease or symptom associated with a disease prior to affliction with such disease. Such prevention or reduction of disease severity prior to affliction refers to the administration of the pharmaceutical composition to the subject at a time of administration that is not afflicted with the disease. "preventing" also refers to preventing the recurrence of a disease or one or more symptoms associated with such a disease. "treatment" and "therapeutically" refer to the act of treating, as "treating" is defined above.

"traumatic brain injury" or "TBI" as used interchangeably herein refers to a complex injury with a broad spectrum of symptoms and disabilities. TBI is many times an acute event similar to other injuries. TBI can be classified as "mild", "moderate" or "severe". The causes of TBI are diverse and include, for example, physical shaking of a person, car accidents, firearm injury, cerebrovascular accidents (e.g., stroke), falls, explosions or shockwaves, and other types of blunt force trauma. Other causes of TBI include ingestion and/or exposure to one or more chemicals or toxins (such as fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (such as carbon monoxide, hydrogen sulfide and cyanide), organometallics (such as methyl mercury, tetraethyl lead and organotin), one or more drugs of abuse, or combinations thereof). Alternatively, TBI may occur in a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, one or more viruses, meningitis, hydrocephalus, hypoxia, or any combination thereof. Young and elderly are the age group with the highest risk of TBI. In certain embodiments herein, traumatic brain injury or TBI does not include, and specifically excludes, cerebrovascular accidents, such as stroke.

As used herein, "mild TBI" refers to brain injury where loss of consciousness is brief and is typically a few seconds or minutes and/or disorganized and disoriented for less than 1 hour. Mild TBI is also known as concussion, mild head trauma, mild TBI, mild brain injury and mild head injury. While MRI and CT scans are often normal, individuals with mild TBI may have cognitive problems such as headache, thought difficulties, memory problems, attention deficit, mood swings, and depression.

Mild TBI is the most prevalent and is often missed at the time of initial injury. Typically, the subject has a glasgow coma scale number between 13-15 (such as 13-15 or 14-15). Fifteen percent (15%) of mild TBI patients have symptoms that persist for 3 months or longer. Mild TBI is defined as the result of forceful movements of the head or impact forces that result in brief changes in mental state (confusion, disorientation or memory loss) or loss of consciousness for less than 30 minutes. Common symptoms of mild TBI include fatigue, headache, visual impairment, memory loss, poor concentration/concentration, sleep disturbance, dizziness/loss of balance, stress mood disorders, depressed mood and epilepsy. Other symptoms associated with mild TBI include nausea, loss of smell, sensitivity to light and sound, mood changes, loss or confusion, and/or thought retardation.

As used herein, "moderate TBI" refers to brain injury in which loss of consciousness and/or confusion and disorientation is between 1 to 24 hours and the subject has a glasgow coma scale number between 9-13 (such as 9-12 or 9-13). Individuals with moderate TBI have abnormal brain imaging results. As used herein, "severe TBI" refers to brain injury where consciousness is lost for more than 24 hours and memory loss is remembered for longer than 24 hours after injury or penetrating skull injury and subjects have a glasgow coma scale number between 3-8. Deficits range from higher levels of cognitive impairment to comatose states. Survivors may have limited arm or leg function, speech or language abnormalities, loss of mental capacity, or emotional problems. Individuals with severe injury may be left unresponsive for extended periods of time. For many people with severe TBI, long-term rehabilitation is often required to maximize function and independence.

As used herein, "moderate to severe" TBI is meant to include a spectrum of moderate to severe brain injury, and thus includes a combination of moderate TBI alone, severe TBI alone, and moderate to severe TBI. Subjects with moderate to severe TBI have a glasgow coma scale number of 3-13 (e.g., 3-12 or 3-13). For example, in certain clinical situations, a subject may be initially diagnosed as having moderate TBI, but over time (minutes, hours, or days) the subject may develop severe TBI (e.g., in the presence of cerebral hemorrhage). Such subjects are examples of patients that may be classified as "moderate to severe". Common symptoms of moderate to severe TBI include cognitive deficits including attention, concentration, distraction, memory, difficulty in operating speed, confusion, sustained speech, impulsions, speech processing and/or "executive function", unintelligibility of spoken words (sensory aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, high or slow speech speed, reading problems, writing problems, interpreting touch, temperature, movement, limb position and fine discrimination difficulties, integrating or patterning sensory impressions into psychologically meaningful data, partial or total vision loss, eye muscle weakness and double vision (double vision), blurred vision, problems of distance determinations, involuntary eye movements (nystagmus), intolerance of light (photophobia), hearing (such as hearing loss or loss, presence of a chime in the ear (tinnitus), Increased sensitivity to sound), loss or attenuation of smell (hyposmia), loss or attenuation of taste, convulsions associated with epilepsy, which can be of several types and can involve consciousness, sensory perception or motor movement, disruption of bowel and bladder control, insomnia, loss of endurance, altered appetite, thermoregulation, menstrual difficulties, dependent behavior, emotional ability, lack of motivation, irritability, aggression, depression, disinhibition or rejection/lack of consciousness.

"variants" are used herein to describe peptides or polypeptides that differ in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or to promote an immune response. Variants are also used herein to describe proteins having substantially the same amino acid sequence as a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., the replacement of an amino acid with a different amino acid of similar characteristics (e.g., hydrophilicity, extent and distribution of charged regions) are recognized in the art as typically involving minor changes. As understood in the art, these minor changes can be identified in part by considering the hydropathic index of amino acids. Kyte et al, J.mol.biol.157:105-132 (1982). The hydropathic index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids of similar hydrophobicity indices may be substituted and still retain protein function. In one aspect, amino acids with a hydrophobicity index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows the calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101 is incorporated by reference herein in its entirety. As is understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity (e.g., immunogenicity). Substitutions may be made with amino acids having hydrophilicity values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the particular side chain of that amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties. "variant" may also be used to refer to an antigen-reactive fragment of an anti-cTnI antibody that differs in amino acid sequence from the corresponding fragment of the anti-cTnI antibody, but is still antigen-reactive and can compete with the corresponding fragment of the anti-cTnI antibody for binding to cTnI. "variant" may also be used to describe a polypeptide or fragment thereof that has been differentially processed (such as by proteolysis, phosphorylation, or other post-translational modification), but still retains its antigenic reactivity.

"vector" is used herein to describe a nucleic acid molecule that can transport another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors suitable for use in recombinant DNA techniques are often in the form of plasmids. "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions, may be used. In this regard, RNA versions of the vectors (including RNA viral vectors) may also be used in the context of the present disclosure.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure will have the meanings that are commonly understood by one of ordinary skill in the art. For example, any nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The meaning and scope of the terms should be clear; however, if there is any implicit ambiguity, the definitions provided herein take precedence over any dictionary or foreign definition. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

2. Methods for using cardiac troponin i (ctni) to aid in the diagnosis and assessment of whether a human subject suffers from or may suffer from (or has actual or suspected) head injury

The present disclosure also relates to, among other methods, methods of using cardiac troponin i (cTnI) levels or changes in cTnI levels to aid in the diagnosis and assessment of whether a human subject suffers from or is likely to suffer from (or has actual or suspected) head injury. In particular, the methods described herein may assist in determining the extent of traumatic brain injury in a human subject with actual or suspected head injury, e.g., determining whether the subject has mild traumatic brain injury or moderate, severe, or moderate to severe traumatic brain injury. As used herein, "determining whether a subject has mild traumatic brain injury or moderate, severe, or moderate to severe traumatic brain injury" refers to the fact that: the methods described above can be used, for example, in conjunction with other information (e.g., clinical assessment data) to determine that a subject is more likely to have mild traumatic brain injury, moderate, severe, or moderate to severe traumatic brain injury, or no traumatic brain injury. The methods may comprise assaying a sample obtained from a human subject within about 24 hours, e.g., within about 2 hours, after an actual or suspected head injury to measure or detect the level of cardiac troponin i (ctni) in the sample and determine whether the subject suffers from mild, moderate, severe, moderate to severe Traumatic Brain Injury (TBI) or is free of TBI. In some embodiments, the subject is determined to be (1) moderate, severe, or moderate to severe TBI when the level of cTnI in the sample is above a cTnI reference level, or (2) mild TBI when the level of cTnI in the sample is below a cTnI reference level. The sample may be a biological sample. In some aspects, the biological sample is a whole blood sample. In other aspects, the biological sample is a serum sample. In other aspects, the biological sample is a plasma sample.

In some embodiments, the method can include obtaining a sample within about 24 hours, e.g., within about 2 hours, of an actual or suspected injury in the subject and contacting the sample with an antibody to cTnI such that a complex of the antibody and cTnI is formed. The method further comprises detecting the antibody-cTnI complex produced.

In some embodiments, the time period may be within about 0 minutes, within about 1 minute, within about 2 minutes, within about 3 minutes, within about 4 minutes, within about 5 minutes, within about 6 minutes, within about 7 minutes, within about 8 minutes, within about 9 minutes, within about 10 minutes, within about 11 minutes, within about 12 minutes, within about 13 minutes, within about 14 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours of a suspected head injury, Within about 23 hours or within about 24 hours, a sample is obtained or taken from the subject.

In some embodiments, the sample is taken from the human subject within about 2 hours after the head (actual) injury or suspected injury. For example, a sample may be taken from a human subject within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 2 hours of a head injury or suspected injury. In some embodiments, the onset of cTnI manifests itself within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 2 hours of the head injury.

In some embodiments, at one or more time points, the subject may receive a glasgow coma scale score before or after the determination of the cardiac troponin level. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on the glasgow coma scale score. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on abnormal head CT. In some embodiments, the subject receives a CT scan before or after performing the assay. In some embodiments, the subject's head CT is normal.

In some embodiments, the reference level of cTnI is associated with a subject with moderate, severe, or moderate to severe traumatic brain injury. In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 3-12 (moderate to severe TBI). In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 3-8 (severe TBI). In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 9-13 (moderate TBI). In some embodiments, the subject is suspected of having mild traumatic brain injury based on the glasgow coma scale score. In some embodiments, the reference level of cTnI is associated with a subject having mild traumatic brain injury. In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 13-15 (mild TBI).

In general, the cTnI reference level may also be used as a benchmark to assess the results obtained in determining the cTnI of a test sample. In general, when making such a comparison, a reference level of cTnI is obtained by running a particular assay under appropriate conditions a sufficient number of times to correlate or correlate the presence, amount, or concentration of the analyte with a particular stage or endpoint of TBI or with a particular indication. Typically, the reference level of cTnI is obtained by an assay of a reference subject (or population of subjects). The measured cTnI may include fragments thereof, degradation products thereof, and/or enzymatic cleavage products thereof.

In certain embodiments, the reference level can be associated with a control subject that has not suffered a head injury.

In some embodiments, the cTnI reference level is determined by an assay having a sensitivity between at least about 65% and about 100% and a specificity between at least about 30% and about 100%. In some embodiments, the sensitivity is between at least about 65% to about 100%, at least about 65% to at least about 99%, at least about 65% to at least about 95%, at least about 65% to at least about 90%, at least about 65% to at least about 85%, at least about 65% to at least about 80%, at least about 65% to at least about 75%, at least about 65% to at least about 70%, at least about 75% to about 100%, at least about 75% to at least about 99%, at least about 75% to at least about 95%, at least about 75% to at least about 90%, at least about 75% to at least about 85%, at least about 75% to at least about 80%, at least about 85% to about 100%, at least about 85% to at least about 99%, at least about 85% to at least about 95%, at least about 85% to at least about 90%, at least about 95% to about 100%, or at least about 95% to at least about 99%. In some embodiments, the sensitivity is at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% to about 100%, between at least about 30% to about 99%, between at least about 30% to about 95%, between at least about 30% to about 90%, between at least about 30% to about 85%, between at least about 30% to about 80%, between at least about 30% to about 75%, between at least about 30% to about 70%, between at least about 30% to about 60%, between at least about 30% to about 50%, between at least about 40% to about 100%, between at least about 40% to about 99%, between at least about 40% to about 95%, between at least about 40% to about 90%, between at least about 40% to about 85%, between at least about 40% to about 80%, between at least about 40% to about 75%, between at least about 40% to about 70%, between at least about 40% to about 60%, between at least about 40% to about 50%, between at least about 50% to about 100%, at least about 50% to about 99%, at least about 50% to about 95%, at least about 50% to about 90%, at least about 50% to about 85%, at least about 50% to about 80%, at least about 50% to about 75%, at least about 50% to about 70%, at least about 50% to about 60%, at least about 60% to about 100%, at least about 60% to about 99%, at least about 60% to about 95%, at least about 60% to about 90%, at least about 60% to about 85%, at least about 60% to about 80%, at least about 60% to about 75%, at least about 60% to about 70%, at least about 70% to about 100%, at least about 70% to about 99%, at least about 70% to about 95%, at least about 70% to about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%. In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.0. For example, the sensitivity is at least about 99% and the specificity is at least about 75%, the sensitivity is at least about 99% and the specificity is at least about 99%, or the sensitivity is at least about 100% and the specificity is at least about 100%.

In some embodiments, the amount of cardiac troponin I in the sample is from about 1pg/mL to about 50pg/mL, from about 1pg/mL to about 45pg/mL, from about 1pg/mL to about 40pg/mL, from about 1pg/mL to about 35pg/mL, from about 1pg/mL to about 30pg/mL, from about 1pg/mL to about 25pg/mL, from about 1pg/mL to about 20pg/mL, from about 1pg/mL to about 15pg/mL, from about 1pg/mL to about 10pg/mL, from about 1pg/mL to about 9pg/mL, from about 1pg/mL to about 8pg/mL, from about 1pg/mL to about 7pg/mL, from about 1pg/mL to about 6pg/mL, from about 1pg/mL to about 5pg/mL, from about 1pg/mL to about 4 mL/mL About 1pg/mL to about 3pg/mL, about 1pg/mL to about 2pg/mL, about 1pg/mL to about 1.5pg/mL, about 1.5pg/mL to about 50pg/mL, about 1.5pg/mL to about 45pg/mL, about 1.5pg/mL to about 40pg/mL, about 1.5pg/mL to about 35pg/mL, about 1.5pg/mL to about 30pg/mL, about 1.5pg/mL to about 25pg/mL, about 1.5pg/mL to about 20pg/mL, about 1.5pg/mL to about 15pg/mL, about 1.5pg/mL to about 10pg/mL, about 1.5pg/mL to about 9pg/mL, about 1.5pg/mL to about 8pg/mL, about 1.5pg/mL to about 6 mL, about 1.5pg/mL to about 5pg/mL, about 1.5pg/mL to about 6pg/mL About 1.5pg/mL to about 4pg/mL, about 1.5pg/mL to about 3pg/mL, about 1.5pg/mL to about 2pg/mL, about 2pg/mL to about 50pg/mL, about 2pg/mL to about 45pg/mL, about 2pg/mL to about 40pg/mL, about 2pg/mL to about 35pg/mL, about 2pg/mL to about 30pg/mL, about 2pg/mL to about 25pg/mL, about 2pg/mL to about 20pg/mL, about 2pg/mL to about 15pg/mL, about 2pg/mL to about 10pg/mL, about 2pg/mL to about 9pg/mL, about 2pg/mL to about 8pg/mL, about 2pg/mL to about 7pg/mL, about 2pg/mL to about 6pg/mL, about 2 mL to about 5 mL, about 2pg/mL to about 20pg/mL, about 2pg/mL to about 15pg/mL, about 2pg/mL, about 6pg/mL, about 2pg/mL to about 6pg/mL, about 2pg/mL, about 5, About 2pg/mL to about 4pg/mL, about 2pg/mL to about 3pg/mL, about 3pg/mL to about 50pg/mL, about 3pg/mL to about 45pg/mL, about 3pg/mL to about 40pg/mL, about 3pg/mL to about 35pg/mL, about 3pg/mL to about 30pg/mL, about 3pg/mL to about 25pg/mL, about 3pg/mL to about 20pg/mL, about 3pg/mL to about 15pg/mL, about 3pg/mL to about 10pg/mL, about 3pg/mL to about 9pg/mL, about 3pg/mL to about 8pg/mL, about 3pg/mL to about 7pg/mL, about 3pg/mL to about 6pg/mL, about 3pg/mL to about 5pg/mL, about 3pg/mL to about 4 mL, about 3pg/mL to about 5pg/mL, about 3pg/mL to about 4 mL, About 4pg/mL to about 50pg/mL, about 4pg/mL to about 45pg/mL, about 4pg/mL to about 40pg/mL, about 4pg/mL to about 35pg/mL, about 4pg/mL to about 30pg/mL, about 4pg/mL to about 25pg/mL, about 4pg/mL to about 20pg/mL, about 4pg/mL to about 15pg/mL, about 4pg/mL to about 10pg/mL, about 4pg/mL to about 9pg/mL, about 4pg/mL to about 8pg/mL, about 4pg/mL to about 7pg/mL, about 4pg/mL to about 6pg/mL, about 4pg/mL to about 5pg/mL, about 5pg/mL to about 50pg/mL, about 5pg/mL to about 45pg/mL, about 40 mL to about 40 mL/mL, About 5pg/mL to about 35pg/mL, about 5pg/mL to about 30pg/mL, about 5pg/mL to about 25pg/mL, about 5pg/mL to about 20pg/mL, about 5pg/mL to about 15pg/mL, about 5pg/mL to about 10pg/mL, about 5pg/mL to about 9pg/mL, about 5pg/mL to about 8pg/mL, about 5pg/mL to about 7pg/mL, about 5pg/mL to about 6pg/mL, about 6pg/mL to about 50pg/mL, about 6pg/mL to about 45pg/mL, about 6pg/mL to about 40pg/mL, about 6pg/mL to about 35pg/mL, about 6pg/mL to about 30pg/mL, about 6pg/mL to about 25pg/mL, about 6pg/mL to about 20 mL, About 6pg/mL to about 15pg/mL, about 6pg/mL to about 10pg/mL, about 6pg/mL to about 9pg/mL, about 6pg/mL to about 8pg/mL, about 6pg/mL to about 7pg/mL, about 7pg/mL to about 50pg/mL, about 7pg/mL to about 45pg/mL, about 7pg/mL to about 40pg/mL, about 7pg/mL to about 35pg/mL, about 7pg/mL to about 30pg/mL, about 7pg/mL to about 25pg/mL, about 7pg/mL to about 20pg/mL, about 7pg/mL to about 15pg/mL, about 7pg/mL to about 10pg/mL, about 7pg/mL to about 9pg/mL, about 7pg/mL to about 8pg/mL, about 50 mL to about 8 mL, about 7pg/mL to about 10pg/mL, about 7pg/mL to about 9pg/mL, about 7pg/mL to about 8pg/mL, about 8pg/mL to about 50 mL, About 8pg/mL to about 45pg/mL, about 8pg/mL to about 40pg/mL, about 8pg/mL to about 35pg/mL, about 8pg/mL to about 30pg/mL, about 8pg/mL to about 25pg/mL, about 8pg/mL to about 20pg/mL, about 8pg/mL to about 15pg/mL, about 8pg/mL to about 10pg/mL, about 8pg/mL to about 9pg/mL, about 9pg/mL to about 50pg/mL, about 9pg/mL to about 45pg/mL, about 9pg/mL to about 40pg/mL, about 9pg/mL to about 35pg/mL, about 9pg/mL to about 30pg/mL, about 9pg/mL to about 25pg/mL, about 9pg/mL to about 20pg/mL, about 9pg/mL to about 15pg/mL, about, About 9pg/mL to about 10pg/mL, about 10pg/mL to about 50pg/mL, about 10pg/mL to about 45pg/mL, about 10pg/mL to about 40pg/mL, about 10pg/mL to about 35pg/mL, about 10pg/mL to about 30pg/mL, about 10pg/mL to about 25pg/mL, about 10pg/mL to about 20pg/mL, about 10pg/mL to about 15pg/mL, about 20pg/mL to about 50pg/mL, about 20pg/mL to about 45pg/mL, about 20pg/mL to about 40pg/mL, about 20pg/mL to about 35pg/mL, about 20pg/mL to about 30pg/mL, or about 20pg/mL to about 25 pg/mL. In some embodiments, the amount of cTnI can be at least about 0.5pg/mL, at least about 1.0pg/mL, at least about 1.5pg/mL, at least about 2.0pg/mL, at least about 2.5pg/mL, at least about 3.0pg/mL, at least about 4.0pg/mL, at least about 5.0pg/mL, at least about 6.0pg/mL, at least about 7.0pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10pg/mL, at least about 15pg/mL, at least about 20pg/mL, at least about 25pg/mL, at least about 30pg/mL, at least about 35pg/mL, at least about 40pg/mL, at least about 45pg/mL, or at least about 50 pg/mL.

In addition to performing the above-described methods, one skilled in the art (e.g., a physician) will also know and understand how to perform additional tests to detect or assess other co-morbidities (e.g., other diseases, disorders, or conditions besides TBI). Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

In one embodiment, to confirm that a change in the amount or level of cTnI in the methods described herein is attributable to head injury or suspected head injury in the subject, rather than the outcome of an acute cardiac syndrome (e.g., myocardial infarction, heart failure, etc.), a physician or other health care provider may perform or conduct one or more additional tests or procedures to confirm the absence of an acute cardiac syndrome. Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

In some embodiments, the method further comprises treating a human subject assessed as having moderate, severe, or moderate to severe traumatic brain injury with a traumatic brain injury treatment as described below. In some embodiments, the method further comprises monitoring a human subject assessed as having mild traumatic brain injury as described below. In some embodiments, the method further comprises requiring additional testing to obtain further clinical information about the traumatic brain injury. In some embodiments, the method comprises treating a human subject assessed as having mild, moderate, severe, or moderate to severe brain injury with a cardioprotective treatment to protect the heart, as described below.

The nature of the assay employed in the methods described herein is not critical, and the assay may be any assay known in the art, for example, immunoassay, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, or protein immunostaining, electrophoretic analysis, protein assay, competitive binding assay, functional protein assay, or chromatography or spectroscopy, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, the assay may be performed in a clinical chemistry format, such as would be known to one of ordinary skill in the art. Such assays are described in further detail in sections 11-13 herein. As known in the art, the values (e.g., reference levels, cut-off values, threshold values, specificity, sensitivity, concentration of calibrators and/or controls, etc.) used in assays employing particular sample types (e.g., immunoassays such as those using serum or point-of-care devices employing whole blood) can be normalized using techniques known in the art, e.g., assays, and so forth to other assay formats. For example, one way in which assay normalization can be performed is to apply a factor to the calibrator used in the assay to make the sample concentration reading higher or lower to obtain a slope consistent with the comparison method. Other methods of normalizing the results obtained in one assay to another are well known and described in the literature (see, e.g., David Wild, Handbook of immunoassays, 4 th edition, chapter 3.5, page 315-322, the contents of which are incorporated herein by reference).

3. Method for using cardiac troponin i (ctni) for assisting in determining whether to perform a CT scan on a human subject likely to suffer from or having suffered (or having actual or suspected) a head injury

The present disclosure relates to, among other methods, a method of assisting in determining whether to perform a computed tomography (CT scan) of a human subject who has suffered or may suffer (or has actual or suspected) a head injury. As used herein, "determining whether to perform a CT scan on a human subject" refers to the fact that: the foregoing methods may be used, for example, along with other information (e.g., clinical assessment data) to determine that a subject is more likely to have a positive head CT scan. In particular, such a method may comprise the steps of: (a) performing an assay on a sample obtained from the subject within about 24 hours, e.g., within about 2 hours, after the actual or suspected head injury to measure or detect the level of cardiac troponin i (ctni) in the sample; and (b) performing a CT scan on the subject when the level of cTnI in the sample is above the cTnI reference level; when the level of cTnI in the sample is below the cTnI reference level, no CT scan is performed on the subject. The sample may be a biological sample.

In some embodiments, the method can include obtaining a sample within about 24 hours, e.g., within about 2 hours, of an actual or suspected injury in the subject and contacting the sample with an antibody to cTnI such that a complex of the antibody and cTnI is formed. The method further comprises detecting the antibody-cTnI complex produced.

In some embodiments, the time period may be within about 0 minutes, within about 1 minute, within about 2 minutes, within about 3 minutes, within about 4 minutes, within about 5 minutes, within about 6 minutes, within about 7 minutes, within about 8 minutes, within about 9 minutes, within about 10 minutes, within about 11 minutes, within about 12 minutes, within about 13 minutes, within about 14 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 19 hours of an actual or suspected head injury, Within about 23 hours or within about 24 hours, a sample is obtained or taken from the subject.

In some embodiments, the sample is obtained from the human subject within about 2 hours after the head injury or suspected injury. For example, a sample may be taken from a human subject within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 2 hours of a head injury or suspected injury. In some embodiments, the onset of cTnI manifests itself within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 2 hours of the head injury.

In some embodiments, the subject receives a CT scan before or after performing the assay. In some embodiments, the subject is suspected of having traumatic brain injury based on a CT scan. In some embodiments, the cTnI reference level is associated with a positive head CT scan.

In general, the cTnI reference level may also be used as a benchmark to assess the results obtained in determining the cTnI of a test sample. In general, when making such a comparison, a reference level of cTnI is obtained by running a particular assay under appropriate conditions a sufficient number of times to correlate or correlate the presence, amount, or concentration of the analyte with a particular stage or endpoint of TBI or with a particular indication. Typically, the reference level of cTnI is obtained by an assay of a reference subject (or population of subjects). The measured cTnI may include fragments thereof, degradation products thereof, and/or enzymatic cleavage products thereof.

In some embodiments, the cTnI reference level is determined by an assay having a sensitivity between at least about 65% and about 100% and a specificity between at least about 30% and about 100%. In some embodiments, the sensitivity is between at least about 65% to about 100%, at least about 65% to at least about 99%, at least about 65% to at least about 95%, at least about 65% to at least about 90%, at least about 65% to at least about 85%, at least about 65% to at least about 80%, at least about 65% to at least about 75%, at least about 65% to at least about 70%, at least about 75% to about 100%, at least about 75% to at least about 99%, at least about 75% to at least about 95%, at least about 75% to at least about 90%, at least about 75% to at least about 85%, at least about 75% to at least about 80%, at least about 85% to about 100%, at least about 85% to at least about 99%, at least about 85% to at least about 95%, at least about 85% to at least about 90%, at least about 95% to about 100%, or at least about 95% to at least about 99%. In some embodiments, the sensitivity is at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% to about 100%, between at least about 30% to about 99%, between at least about 30% to about 95%, between at least about 30% to about 90%, between at least about 30% to about 85%, between at least about 30% to about 80%, between at least about 30% to about 75%, between at least about 30% to about 70%, between at least about 30% to about 60%, between at least about 30% to about 50%, between at least about 40% to about 100%, between at least about 40% to about 99%, between at least about 40% to about 95%, between at least about 40% to about 90%, between at least about 40% to about 85%, between at least about 40% to about 80%, between at least about 40% to about 75%, between at least about 40% to about 70%, between at least about 40% to about 60%, between at least about 40% to about 50%, between at least about 50% to about 100%, at least about 50% to about 99%, at least about 50% to about 95%, at least about 50% to about 90%, at least about 50% to about 85%, at least about 50% to about 80%, at least about 50% to about 75%, at least about 50% to about 70%, at least about 50% to about 60%, at least about 60% to about 100%, at least about 60% to about 99%, at least about 60% to about 95%, at least about 60% to about 90%, at least about 60% to about 85%, at least about 60% to about 80%, at least about 60% to about 75%, at least about 60% to about 70%, at least about 70% to about 100%, at least about 70% to about 99%, at least about 70% to about 95%, at least about 70% to about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%. In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.0. For example, the sensitivity is at least about 99% and the specificity is at least about 75%, the sensitivity is at least about 99% and the specificity is at least about 99%, or the sensitivity is at least about 100% and the specificity is at least about 100%.

In some embodiments, the amount of cardiac troponin I in the sample is from about 1pg/mL to about 50pg/mL, from about 1pg/mL to about 45pg/mL, from about 1pg/mL to about 40pg/mL, from about 1pg/mL to about 35pg/mL, from about 1pg/mL to about 30pg/mL, from about 1pg/mL to about 25pg/mL, from about 1pg/mL to about 20pg/mL, from about 1pg/mL to about 15pg/mL, from about 1pg/mL to about 10pg/mL, from about 1pg/mL to about 9pg/mL, from about 1pg/mL to about 8pg/mL, from about 1pg/mL to about 7pg/mL, from about 1pg/mL to about 6pg/mL, from about 1pg/mL to about 5pg/mL, from about 1pg/mL to about 4pg/mL, about 1pg/mL to about 3pg/mL, about 1pg/mL to about 2pg/mL, about 1pg/mL to about 1.5pg/mL, about 1.5pg/mL to about 50pg/mL, about 1.5pg/mL to about 45pg/mL, about 1.5pg/mL to about 40pg/mL, about 1.5pg/mL to about 35pg/mL, about 1.5pg/mL to about 30pg/mL, about 1.5pg/mL to about 25pg/mL, about 1.5pg/mL to about 20pg/mL, about 1.5pg/mL to about 15pg/mL, about 1.5pg/mL to about 10pg/mL, about 1.5pg/mL to about 9pg/mL, about 1.5pg/mL to about 8pg/mL, about 1.5pg/mL to about 6pg/mL, about 1.5pg/mL to about 5pg/mL, about 1.5pg/mL to about 4pg/mL, about 1.5pg/mL to about 3pg/mL, about 1.5pg/mL to about 2pg/mL, about 2pg/mL to about 50pg/mL, about 2pg/mL to about 45pg/mL, about 2pg/mL to about 40pg/mL, about 2pg/mL to about 35pg/mL, about 2pg/mL to about 30pg/mL, about 2pg/mL to about 25pg/mL, about 2pg/mL to about 20pg/mL, about 2pg/mL to about 15pg/mL, about 2pg/mL to about 10pg/mL, about 2pg/mL to about 9pg/mL, about 2pg/mL to about 8pg/mL, about 2pg/mL to about 7pg/mL, about 2pg/mL to about 6pg/mL, about 2 mL to about 5 mL, about 2pg/mL to about 4pg/mL, about 2pg/mL to about 3pg/mL, about 3pg/mL to about 50pg/mL, about 3pg/mL to about 45pg/mL, about 3pg/mL to about 40pg/mL, about 3pg/mL to about 35pg/mL, about 3pg/mL to about 30pg/mL, about 3pg/mL to about 25pg/mL, about 3pg/mL to about 20pg/mL, about 3pg/mL to about 15pg/mL, about 3pg/mL to about 10pg/mL, about 3pg/mL to about 9pg/mL, about 3pg/mL to about 8pg/mL, about 3pg/mL to about 7pg/mL, about 3pg/mL to about 6pg/mL, about 3pg/mL to about 5pg/mL, about 3pg/mL to about 4 mL, about 4pg/mL to about 50pg/mL, about 4pg/mL to about 45pg/mL, about 4pg/mL to about 40pg/mL, about 4pg/mL to about 35pg/mL, about 4pg/mL to about 30pg/mL, about 4pg/mL to about 25pg/mL, about 4pg/mL to about 20pg/mL, about 4pg/mL to about 15pg/mL, about 4pg/mL to about 10pg/mL, about 4pg/mL to about 9pg/mL, about 4pg/mL to about 8pg/mL, about 4pg/mL to about 7pg/mL, about 4pg/mL to about 6pg/mL, about 4pg/mL to about 5pg/mL, about 5pg/mL to about 50pg/mL, about 5pg/mL to about 45pg/mL, about 40pg/mL, about 5pg/mL to about 35pg/mL, about 5pg/mL to about 30pg/mL, about 5pg/mL to about 25pg/mL, about 5pg/mL to about 20pg/mL, about 5pg/mL to about 15pg/mL, about 5pg/mL to about 10pg/mL, about 5pg/mL to about 9pg/mL, about 5pg/mL to about 8pg/mL, about 5pg/mL to about 7pg/mL, about 5pg/mL to about 6pg/mL, about 6pg/mL to about 50pg/mL, about 6pg/mL to about 45pg/mL, about 6pg/mL to about 40pg/mL, about 6pg/mL to about 35pg/mL, about 6pg/mL to about 30pg/mL, about 6pg/mL to about 25pg/mL, about 6pg/mL to about 20 mL, about 6pg/mL to about 15pg/mL, about 6pg/mL to about 10pg/mL, about 6pg/mL to about 9pg/mL, about 6pg/mL to about 8pg/mL, about 6pg/mL to about 7pg/mL, about 7pg/mL to about 50pg/mL, about 7pg/mL to about 45pg/mL, about 7pg/mL to about 40pg/mL, about 7pg/mL to about 35pg/mL, about 7pg/mL to about 30pg/mL, about 7pg/mL to about 25pg/mL, about 7pg/mL to about 20pg/mL, about 7pg/mL to about 15pg/mL, about 7pg/mL to about 10pg/mL, about 7pg/mL to about 9pg/mL, about 7pg/mL to about 8pg/mL, about 50 mL to about 8 mL, about 8pg/mL to about 45pg/mL, about 8pg/mL to about 40pg/mL, about 8pg/mL to about 35pg/mL, about 8pg/mL to about 30pg/mL, about 8pg/mL to about 25pg/mL, about 8pg/mL to about 20pg/mL, about 8pg/mL to about 15pg/mL, about 8pg/mL to about 10pg/mL, about 8pg/mL to about 9pg/mL, about 9pg/mL to about 50pg/mL, about 9pg/mL to about 45pg/mL, about 9pg/mL to about 40pg/mL, about 9pg/mL to about 35pg/mL, about 9pg/mL to about 30pg/mL, about 9pg/mL to about 25pg/mL, about 9pg/mL to about 20pg/mL, about 9pg/mL to about 15 mL, about 9pg/mL to about 10pg/mL, about 10pg/mL to about 50pg/mL, about 10pg/mL to about 45pg/mL, about 10pg/mL to about 40pg/mL, about 10pg/mL to about 35pg/mL, about 10pg/mL to about 30pg/mL, about 10pg/mL to about 25pg/mL, about 10pg/mL to about 20pg/mL, about 10pg/mL to about 15pg/mL, about 20pg/mL to about 50pg/mL, about 20pg/mL to about 45pg/mL, about 20pg/mL to about 40pg/mL, about 20pg/mL to about 35pg/mL, about 20pg/mL to about 30pg/mL, or about 20pg/mL to about 25 pg/mL. In some embodiments, the amount of cTnI may be at least about 0.5pg/mL, at least about 1.0pg/mL, at least about 1.5pg/mL, at least about 2.0pg/mL, at least about 2.5pg/mL, at least about 3.0pg/mL, at least about 4.0pg/mL, at least about 5.0pg/mL, at least about 6.0pg/mL, at least about 7.0pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10pg/mL, at least about 15pg/mL, at least about 20pg/mL, at least about 25pg/mL, at least about 30pg/mL, at least about 35pg/mL, at least about 40pg/mL, at least about 45pg/mL, or at least about 50 pg/mL.

In addition to performing the above-described methods, one skilled in the art (e.g., a physician) will also know and understand how to perform additional tests to detect or assess other co-morbidities (e.g., other diseases, disorders, or conditions besides TBI). Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

In one embodiment, to confirm that a change in the amount or level of cTnI in the methods described herein is attributable to head injury or suspected head injury in the subject, rather than the outcome of an acute cardiac syndrome (e.g., myocardial infarction, heart failure, etc.), a physician or other health care provider may perform or conduct one or more additional tests or procedures to confirm the absence of an acute cardiac syndrome. Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

In some embodiments, the method further comprises treating and/or monitoring the human subject with a traumatic brain injury treatment as described below. In some embodiments, the method further comprises requiring additional testing to obtain further clinical information about the traumatic brain injury. In some embodiments, the method comprises treating a human subject assessed as having mild, moderate, severe, or moderate to severe brain injury with a cardioprotective treatment to protect the heart, as described below.

The nature of the assay employed in the methods described herein is not critical, and the assay may be any assay known in the art, for example, immunoassay, protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining, or spectroscopic methods, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, the assay may be performed in a clinical chemistry format, such as would be known to one skilled in the art. Such assays are described in further detail in sections 11-13 herein.

4. Methods for aiding in the diagnosis and assessment of whether a human subject is likely to suffer from (or has had actual or suspected) a head injury based on changes in the level of cardiac troponin I (cTnI)

The present disclosure relates to, among other methods, a method of aiding diagnosis and assessment of whether a human subject suffers from or may suffer from (or has actual or suspected of suffering from) a head injury. The methods can assist in determining the extent of traumatic brain injury in a human subject with actual or suspected head injury, e.g., determining whether the subject has mild traumatic brain injury or moderate, severe, or moderate to severe traumatic brain injury. As used herein, "determining whether a subject has mild traumatic brain injury or moderate, severe, or moderate to severe traumatic brain injury" refers to the fact that: the foregoing methods may be used, for example, in conjunction with other information (e.g., clinical assessment data) to determine that a subject is more likely to have mild traumatic brain injury or moderate, severe, or moderate to severe traumatic brain injury. The method may comprise performing an assay on at least two samples obtained from a subject, a first sample being obtained from the subject within about 24 hours, e.g., within about 2 hours, after a head injury or suspected injury, a second sample being obtained from the subject from about 3 to about 6 hours after the first sample; detecting cardiac troponin i (ctni) in the at least two samples; and determining whether the subject suffers from mild or moderate, severe, moderate to severe Traumatic Brain Injury (TBI). Determining that the subject is (1) a moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI decreases or increases by at least one absolute amount from the first sample to the second sample, or (2) a mild traumatic brain injury when the level of cTnI does not decrease or increase by at least one absolute amount from the first sample to the second sample. The sample may be a biological sample.

In one alternative, the method may comprise performing an assay on at least two samples obtained from the subject, a first sample obtained from the subject within about 24 hours, e.g., within about 2 hours, after the head injury or suspected injury, and a second sample obtained from the subject from about 3 to about 6 hours after the first sample; detecting cTnI in the at least two samples; and determining whether the subject suffers from mild or moderate, severe, moderate to severe Traumatic Brain Injury (TBI), wherein the subject is determined to be (1) moderate, severe, or moderate to severe traumatic brain injury when the level of cTnI decreases or increases by at least a first absolute amount from the first sample to the second sample, or (2) mild traumatic brain injury when the level of cTnI does not decrease or increase by at least a second absolute amount from the first sample to the second sample. The sample may be a biological sample

In some embodiments, the method can include contacting the sample with an antibody directed against cTnI such that a complex of the antibody and cTnI is formed. The method further comprises detecting the generated antibody-cTnI complex to determine the level of cTnI in each of the first and second samples. The onset of cTnI appearance manifests within about 0 to about 2 hours after the onset of the suspected injury. In some embodiments, the cTnI begins to appear within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 2 hours after the head injury.

In some embodiments, the first sample is obtained at a first time point within about 24 hours of the suspected injury and the second sample is obtained at a second time point, or optionally a third time point or a fourth time point, after the first time point. In some embodiments, the first sample is taken within about 24 hours after the suspected injury and the second sample is taken within about 3 hours to about 6 hours after the first sample. In some embodiments, the time period may be within about 0 minutes, within about 1 minute, within about 2 minutes, within about 3 minutes, within about 4 minutes, within about 5 minutes, within about 6 minutes, within about 7 minutes, within about 8 minutes, within about 9 minutes, within about 10 minutes, within about 11 minutes, within about 12 minutes, within about 13 minutes, within about 14 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 19 hours of an actual or suspected head injury, Within about 23 hours or within about 24 hours, a first sample is obtained or taken from the subject.

In some embodiments, the first sample is obtained at a first time point within about 2 hours of the suspected injury and the second sample is obtained at a second time point, or optionally a third time point or a fourth time point, after the first time point. In some embodiments, the first sample is taken within about 2 hours after the suspected injury and the second sample is taken within about 3 hours to about 6 hours after the first sample. In some embodiments, the first sample is taken from about 0 to about 2 hours after the head injury or suspected injury. For example, the first sample may be about 0 to about 2 hours, about 0 hours to about 90 minutes, about 0 hours to about 60 minutes, about 0 hours to about 45 minutes, about 0 hours to about 30 minutes, about 0 hours to about 20 minutes, about 0 hours to about 15 minutes, about 0 hours to about 10 minutes, about 0 hours to about 5 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 45 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 45 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 15 minutes, about 15 minutes to about 90 minutes, about 15 minutes to about 60 minutes, about 15 minutes to about 45 minutes, about 0 hours to about 20 minutes, about 5 minutes to about 15 minutes, about 15 minutes to about 90 minutes, about 15 minutes to about 60 minutes, about 45 minutes, about 15 minutes to about 45 minutes, about, Between about 15 minutes to about 30 minutes, about 15 minutes to about 20 minutes, about 20 minutes to about 90 minutes, about 20 minutes to about 60 minutes, about 20 minutes to about 45 minutes, or about 20 minutes to about 30 minutes. For example, the first sample may be taken from the human subject within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 2 hours of the head injury or suspected injury.

In some embodiments, the second sample is taken from about 1 hour to about 10 hours after the first time point, e.g., from about 3 hours to about 6 hours after the first time point. In some embodiments, the second sample is taken about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours after the first sample.

In some embodiments, at one or more time points, the subject may receive a glasgow coma scale score before or after the determination of the cardiac troponin level. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on the glasgow coma scale score. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on abnormal head CT. In some embodiments, the subject receives a CT scan before or after performing the assay. In some embodiments, the subject's head CT is normal.

In some embodiments, the reference level of cTnI is associated with a subject having moderate, severe, or moderate to severe traumatic brain injury. In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 3-12 (moderate to severe TBI). In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 3-8 (severe TBI). In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 9-13 (moderate TBI). In some embodiments, the subject is suspected of having mild traumatic brain injury based on the glasgow coma scale score. In some embodiments, the reference level of cTnI is associated with a subject with mild traumatic brain injury. In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 13-15 (mild TBI).

In some embodiments, the absolute amount can be determined by an assay having a sensitivity between at least about 65% and 100% and a specificity between at least about 65% and 100%. For example, absolute amounts can be determined by assays with a sensitivity between at least about 80% and 100% and a specificity between at least about 65% and 100%. In some embodiments, the sensitivity is at least about 65.0%, the sensitivity is at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. In some embodiments, the specificity is at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. For example, the sensitivity is at least about 100% and the specificity is at least about 75%, the sensitivity is at least about 99% and the specificity is at least about 99%, or the sensitivity is at least about 87% and the specificity is at least about 95%.

In some embodiments, the absolute amount of cardiac troponin I in the sample is from about 1pg/mL to about 50pg/mL, from about 1pg/mL to about 45pg/mL, from about 1pg/mL to about 40pg/mL, from about 1pg/mL to about 35pg/mL, from about 1pg/mL to about 30pg/mL, from about 1pg/mL to about 25pg/mL, from about 1pg/mL to about 20pg/mL, from about 1pg/mL to about 15pg/mL, from about 1pg/mL to about 10pg/mL, from about 1pg/mL to about 9pg/mL, from about 1pg/mL to about 8pg/mL, from about 1pg/mL to about 7pg/mL, from about 1pg/mL to about 6/mL, from about 1pg/mL to about 5pg/mL, from about 1pg/mL to about 4 mL, about 1pg/mL to about 3pg/mL, about 1pg/mL to about 2pg/mL, about 1pg/mL to about 1.5pg/mL, about 1.5pg/mL to about 50pg/mL, about 1.5pg/mL to about 45pg/mL, about 1.5pg/mL to about 40pg/mL, about 1.5pg/mL to about 35pg/mL, about 1.5pg/mL to about 30pg/mL, about 1.5pg/mL to about 25pg/mL, about 1.5pg/mL to about 20pg/mL, about 1.5pg/mL to about 15pg/mL, about 1.5pg/mL to about 10pg/mL, about 1.5pg/mL to about 9pg/mL, about 1.5pg/mL to about 8pg/mL, about 1.5pg/mL to about 6pg/mL, about 1.5pg/mL to about 5pg/mL, about 1.5pg/mL to about 4pg/mL, about 1.5pg/mL to about 3pg/mL, about 1.5pg/mL to about 2pg/mL, about 2pg/mL to about 50pg/mL, about 2pg/mL to about 45pg/mL, about 2pg/mL to about 40pg/mL, about 2pg/mL to about 35pg/mL, about 2pg/mL to about 30pg/mL, about 2pg/mL to about 25pg/mL, about 2pg/mL to about 20pg/mL, about 2pg/mL to about 15pg/mL, about 2pg/mL to about 10pg/mL, about 2pg/mL to about 9pg/mL, about 2pg/mL to about 8pg/mL, about 2pg/mL to about 7pg/mL, about 2pg/mL to about 6pg/mL, about 2 mL to about 5 mL, about 2pg/mL to about 4pg/mL, about 2pg/mL to about 3pg/mL, about 3pg/mL to about 50pg/mL, about 3pg/mL to about 45pg/mL, about 3pg/mL to about 40pg/mL, about 3pg/mL to about 35pg/mL, about 3pg/mL to about 30pg/mL, about 3pg/mL to about 25pg/mL, about 3pg/mL to about 20pg/mL, about 3pg/mL to about 15pg/mL, about 3pg/mL to about 10pg/mL, about 3pg/mL to about 9pg/mL, about 3pg/mL to about 8pg/mL, about 3pg/mL to about 7pg/mL, about 3pg/mL to about 6pg/mL, about 3pg/mL to about 5pg/mL, about 3pg/mL to about 4 mL, about 4pg/mL to about 50pg/mL, about 4pg/mL to about 45pg/mL, about 4pg/mL to about 40pg/mL, about 4pg/mL to about 35pg/mL, about 4pg/mL to about 30pg/mL, about 4pg/mL to about 25pg/mL, about 4pg/mL to about 20pg/mL, about 4pg/mL to about 15pg/mL, about 4pg/mL to about 10pg/mL, about 4pg/mL to about 9pg/mL, about 4pg/mL to about 8pg/mL, about 4pg/mL to about 7pg/mL, about 4pg/mL to about 6pg/mL, about 4pg/mL to about 5pg/mL, about 5pg/mL to about 50pg/mL, about 5pg/mL to about 45pg/mL, about 40pg/mL, about 5pg/mL to about 35pg/mL, about 5pg/mL to about 30pg/mL, about 5pg/mL to about 25pg/mL, about 5pg/mL to about 20pg/mL, about 5pg/mL to about 15pg/mL, about 5pg/mL to about 10pg/mL, about 5pg/mL to about 9pg/mL, about 5pg/mL to about 8pg/mL, about 5pg/mL to about 7pg/mL, about 5pg/mL to about 6pg/mL, about 6pg/mL to about 50pg/mL, about 6pg/mL to about 45pg/mL, about 6pg/mL to about 40pg/mL, about 6pg/mL to about 35pg/mL, about 6pg/mL to about 30pg/mL, about 6pg/mL to about 25pg/mL, about 6pg/mL to about 20 mL, about 6pg/mL to about 15pg/mL, about 6pg/mL to about 10pg/mL, about 6pg/mL to about 9pg/mL, about 6pg/mL to about 8pg/mL, about 6pg/mL to about 7pg/mL, about 7pg/mL to about 50pg/mL, about 7pg/mL to about 45pg/mL, about 7pg/mL to about 40pg/mL, about 7pg/mL to about 35pg/mL, about 7pg/mL to about 30pg/mL, about 7pg/mL to about 25pg/mL, about 7pg/mL to about 20pg/mL, about 7pg/mL to about 15pg/mL, about 7pg/mL to about 10pg/mL, about 7pg/mL to about 9pg/mL, about 7pg/mL to about 8pg/mL, about 50 mL to about 8 mL, about 8pg/mL to about 45pg/mL, about 8pg/mL to about 40pg/mL, about 8pg/mL to about 35pg/mL, about 8pg/mL to about 30pg/mL, about 8pg/mL to about 25pg/mL, about 8pg/mL to about 20pg/mL, about 8pg/mL to about 15pg/mL, about 8pg/mL to about 10pg/mL, about 8pg/mL to about 9pg/mL, about 9pg/mL to about 50pg/mL, about 9pg/mL to about 45pg/mL, about 9pg/mL to about 40pg/mL, about 9pg/mL to about 35pg/mL, about 9pg/mL to about 30pg/mL, about 9pg/mL to about 25pg/mL, about 9pg/mL to about 20pg/mL, about 9pg/mL to about 15 mL, about 9pg/mL to about 10pg/mL, about 10pg/mL to about 50pg/mL, about 10pg/mL to about 45pg/mL, about 10pg/mL to about 40pg/mL, about 10pg/mL to about 35pg/mL, about 10pg/mL to about 30pg/mL, about 10pg/mL to about 25pg/mL, about 10pg/mL to about 20pg/mL, about 10pg/mL to about 15pg/mL, about 20pg/mL to about 50pg/mL, about 20pg/mL to about 45pg/mL, about 20pg/mL to about 40pg/mL, about 20pg/mL to about 35pg/mL, about 20pg/mL to about 30pg/mL, or about 20pg/mL to about 25 pg/mL. In some embodiments, the absolute amount may be at least about 0.5pg/mL, at least about 1.0pg/mL, at least about 1.5pg/mL, at least about 2.0pg/mL, at least about 2.5pg/mL, at least about 3.0pg/mL, at least about 4.0pg/mL, at least about 5.0pg/mL, at least about 6.0pg/mL, at least about 7.0pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10pg/mL, at least about 15pg/mL, at least about 20pg/mL, at least about 25pg/mL, at least about 30pg/mL, at least about 35pg/mL, at least about 40pg/mL, at least about 45pg/mL, or at least about 50 pg/mL.

In addition to performing the above-described methods, one skilled in the art (e.g., a physician) will also know and understand how to perform additional tests to detect or assess other co-morbidities (e.g., other diseases, disorders, or conditions besides TBI). Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

In one embodiment, to confirm that a change in the amount or level of cTnI in the methods described herein is attributable to head injury or suspected head injury in the subject, rather than the outcome of an acute cardiac syndrome (e.g., myocardial infarction, heart failure, etc.), a physician or other health care provider may perform or conduct one or more additional tests or procedures to confirm the absence of an acute cardiac syndrome. Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

In some embodiments, the method further comprises treating a human subject assessed as having moderate, severe, or moderate to severe traumatic brain injury with a traumatic brain injury treatment as described below. In some embodiments, the method further comprises monitoring a human subject assessed as having mild traumatic brain injury as described below. In some embodiments, the method further comprises requiring additional testing to obtain further clinical information about the traumatic brain injury. In some embodiments, the method comprises treating a human subject assessed as having mild, moderate, severe, or moderate to severe brain injury with a cardioprotective treatment to protect the heart, as described below.

The nature of the assay employed in the methods described herein is not critical, and the assay may be any assay known in the art, for example, immunoassay, protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining, or spectroscopic methods, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, the assay may be performed in a clinical chemistry format, such as would be known to one skilled in the art. Such assays are described in further detail in sections 11-13 herein.

5. Method for assisted determination of whether to perform a CT scan on a human subject likely to suffer from or having suffered (or having had actual or suspected) a head injury based on the level of cardiac troponin i (ctni)

The present disclosure relates to, among other methods, a method of aiding in determining whether to perform a Computed Tomography (CT) scan on a human subject suffering from or likely to suffer from (or having actual or suspected) head injury, as used herein, "determining whether to perform a CT scan on a human subject" refers to the fact that: the foregoing methods may be used, for example, along with other information (e.g., clinical assessment data) to determine that a subject is more likely to have a positive head CT scan. In particular, such a method may comprise the steps of: performing an assay on at least two samples obtained from the subject, a first sample obtained from the subject within about 24 hours, e.g., within about 2 hours, of a suspected injury, and a second first sample obtained from the subject about 3 to about 6 hours after the first sample; detecting cardiac troponin i (ctni) in the at least two samples; and performing a CT scan on the subject when the level of cTnI decreases or increases by at least one absolute amount from the first sample to the second sample; when the level of cTnI does not decrease or increase by at least one absolute amount from the first sample to the second sample, no CT scan is performed on the subject. The sample may be a biological sample.

In some embodiments, the method can include contacting the sample with an antibody directed against cTnI such that a complex of the antibody and cTnI is formed. The method further comprises detecting the generated antibody-cTnI complex to determine the level of cTnI in each of the first and second samples. cTnI begins to appear within about 0 to about 24 hours, e.g., within about 2 hours, after the suspected injury occurs. In some embodiments, the onset of cTnI manifests itself within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 2 hours of the head injury.

In some embodiments, the first sample is obtained at a first time point within about 24 hours of the suspected injury and the second sample is obtained at a second time point, or optionally a third time point or a fourth time point, after the first time point. In some embodiments, the first sample is taken within about 24 hours after the suspected injury and the second sample is taken within about 3 hours to about 6 hours after the first sample. In some embodiments, the first sample can be within about 0 minutes, within about 1 minute, within about 2 minutes, within about 3 minutes, within about 4 minutes, within about 5 minutes, within about 6 minutes, within about 7 minutes, within about 8 minutes, within about 9 minutes, within about 10 minutes, within about 11 minutes, within about 12 minutes, within about 13 minutes, within about 14 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours of an actual or suspected head injury, Obtained from the subject within about 22 hours, within about 23 hours, or within about 24 hours

In some embodiments, the first sample is taken at a first time point within about 2 hours of the suspected injury and the second sample is taken at a second time point, or optionally a third time point or a fourth time point, after the first time point to determine whether the subject has a positive or negative head CT scan. In some embodiments, the first sample is taken within about 2 hours after the suspected injury and the second sample is taken within about 3 hours to about 6 hours after the first sample. In some embodiments, the first time point is about 0 to about 2 hours after the head injury or suspected injury. For example, the first time point may be between about 0 to about 2 hours, between about 0 hours and about 90 minutes, between about 0 hours and about 60 minutes, between about 0 hours and about 45 minutes, between about 0 hours and about 30 minutes, between about 0 hours and about 20 minutes, between about 0 hours and about 15 minutes, between about 0 hours and about 10 minutes, between about 0 hours and about 5 minutes, between about 5 minutes and about 90 minutes, between about 5 minutes and about 60 minutes, between about 5 minutes and about 45 minutes, between about 5 minutes and about 30 minutes, between about 5 minutes and about 20 minutes, between about 5 minutes and about 15 minutes, between about 5 minutes and about 10 minutes, between about 10 minutes and about 90 minutes, between about 10 minutes and about 60 minutes, between about 10 minutes and about 45 minutes, between about 10 minutes and about 30 minutes, between about 10 minutes and about 20 minutes, after suspected injury, Between about 10 minutes and about 15 minutes, between about 15 minutes and about 90 minutes, between about 15 minutes and about 60 minutes, between about 15 minutes and about 45 minutes, between about 15 minutes and about 30 minutes, between about 15 minutes and about 20 minutes, between about 20 minutes and about 90 minutes, between about 20 minutes and about 60 minutes, between about 20 minutes and about 45 minutes, or between about 20 minutes and about 30 minutes.

In some embodiments, the second time point, or optionally the third time point or the fourth time point, is about 1 hour to about 10 hours after the first time point, for example about 3 hours to about 6 hours after the first time point. In some embodiments, the second time point is about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours after the first time point.

In some embodiments, the absolute amount can be determined by an assay having a sensitivity between at least about 65% and 100% and a specificity between at least about 65% and 100%. For example, the absolute amount can be determined by an assay having a sensitivity between at least about 80% and 100% and a specificity between at least about 65% and 100%. In some embodiments, the sensitivity is at least about 65.0%, the sensitivity is at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. In some embodiments, the specificity is at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%. For example, a sensitivity of at least about 100% and a specificity of at least about 75%, a sensitivity of at least about 99% and a specificity of at least about 99%, or a sensitivity of at least about 87% and a specificity of at least about 95%.

In some embodiments, the absolute amount of cardiac troponin I in the sample is from about 1pg/mL to about 50pg/mL, from about 1pg/mL to about 45pg/mL, from about 1pg/mL to about 40pg/mL, from about 1pg/mL to about 35pg/mL, from about 1pg/mL to about 30pg/mL, from about 1pg/mL to about 25pg/mL, from about 1pg/mL to about 20pg/mL, from about 1pg/mL to about 15pg/mL, from about 1pg/mL to about 10pg/mL, from about 1pg/mL to about 9pg/mL, from about 1pg/mL to about 8pg/mL, from about 1pg/mL to about 7pg/mL, from about 1pg/mL to about 6/mL, from about 1pg/mL to about 5pg/mL, from about 1pg/mL to about 4 mL, about 1pg/mL to about 3pg/mL, about 1pg/mL to about 2pg/mL, about 1pg/mL to about 1.5pg/mL, about 1.5pg/mL to about 50pg/mL, about 1.5pg/mL to about 45pg/mL, about 1.5pg/mL to about 40pg/mL, about 1.5pg/mL to about 35pg/mL, about 1.5pg/mL to about 30pg/mL, about 1.5pg/mL to about 25pg/mL, about 1.5pg/mL to about 20pg/mL, about 1.5pg/mL to about 15pg/mL, about 1.5pg/mL to about 10pg/mL, about 1.5pg/mL to about 9pg/mL, about 1.5pg/mL to about 8pg/mL, about 1.5pg/mL to about 6pg/mL, about 1.5pg/mL to about 5pg/mL, about 1.5pg/mL to about 4pg/mL, about 1.5pg/mL to about 3pg/mL, about 1.5pg/mL to about 2pg/mL, about 2pg/mL to about 50pg/mL, about 2pg/mL to about 45pg/mL, about 2pg/mL to about 40pg/mL, about 2pg/mL to about 35pg/mL, about 2pg/mL to about 30pg/mL, about 2pg/mL to about 25pg/mL, about 2pg/mL to about 20pg/mL, about 2pg/mL to about 15pg/mL, about 2pg/mL to about 10pg/mL, about 2pg/mL to about 9pg/mL, about 2pg/mL to about 8pg/mL, about 2pg/mL to about 7pg/mL, about 2pg/mL to about 6pg/mL, about 2 mL to about 5 mL, about 2pg/mL to about 4pg/mL, about 2pg/mL to about 3pg/mL, about 3pg/mL to about 50pg/mL, about 3pg/mL to about 45pg/mL, about 3pg/mL to about 40pg/mL, about 3pg/mL to about 35pg/mL, about 3pg/mL to about 30pg/mL, about 3pg/mL to about 25pg/mL, about 3pg/mL to about 20pg/mL, about 3pg/mL to about 15pg/mL, about 3pg/mL to about 10pg/mL, about 3pg/mL to about 9pg/mL, about 3pg/mL to about 8pg/mL, about 3pg/mL to about 7pg/mL, about 3pg/mL to about 6pg/mL, about 3pg/mL to about 5pg/mL, about 3pg/mL to about 4 mL, about 4pg/mL to about 50pg/mL, about 4pg/mL to about 45pg/mL, about 4pg/mL to about 40pg/mL, about 4pg/mL to about 35pg/mL, about 4pg/mL to about 30pg/mL, about 4pg/mL to about 25pg/mL, about 4pg/mL to about 20pg/mL, about 4pg/mL to about 15pg/mL, about 4pg/mL to about 10pg/mL, about 4pg/mL to about 9pg/mL, about 4pg/mL to about 8pg/mL, about 4pg/mL to about 7pg/mL, about 4pg/mL to about 6pg/mL, about 4pg/mL to about 5pg/mL, about 5pg/mL to about 50pg/mL, about 5pg/mL to about 45pg/mL, about 40pg/mL, about 5pg/mL to about 35pg/mL, about 5pg/mL to about 30pg/mL, about 5pg/mL to about 25pg/mL, about 5pg/mL to about 20pg/mL, about 5pg/mL to about 15pg/mL, about 5pg/mL to about 10pg/mL, about 5pg/mL to about 9pg/mL, about 5pg/mL to about 8pg/mL, about 5pg/mL to about 7pg/mL, about 5pg/mL to about 6pg/mL, about 6pg/mL to about 50pg/mL, about 6pg/mL to about 45pg/mL, about 6pg/mL to about 40pg/mL, about 6pg/mL to about 35pg/mL, about 6pg/mL to about 30pg/mL, about 6pg/mL to about 25pg/mL, about 6pg/mL to about 20 mL, about 6pg/mL to about 15pg/mL, about 6pg/mL to about 10pg/mL, about 6pg/mL to about 9pg/mL, about 6pg/mL to about 8pg/mL, about 6pg/mL to about 7pg/mL, about 7pg/mL to about 50pg/mL, about 7pg/mL to about 45pg/mL, about 7pg/mL to about 40pg/mL, about 7pg/mL to about 35pg/mL, about 7pg/mL to about 30pg/mL, about 7pg/mL to about 25pg/mL, about 7pg/mL to about 20pg/mL, about 7pg/mL to about 15pg/mL, about 7pg/mL to about 10pg/mL, about 7pg/mL to about 9pg/mL, about 7pg/mL to about 8pg/mL, about 50 mL to about 8 mL, about 8pg/mL to about 45pg/mL, about 8pg/mL to about 40pg/mL, about 8pg/mL to about 35pg/mL, about 8pg/mL to about 30pg/mL, about 8pg/mL to about 25pg/mL, about 8pg/mL to about 20pg/mL, about 8pg/mL to about 15pg/mL, about 8pg/mL to about 10pg/mL, about 8pg/mL to about 9pg/mL, about 9pg/mL to about 50pg/mL, about 9pg/mL to about 45pg/mL, about 9pg/mL to about 40pg/mL, about 9pg/mL to about 35pg/mL, about 9pg/mL to about 30pg/mL, about 9pg/mL to about 25pg/mL, about 9pg/mL to about 20pg/mL, about 9pg/mL to about 15 mL, about 9pg/mL to about 10pg/mL, about 10pg/mL to about 50pg/mL, about 10pg/mL to about 45pg/mL, about 10pg/mL to about 40pg/mL, about 10pg/mL to about 35pg/mL, about 10pg/mL to about 30pg/mL, about 10pg/mL to about 25pg/mL, about 10pg/mL to about 20pg/mL, about 10pg/mL to about 15pg/mL, about 20pg/mL to about 50pg/mL, about 20pg/mL to about 45pg/mL, about 20pg/mL to about 40pg/mL, about 20pg/mL to about 35pg/mL, about 20pg/mL to about 30pg/mL, or about 20pg/mL to about 25 pg/mL. In some embodiments, the absolute amount may be at least about 0.5pg/mL, at least about 1.0pg/mL, at least about 1.5pg/mL, at least about 2.0pg/mL, at least about 2.5pg/mL, at least about 3.0pg/mL, at least about 4.0pg/mL, at least about 5.0pg/mL, at least about 6.0pg/mL, at least about 7.0pg/mL, at least about 8.0, pg/mL, at least about 9.0pg/mL, at least about 10pg/mL, at least about 15pg/mL, at least about 20pg/mL, at least about 25pg/mL, at least about 30pg/mL, at least about 35pg/mL, at least about 40pg/mL, at least about 45pg/mL, or at least about 50 pg/mL.

In addition to performing the above-described methods, one skilled in the art (e.g., a physician) will also know and understand how to perform additional tests to detect or assess other co-morbidities (e.g., other diseases, disorders, or conditions besides TBI). Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

In one embodiment, to confirm that a change in the amount or level of cTnI in the methods described herein is attributable to head injury or suspected head injury in the subject, rather than the outcome of an acute cardiac syndrome (e.g., myocardial infarction, heart failure, etc.), a physician or other health care provider may perform or conduct one or more additional tests or procedures to confirm the absence of an acute cardiac syndrome. Such additional tests or procedures include one or more of the following: electrocardiography, Complete Blood Cell (CBC) counting, integrated metabolic testing, lipid profiling (e.g., to determine HDL, LDL, triglycerides, etc.), angiography, one or more assays to detect or determine the level of one or more of c-reactive protein (CRP), brain natriuretic peptide, plasma ceramide, etc.

In some embodiments, the method further comprises treating a human subject determined to undergo a CT scan with a traumatic brain injury treatment, as described below. In some embodiments, the method further comprises monitoring a human subject determined to undergo a CT scan as described below. In some embodiments, the method further comprises requiring additional testing to obtain further clinical information about the traumatic brain injury. In some embodiments, the method comprises treating a human subject assessed as having mild, moderate, severe, or moderate to severe brain injury with a cardioprotective treatment to protect the heart, as described below.

The nature of the assay employed in the methods described herein is not critical, and the assay may be any assay known in the art, for example, immunoassay, protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining, or spectroscopic methods, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, the assay may be performed in a clinical chemistry format, such as would be known to one skilled in the art. Such assays are described in further detail in sections 11-13 herein.

6. Method of using cardiac troponin i (ctni) to assist in predicting or predicting outcome in human subjects with mild traumatic brain injury

The present disclosure relates, among other methods, to a method for aiding in predicting (or predicting) the outcome of a human subject with mild Traumatic Brain Injury (TBI), e.g., determining whether the subject will have an adverse outcome or a favorable outcome. As used herein, the phrase "determining whether a subject has a favorable outcome" refers to the fact that: the foregoing methods may be used, for example, along with other information (e.g., clinical assessment data) to determine a positive outcome in which a subject is more likely to have a mild TBI. Further, as used herein, the phrase "determining whether a subject has an adverse outcome" refers to the fact that: the foregoing methods may be used, for example, in conjunction with other information (e.g., clinical assessment data) to determine an adverse or negative outcome in which the subject is more likely to have mild TBI. As described above, the methods described herein can be used to determine whether a subject diagnosed with mild TBI is more likely to have (1) a favorable outcome (optionally, the favorable outcome may be complete recovery of the subject and not continue to experience one or more symptoms of mild TBI); or (2) an adverse outcome (optionally, the adverse outcome may be one or more symptoms that the subject has not fully recovered and continues to experience mild TBI).

Alternatively, and optionally, favorable outcome may refer to a subject more likely to suffer no more than one symptom of postconcussion syndrome due to mild TBI, for example: (a) physical difficulties (e.g., headache, dizziness, fatigue, sensitivity to light noise and light, etc.); (b) cognitive difficulties (e.g., inattention, memory problems, restlessness, etc.); (c) mood difficulties (e.g., personality changes, irritability, depression, apathy, etc.); or (d) difficulty in sleeping (e.g., insomnia, etc.). Alternatively, and optionally, subjects with adverse outcome are more likely to suffer from more than one postconcussion syndrome symptom, such as: (a) physical difficulties (e.g., headache, dizziness, fatigue, sensitivity to light noise and light, etc.); (b) cognitive difficulties (e.g., inattention, memory problems, restlessness, etc.); (c) mood difficulties (e.g., personality changes, irritability, depression, apathy, etc.); or (d) difficulty sleeping (e.g., insomnia, etc.); or (e) any combination of (a) - (d). Alternatively, and optionally, adverse outcome may also refer to a subject exhibiting one or more symptoms of mild TBI. Alternatively, and optionally, adverse outcome may also refer to an exacerbation of the subject from mild TBI to moderate, moderate to severe, or severe. Furthermore, subjects with favorable outcomes may have a GOSE score of 5 or higher, while subjects with unfavorable outcomes may have a GOSE score of less than 5.

At the time of this disclosure, it is known in the art that levels of cTnI are elevated in a subject following severe traumatic injury. Indeed, elevated levels of cTnI in subjects with Severe Traumatic Injury tend to be associated with poor outcome (see, Cai et al, Prognostic Value of cardiac troponin I Following Severe Traumatic Brain Injury (cognitive Value of cardiac troponin I coming over serum neurological Brain infusion); 2015 Abstract of Surgical conference (Academic Surgical Congress Abstracts 2015), incorporated herein by reference). In view of this, the findings in the present disclosure: it is surprising that detecting and/or measuring the level of cTnI in a subject suffering from or likely to suffer from head injury can be used to predict injury outcome and severity in human subjects with mild TBI.

In particular, such a method may comprise the steps of: determining the level of cardiac troponin I in a sample taken from the subject within 28 hours, e.g. within 24 hours, after the head injury, and if the level of cTnI is higher than a cTnI reference level, predicting that the subject has an adverse outcome, e.g. at 1 or 6 months, or has a more severe traumatic brain injury; or if the level of cTnI is below a reference level of cTnI, the subject is predicted to have a favorable outcome, e.g., at 1 month or 6 months, or a reduced severity of traumatic brain injury. The sample may be a biological sample.

In some embodiments, the time period may be within about 0 minutes, within about 1 minute, within about 2 minutes, within about 3 minutes, within about 4 minutes, within about 5 minutes, within about 6 minutes, within about 7 minutes, within about 8 minutes, within about 9 minutes, within about 10 minutes, within about 11 minutes, within about 12 minutes, within about 13 minutes, within about 14 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours of a head injury, The sample is obtained or taken from the subject within about 23 hours, within about 24 hours, within about 25 hours, within about 26 hours, within about 27 hours, or within about 28 hours.

In some embodiments, at one or more time points, the subject may receive a glasgow coma scale score before or after the determination of the cardiac troponin level. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on the glasgow coma scale score. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on abnormal head CT. In some embodiments, the subject receives a CT scan before or after performing the assay. In some embodiments, the subject's head CT is normal.

In some embodiments, the subject receives a GOSE score after performing the assay. In some embodiments, the subject is suspected of having an adverse outcome based on the GOSE score. In some embodiments, the subject has a GOSE score of less than 5 at 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after the suspected injury. In some embodiments, the cTnI reference level is associated with a subject with an adverse outcome. In some embodiments, the cTnI reference level is associated with a GOSE score of 1-5. In some embodiments, the subject is suspected of having a favorable outcome based on the GOSE score. In some embodiments, the cTnI reference level is associated with a subject with a favorable outcome. In some embodiments, the cTnI reference level is associated with a GOSE score of 6-8.

In some embodiments, the reference level of cTnI is associated with a subject having more severe traumatic brain injury, e.g., moderate to severe traumatic brain injury. In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 3-12. In some embodiments, the subject is suspected of having mild traumatic brain injury based on the glasgow coma scale score. In some embodiments, the reference level of cTnI is associated with a subject having less severe traumatic brain injury, e.g., mild traumatic brain injury. In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 13-15.

In general, the cTnI reference level may also be used as a benchmark to assess the results obtained in determining the cTnI of a test sample. In general, when making such a comparison, a reference level of cTnI is obtained by running a particular assay under appropriate conditions a sufficient number of times to correlate or correlate the presence, amount, or concentration of the analyte with a particular stage or endpoint of TBI or with a particular indication. Typically, the reference level of cTnI is obtained by an assay of a reference subject (or population of subjects). The measured cTnI may include fragments thereof, degradation products thereof, and/or enzymatic cleavage products thereof.

In certain embodiments, the reference level can be associated with a control subject that has not suffered a head injury.

In some embodiments, the method may comprise obtaining a sample from a subject and contacting the sample with an antibody to cardiac troponin I such that a complex of the antibody and cardiac troponin I is formed. The method further comprises detecting the antibody-cardiac troponin I complex produced.

In some embodiments, the reference level of cTnI is determined by an assay having a sensitivity of between at least about 65% and about 100% and a specificity of between at least about 30% and about 100%. In some embodiments, the sensitivity is between at least about 65% to about 100%, at least about 65% to at least about 99%, at least about 65% to at least about 95%, at least about 65% to at least about 90%, at least about 65% to at least about 85%, at least about 65% to at least about 80%, at least about 65% to at least about 75%, at least about 65% to at least about 70%, at least about 75% to about 100%, at least about 75% to at least about 99%, at least about 75% to at least about 95%, at least about 75% to at least about 90%, at least about 75% to at least about 85%, at least about 75% to at least about 80%, at least about 80% to about 100%, at least about 80% to at least about 99%, at least about 80% to at least about 95%, at least about 80% to at least about 90%, at least about 85% to about 100%, at least about 85% to at least about 99%, at least about 85% to at least about 95%, at least about 85% to at least about 90%, at least about 95% to about 100%, or at least about 95% to at least about 99%. In some embodiments, the sensitivity is at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 83.0%, at least about 83.3%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% to about 100%, between at least about 30% to about 99%, between at least about 30% to about 95%, between at least about 30% to about 90%, between at least about 30% to about 85%, between at least about 30% to about 80%, between at least about 30% to about 75%, between at least about 30% to about 70%, between at least about 30% to about 60%, between at least about 30% to about 50%, between at least about 40% to about 100%, between at least about 40% to about 99%, between at least about 40% to about 95%, between at least about 40% to about 90%, between at least about 40% to about 85%, between at least about 40% to about 80%, between at least about 40% to about 75%, between at least about 40% to about 70%, between at least about 40% to about 60%, between at least about 40% to about 50%, between at least about 45% to about 100%, at least between about 45% and about 99%, at least between about 45% and about 95%, at least between about 45% and about 90%, at least between about 45% and about 85%, at least between about 45% and about 80%, at least between about 45% and about 75%, at least between about 45% and about 70%, at least between about 45% and about 60%, at least between about 45% and about 50%, at least between about 50% and about 100%, at least between about 50% and about 99%, at least between about 50% and about 95%, at least between about 50% and about 90%, at least between about 50% and about 85%, at least between about 50% and about 80%, at least between about 50% and about 75%, at least between about 50% and about 70%, at least between about 50% and about 60%, at least between about 60% and about 100%, at least between about 60% and about 99%, at least between about 60% and about 95%, at least between about 60% and about 90%, at least about 60% to about 85%, at least about 60% to about 80%, at least about 60% to about 75%, at least about 60% to about 70%, at least about 70% to about 100%, at least about 70% to about 99%, at least about 70% to about 95%, at least about 70% to about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%. In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 49.2%, at least about 50.0%, at least about 54.9%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.0%, at least about 99.9%, at least about 99, or at least about 100.0%. For example, a sensitivity of at least about 99% and a specificity of at least about 75%, a sensitivity of at least about 99% and a specificity of at least about 99%, or a sensitivity of at least about 100% and a specificity of at least about 100%.

In some embodiments, the reference level of cardiac troponin I in the sample is about 1.0pg/mL to about 50.0pg/mL, about 1.5pg/mL to about 50.0pg/mL, about 2.0pg/mL to about 50.0pg/mL, about 2.5pg/mL to about 50.0pg/mL, about 3.0pg/mL to about 50.0pg/mL, about 3.5pg/mL to about 50.0pg/mL, about 4.0pg/mL to about 50.0pg/mL, about 4.5pg/mL to about 50.0pg/mL, about 5.0pg/mL to about 50.0pg/mL, about 5.5pg/mL to about 50.0pg/mL, about 6.0pg/mL to about 50.0pg/mL, about 6.5pg/mL to about 50.0pg/mL, about 7.0pg/mL, about 0 mL to about 50.0 mL, about 0pg/mL, about 8.5pg/mL to about 50.0pg/mL, about 9.0pg/mL to about 50.0pg/mL, about 9.5pg/mL to about 50.0pg/mL, about 10.0pg/mL to about 50.0pg/mL, about 1.0pg/mL to about 40.0pg/mL, about 1.5pg/mL to about 40.0pg/mL, about 2.0pg/mL to about 40.0pg/mL, about 2.5pg/mL to about 40.0pg/mL, about 3.0pg/mL to about 40.0pg/mL, about 3.5pg/mL to about 40.0pg/mL, about 4.0pg/mL to about 40.0pg/mL, about 4.5pg/mL to about 40.0pg/mL, about 5pg/mL to about 40.0pg/mL, about 6.0pg/mL to about 40.0pg/mL, about 7.5pg/mL to about 40.0pg/mL, about 8.0pg/mL to about 40.0pg/mL, about 8.5pg/mL to about 40.0pg/mL, about 9.0pg/mL to about 40.0pg/mL, about 9.5pg/mL to about 40.0pg/mL, about 10.0pg/mL to about 40.0pg/mL, about 1.0pg/mL to about 35.0pg/mL, about 1.5pg/mL to about 35.0pg/mL, about 2.0pg/mL to about 35.0pg/mL, about 2.5pg/mL to about 35.0pg/mL, about 3.0pg/mL to about 35.0pg/mL, about 3.5pg/mL to about 35.0pg/mL, about 4.0pg/mL to about 35.0pg/mL, about 35.0pg/mL to about 6.0pg/mL, about 6.5pg/mL to about 35.0pg/mL, about 7.0pg/mL to about 35.0pg/mL, about 7.5pg/mL to about 35.0pg/mL, about 8.0pg/mL to about 35.0pg/mL, about 8.5pg/mL to about 35.0pg/mL, about 9.0pg/mL to about 35.0pg/mL, about 9.5pg/mL to about 35.0pg/mL, about 10.0pg/mL to about 35.0pg/mL, about 1.0pg/mL to about 30.0pg/mL, about 1.5pg/mL to about 30.0pg/mL, about 2.0pg/mL to about 30.0pg/mL, about 2.5pg/mL to about 30.0pg/mL, about 3.0pg/mL to about 30.0pg/mL, about 4 mL to about 4.0pg/mL, about 5.5pg/mL to about 30.0pg/mL, about 6.0pg/mL to about 30.0pg/mL, about 6.5pg/mL to about 30.0pg/mL, about 7.0pg/mL to about 30.0pg/mL, about 7.5pg/mL to about 30.0pg/mL, about 8.0pg/mL to about 30.0pg/mL, about 8.5pg/mL to about 30.0pg/mL, about 9.0pg/mL to about 30.0pg/mL, about 9.5pg/mL to about 30.0pg/mL, about 10.0pg/mL to about 30.0pg/mL, about 1.0pg/mL to about 25.0pg/mL, about 1.5pg/mL to about 25.0pg/mL, about 2.0pg/mL to about 3.0pg/mL, about 4.5pg/mL to about 25.0pg/mL, about 5.0pg/mL to about 25.0pg/mL, about 5.5pg/mL to about 25.0pg/mL, about 6.0pg/mL to about 25.0pg/mL, about 6.5pg/mL to about 25.0pg/mL, about 7.0pg/mL to about 25.0pg/mL, about 7.5pg/mL to about 25.0pg/mL, about 8.0pg/mL to about 25.0pg/mL, about 8.5pg/mL to about 25.0pg/mL, about 9.0pg/mL to about 25.0pg/mL, about 9.5pg/mL to about 25.0pg/mL, about 10.0pg/mL to about 25.0pg/mL, about 1.0pg/mL to about 24.0pg/mL, about 24.0pg/mL to about 2.0pg/mL, about 24.0pg/mL to about 24.0pg/mL, about 3.5pg/mL to about 24.0pg/mL, about 4.0pg/mL to about 24.0pg/mL, about 4.5pg/mL to about 24.0pg/mL, about 5.0pg/mL to about 24.0pg/mL, about 5.5pg/mL to about 24.0pg/mL, about 6.0pg/mL to about 24.0pg/mL, about 6.5pg/mL to about 24.0pg/mL, about 7.0pg/mL to about 24.0pg/mL, about 7.5pg/mL to about 24.0pg/mL, about 8.0pg/mL to about 24.0pg/mL, about 8.5pg/mL to about 24.0pg/mL, about 9.0pg/mL to about 24.0pg/mL, about 9.5pg/mL to about 1.0pg/mL, about 1.0 mL to about 1.0 mL, about 2.5pg/mL to about 23.0pg/mL, about 3.0pg/mL to about 23.0pg/mL, about 3.5pg/mL to about 23.0pg/mL, about 4.0pg/mL to about 23.0pg/mL, about 4.5pg/mL to about 23.0pg/mL, about 5.0pg/mL to about 23.0pg/mL, about 5.5pg/mL to about 23.0pg/mL, about 6.0pg/mL to about 23.0pg/mL, about 6.5pg/mL to about 23.0pg/mL, about 7.0pg/mL to about 23.0pg/mL, about 7.5pg/mL to about 23.0pg/mL, about 8.0pg/mL to about 23.0pg/mL, about 8.5pg/mL to about 9.0pg/mL, about 10pg/mL to about 23.0pg/mL, about 1.0 mL, about 1.5pg/mL to about 22.0pg/mL, about 2.0pg/mL to about 22.0pg/mL, about 2.5pg/mL to about 22.0pg/mL, about 3.0pg/mL to about 22.0pg/mL, about 3.5pg/mL to about 22.0pg/mL, about 4.0pg/mL to about 22.0pg/mL, about 4.5pg/mL to about 22.0pg/mL, about 5.0pg/mL to about 22.0pg/mL, about 5.5pg/mL to about 22.0pg/mL, about 6.0pg/mL to about 22.0pg/mL, about 6.5pg/mL to about 22.0pg/mL, about 7.0pg/mL to about 7.0pg/mL, about 7.0pg/mL to about 22.0pg/mL, about 22.0pg/mL to about 22.0pg/mL, about 7.0pg/mL to about 22.0pg/mL, about 5pg/mL, about 10.0pg/mL to about 22.0pg/mL, about 1.0pg/mL to about 21.0pg/mL, about 1.5pg/mL to about 21.0pg/mL, about 2.0pg/mL to about 21.0pg/mL, about 2.5pg/mL to about 21.0pg/mL, about 3.0pg/mL to about 21.0pg/mL, about 3.5pg/mL to about 21.0pg/mL, about 4.0pg/mL to about 21.0pg/mL, about 4.5pg/mL to about 21.0pg/mL, about 5.0pg/mL to about 21.0pg/mL, about 5.5pg/mL to about 21.0pg/mL, about 6.0pg/mL to about 21.0pg/mL, about 6.5pg/mL to about 6.0pg/mL, about 6.5pg/mL to about 21.0pg/mL, about 6.0pg/mL, about 7.0pg/mL to about 21.0pg/mL, about 7.0pg/mL, about 8 mL to about 21.0pg/mL, about 9.0pg/mL to about 21.0pg/mL, about 9.5pg/mL to about 21.0pg/mL, about 10.0pg/mL to about 21.0pg/mL, about 1.0pg/mL to about 20.0pg/mL, about 1.5pg/mL to about 20.0pg/mL, about 2.0pg/mL to about 20.0pg/mL, about 2.5pg/mL to about 20.0pg/mL, about 3.0pg/mL to about 20.0pg/mL, about 3.5pg/mL to about 20.0pg/mL, about 4.0pg/mL to about 20.0pg/mL, about 4.5pg/mL to about 20.0pg/mL, about 5.0pg/mL to about 20.0pg/mL, about 5pg/mL to about 5.0pg/mL, about 5pg/mL to about 6.0pg/mL, about 7.0pg/mL, about 6 mL to about 7.0pg/mL, about 8.0pg/mL to about 20.0pg/mL, about 8.5pg/mL to about 20.0pg/mL, about 9.0pg/mL to about 20.0pg/mL, about 9.5pg/mL to about 20.0pg/mL, about 10.0pg/mL to about 20.0pg/mL, about 1.0pg/mL to about 19.0pg/mL, about 1.5pg/mL to about 19.0pg/mL, about 2.0pg/mL to about 19.0pg/mL, about 2.5pg/mL to about 19.0pg/mL, about 3.0pg/mL to about 19.0pg/mL, about 4.0pg/mL to about 19.0pg/mL, about 4.5pg/mL to about 19.0pg/mL, about 6.0pg/mL to about 6.0pg/mL, about 7.0pg/mL to about 19.0pg/mL, about 7.5pg/mL to about 19.0pg/mL, about 8.0pg/mL to about 19.0pg/mL, about 8.5pg/mL to about 19.0pg/mL, about 9.0pg/mL to about 19.0pg/mL, about 9.5pg/mL to about 19.0pg/mL, about 10.0pg/mL to about 19.0pg/mL, about 1.0pg/mL to about 18.0pg/mL, about 1.5pg/mL to about 18.0pg/mL, about 2.0pg/mL to about 18.0pg/mL, about 2.5pg/mL to about 18.0pg/mL, about 3.0pg/mL to about 18.0pg/mL, about 3.5pg/mL to about 3.0pg/mL, about 3.5pg/mL to about 4.0pg/mL, about 4.0 mL, about 4 mL to about 4.0pg/mL, about 6.0pg/mL to about 18.0pg/mL, about 6.5pg/mL to about 18.0pg/mL, about 7.0pg/mL to about 18.0pg/mL, about 7.5pg/mL to about 18.0pg/mL, about 8.0pg/mL to about 18.0pg/mL, about 8.5pg/mL to about 18.0pg/mL, about 9.0pg/mL to about 18.0pg/mL, about 9.5pg/mL to about 18.0pg/mL, about 10.0pg/mL to about 18.0pg/mL, about 1.0pg/mL to about 17.0pg/mL, about 1.5pg/mL to about 17.0pg/mL, about 2.0pg/mL to about 17.0pg/mL, about 2.5pg/mL to about 17.0pg/mL, about 2.0pg/mL to about 17.0pg/mL, about 2.5pg/mL to about 4.0pg/mL, about 4 mL to about 4.0pg/mL, about 5.0pg/mL to about 17.0pg/mL, about 5.5pg/mL to about 17.0pg/mL, about 6.0pg/mL to about 17.0pg/mL, about 6.5pg/mL to about 17.0pg/mL, about 7.0pg/mL to about 17.0pg/mL, about 7.5pg/mL to about 17.0pg/mL, about 8.0pg/mL to about 17.0pg/mL, about 8.5pg/mL to about 17.0pg/mL, about 9.0pg/mL to about 17.0pg/mL, about 9.5pg/mL to about 17.0pg/mL, about 10.0pg/mL to about 17.0pg/mL, about 1.0pg/mL to about 16.0pg/mL, about 1.5pg/mL to about 16.0pg/mL, about 3.0pg/mL to about 16.0pg/mL, about 4.0pg/mL to about 16.0pg/mL, about 4.5pg/mL to about 16.0pg/mL, about 5.0pg/mL to about 16.0pg/mL, about 5.5pg/mL to about 16.0pg/mL, about 6.0pg/mL to about 16.0pg/mL, about 6.5pg/mL to about 16.0pg/mL, about 7.0pg/mL to about 16.0pg/mL, about 7.5pg/mL to about 16.0pg/mL, about 8.0pg/mL to about 16.0pg/mL, about 8.5pg/mL to about 16.0pg/mL, about 9.0pg/mL to about 16.0pg/mL, about 9.5pg/mL to about 16.0pg/mL, about 10.0pg/mL to about 15.0pg/mL, about 1.0pg/mL to about 15.0 mL, about 2 mL, about 2.0pg/mL, about 3.0pg/mL to about 15.0pg/mL, about 3.5pg/mL to about 15.0pg/mL, about 4.0pg/mL to about 15.0pg/mL, about 4.5pg/mL to about 15.0pg/mL, about 5.0pg/mL to about 15.0pg/mL, about 5.5pg/mL to about 15.0pg/mL, about 6.0pg/mL to about 15.0pg/mL, about 6.5pg/mL to about 15.0pg/mL, about 7.0pg/mL to about 15.0pg/mL, about 7.5pg/mL to about 15.0pg/mL, about 8.0pg/mL to about 15.0pg/mL, about 8.5pg/mL to about 15.0pg/mL, about 9.0pg/mL to about 15.0pg/mL, about 10 mL to about 15.0pg/mL, or about 15.0 pg/mL. In some embodiments, the amount of cardiac troponin I in the sample is about 1.0pg/mL, about 1.5pg/mL, about 2.0pg/mL, about 2.5pg/mL, about 3.0pg/mL, about 3.5pg/mL, about 4.0pg/mL, about 4.5pg/mL, about 5.0pg/mL, about 5.5pg/mL, about 5.6pg/mL, about 5.7pg/mL, about 5.8pg/mL, about 5.9pg/mL, about 6.0pg/mL, about 6.5pg/mL, about 7.0pg/mL, about 7.5pg/mL, about 8.0pg/mL, about 8.5.5 pg/mL, about 9.0pg/mL, about 9.5pg/mL, or about 10.0 pg/mL.

In some embodiments, the method further comprises treating a human subject predicted to have an adverse outcome with a traumatic brain injury treatment as described below. In some embodiments, the method further comprises monitoring a human subject predicted to have an adverse outcome as described below. In some embodiments, the method further comprises requiring additional testing to obtain further clinical information regarding mild TBI in human subjects predicted to have adverse outcomes. In some embodiments, the method comprises treating a human subject predicted to have an adverse outcome with a cardioprotective treatment to protect the heart, as described below.

The nature of the assay employed in the methods described herein is not critical, and the assay may be any assay known in the art, for example, immunoassay, protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining, or spectroscopic methods, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assays are described in further detail in sections 11-13 herein. Also, the assay may be performed in a clinical chemistry format, such as would be known to one skilled in the art. For example, a clinical chemistry format may include assays involving one antibody or no antibody. Examples of analyzers that can be used in clinical chemistry formats are described in U.S. patent publication nos. 2016/0320422 and 2015/0112630.

7. Method for aiding in predicting or predicting outcome in human subjects with mild traumatic brain injury based on changes in cardiac troponin I (cTnI) levels

The present disclosure relates, among other methods, to a method for aiding in predicting (or predicting) outcome in a human subject with mild Traumatic Brain Injury (TBI), e.g., determining whether a subject will have adverse or negative or favorable outcome. As used herein, the phrase "determining whether a subject has a favorable outcome" refers to the fact that: the foregoing methods can be used, for example, with other information (e.g., clinical assessment data) to determine a positive outcome in which the subject is more likely to have a mild TBI. Further, as used herein, the phrase "determining whether a subject has an adverse outcome" refers to the fact that: the foregoing methods can be used, for example, with other information (e.g., clinical assessment data) to determine that a subject is more likely to have an adverse or negative outcome of mild TBI.

As described above, the methods described herein can be used to determine whether a subject diagnosed with mild TBI is more likely to have (1) a favorable outcome (optionally, the favorable outcome may be complete recovery of the subject and not continue to experience one or more symptoms of mild TBI); or (2) an adverse outcome (optionally, the adverse outcome may be one or more symptoms that the subject has not fully recovered and continues to experience mild TBI).

Alternatively, and optionally, favorable outcome may refer to a subject more likely to suffer no more than one symptom of postconcussion syndrome due to mild TBI, for example: (a) physical difficulties (e.g., headache, dizziness, fatigue, sensitivity to light noise and light, etc.); (b) cognitive difficulties (e.g., inattention, memory problems, restlessness, etc.); (c) mood difficulties (e.g., personality changes, irritability, depression, apathy, etc.); or (d) difficulty in sleeping (e.g., insomnia, etc.). Alternatively, and optionally, subjects with adverse outcome are more likely to suffer from more than one postconcussion syndrome symptom, such as: (a) physical difficulties (e.g., headache, dizziness, fatigue, sensitivity to light noise and light, etc.); (b) cognitive difficulties (e.g., inattention, memory problems, restlessness, etc.); (c) mood difficulties (e.g., personality changes, irritability, depression, apathy, etc.); or (d) difficulty sleeping (e.g., insomnia, etc.); or (e) any combination of (a) - (d). Alternatively, and optionally, adverse outcome may also refer to a subject exhibiting one or more symptoms of mild TBI. Alternatively, and optionally, adverse outcome may also refer to an exacerbation of the subject from mild TBI to moderate, moderate to severe, or severe. Furthermore, subjects with favorable outcomes may have a GOSE score of 5 or higher, while subjects with unfavorable outcomes may have a GOSE score of less than 5.

The methods disclosed herein may comprise performing at least two or more assays on one or more samples obtained from a human subject at two or more different time points after a head injury and determining the level of cardiac troponin I at each of these time points. A subject is determined to be predicted to have an adverse outcome, e.g., at 6 months, or more severe traumatic brain injury, if the amount or level of cardiac troponin I in the sample increases from the first (or prior) sample to a subsequent (e.g., second) sample. However, if the amount or level of cardiac troponin I in the sample remains the same or decreases from the first (or prior) sample to the second (or subsequent) sample, the subject is determined to be predicted to have a favorable outcome, or less severe traumatic brain injury, at 6 months.

In particular, such a method may comprise the steps of: (a) determining a sample from a human subject to measure or detect a cardiac troponin I level in a first sample and a second sample, wherein the first sample is taken from the human subject at a first time point within about 24 hours after a head injury and the second sample is taken from the human subject about 0 to about 4 hours after the first sample, wherein the sample is a biological sample; (b) determining whether the amount of cardiac troponin I increases or decreases from the first sample to the second sample; and (c) predicting an adverse or negative outcome of the human subject if the detected level of cardiac troponin I is increased from the first sample to the second sample. In some embodiments, a human subject is predicted to have an adverse or negative outcome if at least one absolute amount is increased from the first sample to the second sample. In some embodiments, a human subject is predicted to have an adverse or negative outcome if the detected level of cardiac troponin I increases by at least about 20% from the first sample to the second sample, and a favorable outcome if the detected level of cardiac troponin I remains unchanged, decreases, or increases, e.g., by less than about 20%, from the first sample to the second sample.

The first (or prior) sample may be within 0 minutes, within about 1 minute, within about 2 minutes, within about 3 minutes, within about 4 minutes, within about 5 minutes, within about 6 minutes, within about 7 minutes, within about 8 minutes, within about 9 minutes, within about 10 minutes, within about 11 minutes, within about 12 minutes, within about 13 minutes, within about 14 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours of the head injury, obtained or obtained from the subject within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours. In some embodiments, the (or prior) sample may be obtained within about 0 to about 1 hour, within about 0 to about 2 hours, within about 0 to about 3 hours, within about 0 to about 4 hours, within about 0 to about 5 hours, within about 0 to about 6 hours, within about 0 to about 7 hours, within about 0 to about 8 hours, within about 0 to about 9 hours, within about 0 to about 10 hours, within about 0 to about 11 hours, within about 0 to about 12 hours, within about 0 to about 18 hours, within about 6 to about 12 hours, within about 12 to about 18 hours, within about 18 to about 24 hours, or greater than 24 hours after the suspected injury. The second (or subsequent) sample may be obtained after about 0 to 6 hours from the time the first (or previous) sample was obtained. In some embodiments, the second (or subsequent) sample is obtained from the subject within at least about 30 minutes, within at least about 1 hour, within at least about 2 hours, within at least about 3 hours, within at least about 4 hours, within at least about 5 hours, or within at least about 6 hours of obtaining the first sample from the subject.

In some embodiments, the subject is predicted to have an adverse outcome if the detected level of cardiac troponin I is increased from the first sample to the second sample. For example, a subject may be predicted to have an adverse outcome if the detected level of cardiac troponin I increases by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 250%, at least about 500%, or at least about 1000% from a first sample to a second sample. In some embodiments, a subject may be predicted to have an adverse outcome if the detected level of cardiac troponin I increases by at least about 20% from the first sample to the second sample.

In some embodiments, at one or more time points, the subject may receive a glasgow coma scale score before or after the determination of the cardiac troponin level. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on the glasgow coma scale score. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on abnormal head CT. In some embodiments, the subject receives a CT scan before or after performing the assay. In some embodiments, the subject's head CT is normal.

In some embodiments, the subject receives a GOSE score after performing the assay. In some embodiments, the subject is suspected of having an adverse outcome based on the GOSE score. In some embodiments, the subject has a GOSE score of less than 5 at 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after the suspected injury. In some embodiments, the cTnI reference level is associated with a subject with an adverse outcome. In some embodiments, an increase in the level of cTnI correlates with a GOSE score of 1-5. In some embodiments, the subject is suspected of having a favorable outcome based on the GOSE score. In some embodiments, a decrease or no change in cTnI levels is associated with a subject having a favorable outcome. In some embodiments, a decreased or unchanged level of cTnI is associated with a GOSE score of 6-8.

In some embodiments, an increase in cTnI levels is associated with a subject having more severe traumatic brain injury, e.g., moderate to severe traumatic brain injury. In some embodiments, an increase in cTnI levels is associated with a glasgow coma scale score of 3-12. In some embodiments, the subject is suspected of having mild traumatic brain injury based on the glasgow coma scale score. In some embodiments, a decreased or unchanged level of cTnI is associated with a less severe traumatic brain injury, e.g., a mild traumatic brain injury, in the subject. In some embodiments, a decrease or no change in cTnI levels is associated with a glasgow coma scale score of 13-15.

In some embodiments, the method may comprise obtaining a sample from a subject and contacting the sample with an antibody to cardiac troponin I such that a complex of the antibody and cardiac troponin I is formed. The method further comprises detecting the antibody-cardiac troponin I complex produced.

In some embodiments, the amount of the cardiac troponin I in the first or previous sample is from about 1.0pg/mL to about 50.0pg/mL, from about 1.5pg/mL to about 50.0pg/mL, from about 2.0pg/mL to about 50.0pg/mL, from about 2.5pg/mL to about 50.0pg/mL, from about 3.0pg/mL to about 50.0pg/mL, from about 3.5pg/mL to about 50.0pg/mL, from about 4.0pg/mL to about 50.0pg/mL, from about 4.5pg/mL to about 50.0pg/mL, from about 5.0pg/mL to about 50.0pg/mL, from about 5.5pg/mL to about 50.0pg/mL, from about 6.0pg/mL to about 0pg/mL, from about 0pg/mL to about 50.0pg/mL, from about 0pg/mL, from about 6.0pg/mL to about 0pg/mL, from about, about 8.5pg/mL to about 50.0pg/mL, about 9.0pg/mL to about 50.0pg/mL, about 9.5pg/mL to about 50.0pg/mL, about 10.0pg/mL to about 50.0pg/mL, about 1.0pg/mL to about 40.0pg/mL, about 1.5pg/mL to about 40.0pg/mL, about 2.0pg/mL to about 40.0pg/mL, about 2.5pg/mL to about 40.0pg/mL, about 3.0pg/mL to about 40.0pg/mL, about 3.5pg/mL to about 40.0pg/mL, about 4.0pg/mL to about 40.0pg/mL, about 4.5pg/mL to about 40.0pg/mL, about 5pg/mL to about 40.0pg/mL, about 6.0pg/mL to about 40.0pg/mL, about 7.5pg/mL to about 40.0pg/mL, about 8.0pg/mL to about 40.0pg/mL, about 8.5pg/mL to about 40.0pg/mL, about 9.0pg/mL to about 40.0pg/mL, about 9.5pg/mL to about 40.0pg/mL, about 10.0pg/mL to about 40.0pg/mL, about 1.0pg/mL to about 35.0pg/mL, about 1.5pg/mL to about 35.0pg/mL, about 2.0pg/mL to about 35.0pg/mL, about 2.5pg/mL to about 35.0pg/mL, about 3.0pg/mL to about 35.0pg/mL, about 3.5pg/mL to about 35.0pg/mL, about 4.0pg/mL to about 35.0pg/mL, about 35.0pg/mL to about 6.0pg/mL, about 6.5pg/mL to about 35.0pg/mL, about 7.0pg/mL to about 35.0pg/mL, about 7.5pg/mL to about 35.0pg/mL, about 8.0pg/mL to about 35.0pg/mL, about 8.5pg/mL to about 35.0pg/mL, about 9.0pg/mL to about 35.0pg/mL, about 9.5pg/mL to about 35.0pg/mL, about 10.0pg/mL to about 35.0pg/mL, about 1.0pg/mL to about 30.0pg/mL, about 1.5pg/mL to about 30.0pg/mL, about 2.0pg/mL to about 30.0pg/mL, about 2.5pg/mL to about 30.0pg/mL, about 3.0pg/mL to about 30.0pg/mL, about 4 mL to about 4.0pg/mL, about 5.5pg/mL to about 30.0pg/mL, about 6.0pg/mL to about 30.0pg/mL, about 6.5pg/mL to about 30.0pg/mL, about 7.0pg/mL to about 30.0pg/mL, about 7.5pg/mL to about 30.0pg/mL, about 8.0pg/mL to about 30.0pg/mL, about 8.5pg/mL to about 30.0pg/mL, about 9.0pg/mL to about 30.0pg/mL, about 9.5pg/mL to about 30.0pg/mL, about 10.0pg/mL to about 30.0pg/mL, about 1.0pg/mL to about 25.0pg/mL, about 1.5pg/mL to about 25.0pg/mL, about 2.0pg/mL to about 3.0pg/mL, about 4.5pg/mL to about 25.0pg/mL, about 5.0pg/mL to about 25.0pg/mL, about 5.5pg/mL to about 25.0pg/mL, about 6.0pg/mL to about 25.0pg/mL, about 6.5pg/mL to about 25.0pg/mL, about 7.0pg/mL to about 25.0pg/mL, about 7.5pg/mL to about 25.0pg/mL, about 8.0pg/mL to about 25.0pg/mL, about 8.5pg/mL to about 25.0pg/mL, about 9.0pg/mL to about 25.0pg/mL, about 9.5pg/mL to about 25.0pg/mL, about 10.0pg/mL to about 25.0pg/mL, about 1.0pg/mL to about 24.0pg/mL, about 24.0pg/mL to about 2.0pg/mL, about 24.0pg/mL to about 24.0pg/mL, about 3.5pg/mL to about 24.0pg/mL, 4.0pg/mL to about 24.0pg/mL, about 4.5pg/mL to about 24.0pg/mL, about 5.0pg/mL to about 24.0pg/mL, about 5.5pg/mL to about 24.0pg/mL, about 6.0pg/mL to about 24.0pg/mL, about 6.5pg/mL to about 24.0pg/mL, about 7.0pg/mL to about 24.0pg/mL, about 7.5pg/mL to about 24.0pg/mL, about pg 8.0pg/mL to about 24.0pg/mL, about 8.5pg/mL to about 24.0pg/mL, about 9.0pg/mL to about 24.0pg/mL, about 9.5pg/mL to about 24.0pg/mL, about 1.0 mL to about 1.0pg/mL, about 2.5pg/mL to about 23.0pg/mL, about 3.0pg/mL to about 23.0pg/mL, about 3.5pg/mL to about 23.0pg/mL, about 4.0pg/mL to about 23.0pg/mL, about 4.5pg/mL to about 23.0pg/mL, about 5.0pg/mL to about 23.0pg/mL, about 5.5pg/mL to about 23.0pg/mL, about 6.0pg/mL to about 23.0pg/mL, about 6.5pg/mL to about 23.0pg/mL, about 7.0pg/mL to about 23.0pg/mL, about 7.5pg/mL to about 23.0pg/mL, about 8.0pg/mL to about 23.0pg/mL, about 8.5pg/mL to about 9.0pg/mL, about 10pg/mL to about 23.0pg/mL, about 1.0 mL, about 1.5pg/mL to about 22.0pg/mL, about 2.0pg/mL to about 22.0pg/mL, about 2.5pg/mL to about 22.0pg/mL, about 3.0pg/mL to about 22.0pg/mL, about 3.5pg/mL to about 22.0pg/mL, about 4.0pg/mL to about 22.0pg/mL, about 4.5pg/mL to about 22.0pg/mL, about 5.0pg/mL to about 22.0pg/mL, about 5.5pg/mL to about 22.0pg/mL, about 6.0pg/mL to about 22.0pg/mL, about 6.5pg/mL to about 22.0pg/mL, about 7.0pg/mL to about 7.0pg/mL, about 7.0pg/mL to about 22.0pg/mL, about 22.0pg/mL to about 22.0pg/mL, about 7.0pg/mL to about 22.0pg/mL, about 5pg/mL, about 10.0pg/mL to about 22.0pg/mL, about 1.0pg/mL to about 21.0pg/mL, about 1.5pg/mL to about 21.0pg/mL, about 2.0pg/mL to about 21.0pg/mL, about 2.5pg/mL to about 21.0pg/mL, about 3.0pg/mL to about 21.0pg/mL, about 3.5pg/mL to about 21.0pg/mL, about 4.0pg/mL to about 21.0pg/mL, about 4.5pg/mL to about 21.0pg/mL, about 5.0pg/mL to about 21.0pg/mL, about 5.5pg/mL to about 21.0pg/mL, about 6.0pg/mL to about 21.0pg/mL, about 6.5pg/mL to about 6.0pg/mL, about 6.5pg/mL to about 21.0pg/mL, about 6.0pg/mL, about 7.0pg/mL to about 21.0pg/mL, about 7.0pg/mL, about 8 mL to about 21.0pg/mL, about 9.0pg/mL to about 21.0pg/mL, about 9.5pg/mL to about 21.0pg/mL, about 10.0pg/mL to about 21.0pg/mL, about 1.0pg/mL to about 20.0pg/mL, about 1.5pg/mL to about 20.0pg/mL, about 2.0pg/mL to about 20.0pg/mL, about 2.5pg/mL to about 20.0pg/mL, about 3.0pg/mL to about 20.0pg/mL, about 3.5pg/mL to about 20.0pg/mL, about 4.0pg/mL to about 20.0pg/mL, about 4.5pg/mL to about 20.0pg/mL, about 5.0pg/mL to about 20.0pg/mL, about 5pg/mL to about 5.0pg/mL, about 5pg/mL to about 6.0pg/mL, about 7.0pg/mL, about 6 mL to about 7.0pg/mL, about 8.0pg/mL to about 20.0pg/mL, about 8.5pg/mL to about 20.0pg/mL, about 9.0pg/mL to about 20.0pg/mL, about 9.5pg/mL to about 20.0pg/mL, about 10.0pg/mL to about 20.0pg/mL, about 1.0pg/mL to about 19.0pg/mL, about 1.5pg/mL to about 19.0pg/mL, about 2.0pg/mL to about 19.0pg/mL, about 2.5pg/mL to about 19.0pg/mL, about 3.0pg/mL to about 19.0pg/mL, about 4.0pg/mL to about 19.0pg/mL, about 4.5pg/mL to about 19.0pg/mL, about 6.0pg/mL to about 6.0pg/mL, about 7.0pg/mL to about 19.0pg/mL, about 7.5pg/mL to about 19.0pg/mL, about 8.0pg/mL to about 19.0pg/mL, about 8.5pg/mL to about 19.0pg/mL, about 9.0pg/mL to about 19.0pg/mL, about 9.5pg/mL to about 19.0pg/mL, about 10.0pg/mL to about 19.0pg/mL, about 1.0pg/mL to about 18.0pg/mL, about 1.5pg/mL to about 18.0pg/mL, about 2.0pg/mL to about 18.0pg/mL, about 2.5pg/mL to about 18.0pg/mL, about 3.0pg/mL to about 18.0pg/mL, about 3.5pg/mL to about 3.0pg/mL, about 3.5pg/mL to about 4.0pg/mL, about 4.0 mL, about 4 mL to about 4.0pg/mL, about 6.0pg/mL to about 18.0pg/mL, about 6.5pg/mL to about 18.0pg/mL, about 7.0pg/mL to about 18.0pg/mL, about 7.5pg/mL to about 18.0pg/mL, about 8.0pg/mL to about 18.0pg/mL, about 8.5pg/mL to about 18.0pg/mL, about 9.0pg/mL to about 18.0pg/mL, about 9.5pg/mL to about 18.0pg/mL, about 10.0pg/mL to about 18.0pg/mL, about 1.0pg/mL to about 17.0pg/mL, about 1.5pg/mL to about 17.0pg/mL, about 2.0pg/mL to about 17.0pg/mL, about 2.5pg/mL to about 17.0pg/mL, about 2.0pg/mL to about 17.0pg/mL, about 2.5pg/mL to about 4.0pg/mL, about 4 mL to about 4.0pg/mL, about 5.0pg/mL to about 17.0pg/mL, about 5.5pg/mL to about 17.0pg/mL, about 6.0pg/mL to about 17.0pg/mL, about 6.5pg/mL to about 17.0pg/mL, about 7.0pg/mL to about 17.0pg/mL, about 7.5pg/mL to about 17.0pg/mL, about 8.0pg/mL to about 17.0pg/mL, about 8.5pg/mL to about 17.0pg/mL, about 9.0pg/mL to about 17.0pg/mL, about 9.5pg/mL to about 17.0pg/mL, about 10.0pg/mL to about 17.0pg/mL, about 1.0pg/mL to about 16.0pg/mL, about 1.5pg/mL to about 16.0pg/mL, about 3.0pg/mL to about 16.0pg/mL, about 4.0pg/mL to about 16.0pg/mL, about 4.5pg/mL to about 16.0pg/mL, about 5.0pg/mL to about 16.0pg/mL, about 5.5pg/mL to about 16.0pg/mL, about 6.0pg/mL to about 16.0pg/mL, about 6.5pg/mL to about 16.0pg/mL, about 7.0pg/mL to about 16.0pg/mL, about 7.5pg/mL to about 16.0pg/mL, about 8.0pg/mL to about 16.0pg/mL, about 8.5pg/mL to about 16.0pg/mL, about 9.0pg/mL to about 16.0pg/mL, about 9.5pg/mL to about 16.0pg/mL, about 10.0pg/mL to about 15.0pg/mL, about 1.0pg/mL to about 15.0 mL, about 2 mL, about 2.0pg/mL, about 3.0pg/mL to about 15.0pg/mL, about 3.5pg/mL to about 15.0pg/mL, about 4.0pg/mL to about 15.0pg/mL, about 4.5pg/mL to about 15.0pg/mL, about 5.0pg/mL to about 15.0pg/mL, about 5.5pg/mL to about 15.0pg/mL, about 6.0pg/mL to about 15.0pg/mL, about 6.5pg/mL to about 15.0pg/mL, about 7.0pg/mL to about 15.0pg/mL, about 7.5pg/mL to about 15.0pg/mL, about 8.0pg/mL to about 15.0pg/mL, about 8.5pg/mL to about 15.0pg/mL, about 9.0pg/mL to about 15.0pg/mL, about 10 mL to about 15.0pg/mL, or about 15.0 pg/mL.

In some embodiments, the amount of the cardiac troponin I in the second or subsequent sample is from about 1.0pg/mL to about 50.0pg/mL, from about 1.5pg/mL to about 50.0pg/mL, from about 2.0pg/mL to about 50.0pg/mL, from about 2.5pg/mL to about 50.0pg/mL, from about 3.0pg/mL to about 50.0pg/mL, from about 3.5pg/mL to about 50.0pg/mL, from about 4.0pg/mL to about 50.0pg/mL, from about 4.5pg/mL to about 50.0pg/mL, from about 5.0pg/mL to about 50.0pg/mL, from about 5.5pg/mL to about 50.0pg/mL, from about 6.0pg/mL to about 0pg/mL, from about 0pg/mL to about 50.0pg/mL, from about 0pg/mL to about 0pg/mL, from about 0pg, about 8.5pg/mL to about 50.0pg/mL, about 9.0pg/mL to about 50.0pg/mL, about 9.5pg/mL to about 50.0pg/mL, about 10.0pg/mL to about 50.0pg/mL, about 1.0pg/mL to about 40.0pg/mL, about 1.5pg/mL to about 40.0pg/mL, about 2.0pg/mL to about 40.0pg/mL, about 2.5pg/mL to about 40.0pg/mL, about 3.0pg/mL to about 40.0pg/mL, about 3.5pg/mL to about 40.0pg/mL, about 4.0pg/mL to about 40.0pg/mL, about 4.5pg/mL to about 40.0pg/mL, about 5pg/mL to about 40.0pg/mL, about 6.0pg/mL to about 40.0pg/mL, about 7.5pg/mL to about 40.0pg/mL, about 8.0pg/mL to about 40.0pg/mL, about 8.5pg/mL to about 40.0pg/mL, about 9.0pg/mL to about 40.0pg/mL, about 9.5pg/mL to about 40.0pg/mL, about 10.0pg/mL to about 40.0pg/mL, about 1.0pg/mL to about 35.0pg/mL, about 1.5pg/mL to about 35.0pg/mL, about 2.0pg/mL to about 35.0pg/mL, about 2.5pg/mL to about 35.0pg/mL, about 3.0pg/mL to about 35.0pg/mL, about 3.5pg/mL to about 35.0pg/mL, about 4.0pg/mL to about 35.0pg/mL, about 35.0pg/mL to about 6.0pg/mL, about 6.5pg/mL to about 35.0pg/mL, about 7.0pg/mL to about 35.0pg/mL, about 7.5pg/mL to about 35.0pg/mL, about 8.0pg/mL to about 35.0pg/mL, about 8.5pg/mL to about 35.0pg/mL, about 9.0pg/mL to about 35.0pg/mL, about 9.5pg/mL to about 35.0pg/mL, about 10.0pg/mL to about 35.0pg/mL, about 1.0pg/mL to about 30.0pg/mL, about 1.5pg/mL to about 30.0pg/mL, about 2.0pg/mL to about 30.0pg/mL, about 2.5pg/mL to about 30.0pg/mL, about 3.0pg/mL to about 30.0pg/mL, about 4 mL to about 4.0pg/mL, about 5.5pg/mL to about 30.0pg/mL, about 6.0pg/mL to about 30.0pg/mL, about 6.5pg/mL to about 30.0pg/mL, about 7.0pg/mL to about 30.0pg/mL, about 7.5pg/mL to about 30.0pg/mL, about 8.0pg/mL to about 30.0pg/mL, about 8.5pg/mL to about 30.0pg/mL, about 9.0pg/mL to about 30.0pg/mL, about 9.5pg/mL to about 30.0pg/mL, about 10.0pg/mL to about 30.0pg/mL, about 1.0pg/mL to about 29.0pg/mL, about 1.5pg/mL to about 29.0pg/mL, about 2.0pg/mL to about 29.0pg/mL, about 29.0pg/mL to about 3.0pg/mL, about 4.5pg/mL to about 29.0pg/mL, about 5.0pg/mL to about 29.0pg/mL, about 5.5pg/mL to about 29.0pg/mL, about 6.0pg/mL to about 29.0pg/mL, about 6.5pg/mL to about 29.0pg/mL, about 7.0pg/mL to about 29.0pg/mL, about 7.5pg/mL to about 29.0pg/mL, about 8.0pg/mL to about 29.0pg/mL, about 8.5pg/mL to about 29.0pg/mL, about 9.0pg/mL to about 29.0pg/mL, about 9.5pg/mL to about 29.0pg/mL, about 10.0pg/mL to about 29.0pg/mL, about 1.0pg/mL to about 28.0pg/mL, about 28.0pg/mL to about 28.0pg/mL, about 3.5pg/mL to about 28.0pg/mL, about 4.0pg/mL to about 28.0pg/mL, about 4.5pg/mL to about 28.0pg/mL, about 5.0pg/mL to about 28.0pg/mL, about 5.5pg/mL to about 28.0pg/mL, about 6.0pg/mL to about 28.0pg/mL, about 6.5pg/mL to about 28.0pg/mL, about 7.0pg/mL to about 28.0pg/mL, about 7.5pg/mL to about 28.0pg/mL, about 8.0pg/mL to about 28.0pg/mL, about 8.5pg/mL to about 28.0pg/mL, about 9.0pg/mL to about 28.0pg/mL, about 9.5pg/mL to about 9.0pg/mL, about 0pg/mL to about 0pg/mL, about 0 mL, about 9.5pg/mL to about 0 mL, about 0pg/mL, about 0 pg/0 mL, about 0 pg/about 0 mL to about 2.0pg/mL, about 0 pg/1.0 mL to about 0 mL, about, about 2.5pg/mL to about 27.0pg/mL, about 3.0pg/mL to about 27.0pg/mL, about 3.5pg/mL to about 27.0pg/mL, about 4.0pg/mL to about 27.0pg/mL, about 4.5pg/mL to about 27.0pg/mL, about 5.0pg/mL to about 27.0pg/mL, about 5.5pg/mL to about 27.0pg/mL, about 6.0pg/mL to about 27.0pg/mL, about 6.5pg/mL to about 27.0pg/mL, about 7.0pg/mL to about 27.0pg/mL, about 7.5pg/mL to about 27.0pg/mL, about 8.0pg/mL to about 27.0pg/mL, about 8.5pg/mL to about 9.0pg/mL, about 26 mL, about 0.0pg/mL to about 27.0 mL, about 27.0pg/mL, about 1.5pg/mL to about 26.0pg/mL, about 2.0pg/mL to about 26.0pg/mL, about 2.5pg/mL to about 26.0pg/mL, about 3.0pg/mL to about 26.0pg/mL, about 3.5pg/mL to about 26.0pg/mL, about 4.0pg/mL to about 26.0pg/mL, about 4.5pg/mL to about 26.0pg/mL, about 5.0pg/mL to about 26.0pg/mL, about 5.5pg/mL to about 26.0pg/mL, about 6.0pg/mL to about 26.0pg/mL, about 6.5pg/mL to about 26.0pg/mL, about 7.0pg/mL to about 7.0pg/mL, about 7.5pg/mL to about 26.0pg/mL, about 7.0pg/mL to about 26.0pg/mL, about 26.0pg, about 10.0pg/mL to about 26.0pg/mL, about 1.0pg/mL to about 25.0pg/mL, about 1.5pg/mL to about 25.0pg/mL, about 2.0pg/mL to about 25.0pg/mL, about 2.5pg/mL to about 25.0pg/mL, about 3.0pg/mL to about 25.0pg/mL, about 3.5pg/mL to about 25.0pg/mL, about 4.0pg/mL to about 25.0pg/mL, about 4.5pg/mL to about 25.0pg/mL, about 5.0pg/mL to about 25.0pg/mL, about 5.5pg/mL to about 25.0pg/mL, about 6.0pg/mL to about 5.0pg/mL, about 6.5pg/mL to about 25.0pg/mL, about 6.0pg/mL to about 25.0pg/mL, about 8.0pg/mL, about 8 mL to about 8.0/mL, about 9.0pg/mL to about 25.0pg/mL, about 9.5pg/mL to about 25.0pg/mL, about 10.0pg/mL to about 25.0pg/mL, about 1.0pg/mL to about 24.0pg/mL, about 1.5pg/mL to about 24.0pg/mL, about 2.0pg/mL to about 24.0pg/mL, about 2.5pg/mL to about 24.0pg/mL, about 3.0pg/mL to about 24.0pg/mL, about 3.5pg/mL to about 24.0pg/mL, about 4.0pg/mL to about 24.0pg/mL, about 4.5pg/mL to about 24.0pg/mL, about 5.0pg/mL to about 24.0pg/mL, about 5pg/mL to about 5.0pg/mL, about 5pg/mL to about 6.0 mL, about 6.0pg/mL, about 6.0 mL to about 6.0pg/mL, about 8.0pg/mL to about 24.0pg/mL, about 8.5pg/mL to about 24.0pg/mL, about 9.0pg/mL to about 24.0pg/mL, about 9.5pg/mL to about 24.0pg/mL, about 10.0pg/mL to about 24.0pg/mL, about 1.0pg/mL to about 23.0pg/mL, about 1.5pg/mL to about 23.0pg/mL, about 2.0pg/mL to about 23.0pg/mL, about 2.5pg/mL to about 23.0pg/mL, about 3.0pg/mL to about 23.0pg/mL, about 4.0pg/mL to about 23.0pg/mL, about 4.5pg/mL to about 4.0pg/mL, about 4.5pg/mL to about 23.0pg/mL, about 6.5pg/mL to about 23.0pg/mL, about 6.0pg/mL, about 7.0pg/mL to about 23.0pg/mL, about 7.5pg/mL to about 23.0pg/mL, about 8.0pg/mL to about 23.0pg/mL, about 8.5pg/mL to about 23.0pg/mL, about 9.0pg/mL to about 23.0pg/mL, about 9.5pg/mL to about 23.0pg/mL, about 10.0pg/mL to about 23.0pg/mL, about 1.0pg/mL to about 22.0pg/mL, about 1.5pg/mL to about 22.0pg/mL, about 2.0pg/mL to about 22.0pg/mL, about 2.5pg/mL to about 22.0pg/mL, about 3.0pg/mL to about 22.0pg/mL, about 3.5pg/mL to about 4.0pg/mL, about 22.5 pg/mL to about 22.0pg/mL, about 22.0pg/mL to about 22.0pg/mL, about 4.0pg/mL, about 6.0pg/mL to about 22.0pg/mL, about 6.5pg/mL to about 22.0pg/mL, about 7.0pg/mL to about 22.0pg/mL, about 7.5pg/mL to about 22.0pg/mL, about 8.0pg/mL to about 22.0pg/mL, about 8.5pg/mL to about 22.0pg/mL, about 9.0pg/mL to about 22.0pg/mL, about 9.5pg/mL to about 22.0pg/mL, about 10.0pg/mL to about 22.0pg/mL, about 1.0pg/mL to about 21.0pg/mL, about 1.5pg/mL to about 21.0pg/mL, about 2.0pg/mL to about 21.0pg/mL, about 2.5pg/mL to about 21.0pg/mL, about 2.0pg/mL to about 3.0pg/mL, about 3.0pg/mL to about 3.0pg/mL, about 5.0pg/mL to about 21.0pg/mL, about 5.5pg/mL to about 21.0pg/mL, about 6.0pg/mL to about 21.0pg/mL, about 6.5pg/mL to about 21.0pg/mL, about 7.0pg/mL to about 21.0pg/mL, about 7.5pg/mL to about 21.0pg/mL, about 8.0pg/mL to about 21.0pg/mL, about 8.5pg/mL to about 21.0pg/mL, about 9.0pg/mL to about 21.0pg/mL, about 9.5pg/mL to about 21.0pg/mL, about 10.0pg/mL to about 21.0pg/mL, about 1.0pg/mL to about 20.0pg/mL, about 1.5pg/mL to about 2.0pg/mL, about 3.0 mL to about 3.0pg/mL, about 4.0pg/mL to about 20.0pg/mL, about 4.5pg/mL to about 20.0pg/mL, about 5.0pg/mL to about 20.0pg/mL, about 5.5pg/mL to about 20.0pg/mL, about 6.0pg/mL to about 20.0pg/mL, about 6.5pg/mL to about 20.0pg/mL, about 7.0pg/mL to about 20.0pg/mL, about 7.5pg/mL to about 20.0pg/mL, about 8.0pg/mL to about 20.0pg/mL, about 8.5pg/mL to about 20.0pg/mL, about 9.0pg/mL to about 20.0pg/mL, about 9.5pg/mL to about 20.0pg/mL, about 10.0pg/mL to about 19.0pg/mL, about 1.0pg/mL to about 2.0pg/mL, about 3.0pg/mL to about 19.0pg/mL, about 3.5pg/mL to about 19.0pg/mL, about 4.0pg/mL to about 19.0pg/mL, about 4.5pg/mL to about 19.0pg/mL, about 5.0pg/mL to about 19.0pg/mL, about 5.5pg/mL to about 19.0pg/mL, about 6.0pg/mL to about 19.0pg/mL, about 6.5pg/mL to about 19.0pg/mL, about 7.0pg/mL to about 19.0pg/mL, about 7.5pg/mL to about 19.0pg/mL, about 8.0pg/mL to about 19.0pg/mL, about 8.5pg/mL to about 19.0pg/mL, about 9.0pg/mL to about 10.0pg/mL, about 10.0pg/mL to about 1.0pg/mL, about 2.0pg/mL to about 18.0pg/mL, about 2.5pg/mL to about 18.0pg/mL, about 3.0pg/mL to about 18.0pg/mL, about 3.5pg/mL to about 18.0pg/mL, about 4.0pg/mL to about 18.0pg/mL, about 4.5pg/mL to about 18.0pg/mL, about 5.0pg/mL to about 18.0pg/mL, about 5.5pg/mL to about 18.0pg/mL, about 6.0pg/mL to about 18.0pg/mL, about 6.5pg/mL to about 18.0pg/mL, about 7.0pg/mL to about 18.0pg/mL, about 7.5pg/mL to about 18.0pg/mL, about 8.0pg/mL to about 18.0pg/mL, about 10.0pg/mL, about 1.0pg/mL to about 17.0pg/mL, about 1.5pg/mL to about 17.0pg/mL, about 2.0pg/mL to about 17.0pg/mL, about 2.5pg/mL to about 17.0pg/mL, about 3.0pg/mL to about 17.0pg/mL, about 3.5pg/mL to about 17.0pg/mL, about 4.0pg/mL to about 17.0pg/mL, about 4.5pg/mL to about 17.0pg/mL, about 5.0pg/mL to about 17.0pg/mL, about 5.5pg/mL to about 17.0pg/mL, about 6.0pg/mL to about 17.0pg/mL, about 6.5pg/mL to about 17.0pg/mL, about 7.0pg/mL to about 17.0pg/mL, about 8 mL to about 17.0pg/mL, about 9.5pg/mL to about 17.0pg/mL, about 10.0pg/mL to about 17.0pg/mL, about 1.0pg/mL to about 16.0pg/mL, about 1.5pg/mL to about 16.0pg/mL, about 2.0pg/mL to about 16.0pg/mL, about 2.5pg/mL to about 16.0pg/mL, about 3.0pg/mL to about 16.0pg/mL, about 3.5pg/mL to about 16.0pg/mL, about 4.0pg/mL to about 16.0pg/mL, about 4.5pg/mL to about 16.0pg/mL, about 5.0pg/mL to about 16.0pg/mL, about 5.5pg/mL to about 16.0pg/mL, about 6.0pg/mL to about 16.0pg/mL, about 5pg/mL to about 6.0pg/mL, about 6.0pg/mL to about 16.0 mL, about 16.0pg/mL, about 8.5pg/mL to about 16.0pg/mL, about 9.0pg/mL to about 16.0pg/mL, about 9.5pg/mL to about 16.0pg/mL, about 10.0pg/mL to about 16.0pg/mL, about 1.0pg/mL to about 15.0pg/mL, about 1.5pg/mL to about 15.0pg/mL, about 2.0pg/mL to about 15.0pg/mL, about 2.5pg/mL to about 15.0pg/mL, about 3.0pg/mL to about 15.0pg/mL, about 3.5pg/mL to about 15.0pg/mL, about 4.0pg/mL to about 15.0pg/mL, about 4.5pg/mL to about 15.0pg/mL, about 5pg/mL to about 6.0pg/mL, about 15.0pg/mL, about 7.5pg/mL to about 15.0pg/mL, about 8.0pg/mL to about 15.0pg/mL, about 8.5pg/mL to about 15.0pg/mL, about 9.0pg/mL to about 15.0pg/mL, about 9.5pg/mL to about 15.0pg/mL, or about 10.0pg/mL to about 15.0 pg/mL.

In some embodiments, the absolute amount of cTnI is determined by an assay having a sensitivity of between at least about 65% and about 100% and a specificity of between at least about 30% and about 100%. In some embodiments, the sensitivity is between at least about 65% to about 100%, at least about 65% to at least about 99%, at least about 65% to at least about 95%, at least about 65% to at least about 90%, at least about 65% to at least about 85%, at least about 65% to at least about 80%, at least about 65% to at least about 75%, at least about 65% to at least about 70%, at least about 75% to about 100%, at least about 75% to at least about 99%, at least about 75% to at least about 95%, at least about 75% to at least about 90%, at least about 75% to at least about 85%, at least about 75% to at least about 80%, at least about 85% to about 100%, at least about 85% to at least about 99%, at least about 85% to at least about 95%, at least about 85% to at least about 90%, at least about 95% to about 100%, or at least about 95% to at least about 99%. In some embodiments, the sensitivity is at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 82.4%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% to about 100%, between at least about 30% to about 99%, between at least about 30% to about 95%, between at least about 30% to about 90%, between at least about 30% to about 85%, between at least about 30% to about 80%, between at least about 30% to about 75%, between at least about 30% to about 70%, between at least about 30% to about 60%, between at least about 30% to about 50%, between at least about 40% to about 100%, between at least about 40% to about 99%, between at least about 40% to about 95%, between at least about 40% to about 90%, between at least about 40% to about 85%, between at least about 40% to about 80%, between at least about 40% to about 75%, between at least about 40% to about 70%, between at least about 40% to about 60%, between at least about 40% to about 50%, between at least about 50% to about 100%, at least about 50% to about 99%, at least about 50% to about 95%, at least about 50% to about 90%, at least about 50% to about 85%, at least about 50% to about 80%, at least about 50% to about 75%, at least about 50% to about 70%, at least about 50% to about 60%, at least about 60% to about 100%, at least about 60% to about 99%, at least about 60% to about 95%, at least about 60% to about 90%, at least about 60% to about 85%, at least about 60% to about 80%, at least about 60% to about 75%, at least about 60% to about 70%, at least about 70% to about 100%, at least about 70% to about 99%, at least about 70% to about 95%, at least about 70% to about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%. In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 69.5%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.0%, at least about 99.. For example, a sensitivity of at least about 82.4% and a specificity of at least about 69.5%, a sensitivity of at least about 99% and a specificity of at least about 75%, a sensitivity of at least about 99% and a specificity of at least about 99%, or a sensitivity of at least about 100% and a specificity of at least about 100%.

In some embodiments, the absolute amount may be between at least about 1pg/mL to about 25 pg/mL. In some embodiments, the absolute amount may be between at least about 1pg/mL to about 25pg/mL, at least about 1pg/mL to about 20pg/mL, at least about 1pg/mL to about 15pg/mL, at least about 1pg/mL to about 10pg/mL, at least about 1pg/mL to about 9pg/mL, at least about 1pg/mL to about 8pg/mL, at least about 1pg/mL to about 7pg/mL, at least about 1pg/mL to about 6pg/mL, at least about 1pg/mL to about 5pg/mL, at least about 1pg/mL to about 4pg/mL, at least about 1pg/mL to about 3pg/mL, at least about 1pg/mL to about 2pg/mL, at least between about 1pg/mL to about 1.5pg/mL, at least between about 1.5pg/mL to about 25pg/mL, at least between about 1.5pg/mL to about 20pg/mL, at least between about 1.5pg/mL to about 15pg/mL, at least between about 1.5pg/mL to about 10pg/mL, at least between about 1.5pg/mL to about 9pg/mL, at least between about 1.5pg/mL to about 8pg/mL, at least between about 1.5pg/mL to about 7pg/mL, at least between about 1.5pg/mL to about 6pg/mL, at least between about 1.5pg/mL to about 5pg/mL, at least between about 1.5pg/mL to about 4pg/mL, at least between about 1.5pg/mL to about 3pg/mL, at least between about 1.5pg/mL to about 2 mL, at least between about 2pg/mL to about 25pg/mL, at least between about 2pg/mL to about 20pg/mL, at least between about 2pg/mL to about 15pg/mL, at least between about 2pg/mL to about 10pg/mL, at least between about 2pg/mL to about 9pg/mL, at least between about 2pg/mL to about 8pg/mL, at least between about 2pg/mL to about 7pg/mL, at least between about 2pg/mL to about 6pg/mL, at least between about 2pg/mL to about 5pg/mL, at least between about 2pg/mL to about 4pg/mL, at least between about 2pg/mL to about 3pg/mL, at least between about 3pg/mL to about 25pg/mL, at least between about 3pg/mL to about 20pg/mL, at least between about 3pg/mL to about 15pg/mL, at least between about 3pg/mL to about 10pg/mL, at least between about 3pg/mL to about 9pg/mL, at least between about 3pg/mL to about 8pg/mL, at least between about 3pg/mL to about 7pg/mL, at least between about 3pg/mL to about 6pg/mL, at least between about 3pg/mL to about 5pg/mL, at least between about 3pg/mL to about 4pg/mL, at least between about 4pg/mL to about 25pg/mL, at least between about 4pg/mL to about 20pg/mL, at least between about 4pg/mL to about 15pg/mL, at least between about 4pg/mL to about 10pg/mL, at least between about 4/mL to about 9pg/mL, at least between about 4pg/mL to about 8pg/mL, at least between about 4pg/mL to about 7pg/mL, at least between about 4pg/mL to about 6pg/mL, at least between about 4pg/mL to about 5pg/mL, at least between about 5pg/mL to about 25pg/mL, at least between about 5pg/mL to about 20pg/mL, at least between about 5pg/mL to about 15pg/mL, at least between about 5pg/mL to about 10pg/mL, at least between about 5pg/mL to about 9pg/mL, at least between about 5pg/mL to about 8pg/mL, at least between about 5pg/mL to about 7pg/mL, at least between about 5pg/mL to about 6pg/mL, at least between about 6pg/mL to about 25pg/mL, at least between about 6pg/mL to about 20pg/mL, at least between about 6pg/mL to about 15pg/mL, at least between about 6pg/mL to about 10pg/mL, at least between about 6pg/mL to about 9pg/mL, at least between about 6pg/mL to about 8pg/mL, at least between about 6pg/mL to about 7pg/mL, at least between about 7pg/mL to about 25pg/mL, at least between about 7pg/mL to about 20pg/mL, at least between about 7pg/mL to about 15pg/mL, at least between about 7pg/mL to about 10pg/mL, at least between about 7pg/mL to about 9pg/mL, at least between about 7pg/mL to about 8pg/mL, at least between about 8pg/mL to about 25pg/mL, at least between about 8pg/mL to about 20pg/mL, at least between about 8pg/mL to about 15pg/mL, at least between about 8pg/mL to about 10pg/mL, at least between about 8pg/mL to about 9pg/mL, at least between about 9pg/mL to about 25pg/mL, at least between about 9pg/mL to about 20pg/mL, at least between about 9pg/mL to about 15pg/mL, at least between about 9pg/mL to about 10pg/mL, at least between about 10pg/mL to about 25pg/mL, at least between about 10pg/mL to about 20pg/mL, at least between about 10pg/mL to about 15pg/mL, at least between about 15pg/mL to about 25pg/mL, at least between about 15pg/mL to about 20pg/mL, or at least between about 20pg/mL to about 25 pg/mL. In some embodiments, the absolute amount may be at least about 0.5pg/mL, at least about 1pg/mL, at least about 1.5pg/mL, at least about 2pg/mL, at least about 3pg/mL, at least about 4pg/mL, at least about 5pg/mL, at least about 6pg/mL, at least about 7pg/mL, at least about 8pg/mL, at least about 9pg/mL, at least about 10pg/mL, at least about 11pg/mL, at least about 12pg/mL, at least about 13pg/mL, at least about 14pg/mL, at least about 15pg/mL, at least about 20pg/mL, or at least about 25 pg/mL.

In some embodiments, the absolute amount is between about 1pg/mL to about 50pg/mL, between about 1pg/mL to about 40pg/mL, between about 1pg/mL to about 30pg/mL, between about 1pg/mL to about 20pg/mL, between about 1pg/mL to about 10pg/mL, between about 1pg/mL to about 5pg/mL, between about 2.5pg/mL to about 50pg/mL, between about 2.5pg/mL to about 40pg/mL, between about 2.5pg/mL to about 30pg/mL, between about 2.5pg/mL to about 20pg/mL, between about 2.5pg/mL to about 10pg/mL, between about 2.5pg/mL to about 5pg/mL, between about 5pg/mL to about 50pg/mL, between about 40pg/mL, between about 5pg/mL to about 30pg/mL, between about 5pg/mL to about 20pg/mL, or between about 5pg/mL to about 10 pg/mL. In some embodiments, the absolute amount is at least about 1pg/mL, at least about 2pg/mL, at least about 3pg/mL, at least about 4pg/mL, at least about 5pg/mL, at least about 5.5pg/mL, at least about 5.6pg/mL, at least about 5.7pg/mL at least about 5.8pg/mL at least about 5.9pg/mL, at least about 6pg/mL, at least about 7pg/mL, at least about 8pg/mL, at least about 9pg/mL, at least about 10pg/mL, at least about 20pg/mL, at least about 30pg/mL, at least about 40pg/mL, or at least about 10 pg/mL.

In some embodiments, the method further comprises treating a human subject predicted to have an adverse outcome with a traumatic brain injury treatment as described below. In some embodiments, the method further comprises monitoring a human subject predicted to have an adverse outcome as described below. In some embodiments, the method further comprises requiring additional testing to obtain further clinical information regarding mild TBI in human subjects predicted to have adverse outcomes. In some embodiments, the method comprises treating a human subject predicted to have an adverse outcome with a cardioprotective treatment to protect the heart, as described below.

The nature of the assay employed in the methods described herein is not critical, and the assay may be any assay known in the art, for example, immunoassay, protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining, or spectroscopic methods, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assays are described in further detail in sections 11-13 herein. Also, the assay may be performed in a clinical chemistry format, such as would be known to one skilled in the art. For example, a clinical chemistry format may include assays involving one antibody or no antibody. Examples of analyzers that can be used in clinical chemistry formats are described in U.S. patent publication nos. 2016/0320422 and 2015/0112630.

8. Method of using cardiac troponin i (ctni) and age to assist in predicting or predicting outcome in human subjects with mild traumatic brain injury

The present disclosure relates, among other methods, to a method for aiding in predicting (or predicting) outcome in a human subject with mild Traumatic Brain Injury (TBI), e.g., determining whether a subject will have adverse or negative or favorable outcome. As used herein, the phrase "determining whether a subject has a favorable outcome" refers to the fact that: the foregoing methods can be used, for example, with other information (e.g., clinical assessment data) to determine a positive outcome in which the subject is more likely to have a mild TBI. Further, as used herein, the phrase "determining whether a subject has an adverse outcome" refers to the fact that: the foregoing methods can be used, for example, with other information (e.g., clinical assessment data) to determine that a subject is more likely to have an adverse or negative outcome of mild TBI.

As described above, the methods described herein can be used to determine whether a subject diagnosed with mild TBI is more likely to have (1) a favorable outcome (optionally, the favorable outcome may be complete recovery of the subject and not continue to experience one or more symptoms of mild TBI); or (2) an adverse outcome (optionally, the adverse outcome may be one or more symptoms that the subject has not fully recovered and continues to experience mild TBI).

Alternatively, and optionally, favorable outcome may refer to a subject more likely to suffer no more than one symptom of postconcussion syndrome due to mild TBI, for example: (a) physical difficulties (e.g., headache, dizziness, fatigue, sensitivity to light noise and light, etc.); (b) cognitive difficulties (e.g., inattention, memory problems, restlessness, etc.); (c) mood difficulties (e.g., personality changes, irritability, depression, apathy, etc.); or (d) difficulty in sleeping (e.g., insomnia, etc.). Alternatively, and optionally, subjects with adverse outcome are more likely to suffer from more than one postconcussion syndrome symptom, such as: (a) physical difficulties (e.g., headache, dizziness, fatigue, sensitivity to light noise and light, etc.); (b) cognitive difficulties (e.g., inattention, memory problems, restlessness, etc.); (c) mood difficulties (e.g., personality changes, irritability, depression, apathy, etc.); or (d) difficulty sleeping (e.g., insomnia, etc.); or (e) any combination of (a) - (d). Alternatively, and optionally, adverse outcome may also refer to a subject exhibiting one or more symptoms of mild TBI. Alternatively, and optionally, adverse outcome may also refer to an exacerbation of the subject from mild TBI to moderate, moderate to severe, or severe. Furthermore, subjects with favorable outcomes may have a GOSE score of 5 or higher, while subjects with unfavorable outcomes may have a GOSE score of less than 5.

The methods disclosed herein can include assaying a first sample and a second sample obtained from a human subject following a head injury and determining the age of the subject. A first sample is taken from the subject within about 24 hours after the injury, and a second sample is taken from the subject at two or more different time points within 0 to about 4 hours after the first sample is taken. The assay was used to determine the cardiac troponin I level in each of these samples. Determining that the subject is predicted to have an adverse outcome, e.g. at 6 months, or more severe traumatic brain injury, if the amount or level of cardiac troponin I in the first sample and/or the second sample is higher than a reference level of cardiac troponin I and the age of the subject is lower than a reference age, e.g. between the ages of 40 and 50. However, if the amount or level of cardiac troponin I in the first and second samples is below a reference level of cardiac troponin I and the age of the subject is below a reference age, the subject is determined to be predicted to have a favorable outcome, or less severe traumatic brain injury, at 6 months.

In some embodiments of the above methods, the subject is 18 to 30 years of age. In other embodiments of the above methods, the subject is 31 to 50 years of age. In still other embodiments of the above methods, the subject is 51 to 70 years of age. In still other embodiments of the above methods, the subject is 70 to 100 years of age. In still other embodiments of the above methods, the age is 18 years or less. In still other embodiments of the above methods, the age is 19-50 years. In yet other embodiments, the age is 51 to 70 years. In still other embodiments of the above methods, the age is greater than 70 years. In still other embodiments of the above methods, the subject is 20 to 30 years of age. In still other embodiments of the above methods, the subject is 31 to 40 years of age. In still other embodiments of the above methods, the subject is from 41 to 50 years of age. In still other embodiments of the above methods, the subject is 51 to 60 years of age. In still other embodiments of the above methods, the subject is 61 to 70 years of age. In still other embodiments of the above methods, the subject is 71 to 80 years of age. In still other embodiments of the above methods, the subject is 81 to 90 years of age. In still other embodiments of the above methods, the subject is 91 to 100 years of age.

In some embodiments, the level of cTnI in the samples taken at the first and second time points is combined with the age of the subject in the following formula:

in some embodiments, age is a continuous variable. For example, if weights are assigned based on coefficients, the weight of age may be 1.06, the weight of initial troponin may be 0.25, and the weight of 4 hour troponin may be 4.22. The first (or prior) sample may be within 0 minutes, within about 1 minute, within about 2 minutes, within about 3 minutes, within about 4 minutes, within about 5 minutes, within about 6 minutes, within about 7 minutes, within about 8 minutes, within about 9 minutes, within about 10 minutes, within about 11 minutes, within about 12 minutes, within about 13 minutes, within about 14 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours of the head injury, obtained or obtained from the subject within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours. In some embodiments, the (or prior) sample may be obtained within about 0 to about 1 hour, within about 0 to about 2 hours, within about 0 to about 3 hours, within about 0 to about 4 hours, within about 0 to about 5 hours, within about 0 to about 6 hours, within about 0 to about 7 hours, within about 0 to about 8 hours, within about 0 to about 9 hours, within about 0 to about 10 hours, within about 0 to about 11 hours, within about 0 to about 12 hours, within about 0 to about 18 hours, within about 6 to about 12 hours, within about 12 to about 18 hours, within about 18 to about 24 hours, or greater than 24 hours after injury. The second (or subsequent) sample may be obtained after about 0 to 6 hours from the time the first (or previous) sample was obtained. In some embodiments, the second (or subsequent) sample is obtained from the subject within at least about 30 minutes, within at least about 1 hour, within at least about 2 hours, within at least about 3 hours, within at least about 4 hours, within at least about 5 hours, or within at least about 6 hours of obtaining the first sample from the subject.

In some embodiments, at one or more time points, the subject may receive a glasgow coma scale score before or after the determination of the cardiac troponin level. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on the glasgow coma scale score. In certain embodiments, the subject may be suspected of having mild traumatic brain injury based on abnormal head CT. In some embodiments, the subject receives a CT scan before or after performing the assay. In some embodiments, the subject's head CT is normal.

In some embodiments, the subject receives a GOSE score after performing the assay. In some embodiments, the subject is suspected of having an adverse outcome based on the GOSE score. In some embodiments, the subject has a GOSE score of less than 5 at 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months post-injury. In some embodiments, the cTnI reference level is associated with a subject with an adverse outcome. In some embodiments, the cTnI reference level is associated with a GOSE score of 1-5. In some embodiments, the subject is suspected of having a favorable outcome based on the GOSE score. In some embodiments, the cTnI reference level is associated with a subject having a favorable outcome. In some embodiments, the cTnI reference level is associated with a GOSE score of 6-8.

In some embodiments, the reference level of cTnI is associated with a subject having more severe traumatic brain injury, e.g., moderate to severe traumatic brain injury. In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 3-12. In some embodiments, the subject is suspected of having mild traumatic brain injury based on the glasgow coma scale score. In some embodiments, the reference level of cTnI is associated with a subject having less severe traumatic brain injury, e.g., mild traumatic brain injury. In some embodiments, the cTnI reference level is associated with a glasgow coma scale score of 13-15.

In general, the cTnI reference level may also be used as a benchmark to assess the results obtained in determining the cTnI of a test sample. In general, when making such a comparison, a reference level of cTnI is obtained by running a particular assay under appropriate conditions a sufficient number of times to correlate or correlate the presence, amount, or concentration of the analyte with a particular stage or endpoint of TBI or with a particular indication. Typically, the reference level of cTnI is obtained by an assay of a reference subject (or population of subjects). The measured cTnI may include fragments thereof, degradation products thereof, and/or enzymatic cleavage products thereof.

In certain embodiments, the reference level can be associated with a control subject that has not suffered a head injury.

In some embodiments, the method may comprise obtaining a sample from a subject and contacting the sample with an antibody to cardiac troponin I such that a complex of the antibody and cardiac troponin I is formed. The method further comprises detecting the antibody-cardiac troponin I complex produced.

In some embodiments, the reference level of CTnI is determined by an assay having a sensitivity between at least about 65% and about 100% and a specificity between at least about 30% and about 100%. In some embodiments, the sensitivity is between at least about 65% to about 100%, at least about 65% to at least about 99%, at least about 65% to at least about 95%, at least about 65% to at least about 90%, at least about 65% to at least about 85%, at least about 65% to at least about 80%, at least about 65% to at least about 75%, at least about 65% to at least about 70%, at least about 75% to about 100%, at least about 75% to at least about 99%, at least about 75% to at least about 95%, at least about 75% to at least about 90%, at least about 75% to at least about 85%, at least about 75% to at least about 80%, at least about 85% to about 100%, at least about 85% to at least about 99%, at least about 85% to at least about 95%, at least about 85% to at least about 90%, at least about 95% to about 100%, or at least about 95% to at least about 99%. In some embodiments, the sensitivity is at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 87.5%, at least about 90.0%, at least about 95.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100.0%.

In some embodiments, the specificity is between at least about 30% to about 100%, between at least about 30% to about 99%, between at least about 30% to about 95%, between at least about 30% to about 90%, between at least about 30% to about 85%, between at least about 30% to about 80%, between at least about 30% to about 75%, between at least about 30% to about 70%, between at least about 30% to about 60%, between at least about 30% to about 50%, between at least about 40% to about 100%, between at least about 40% to about 99%, between at least about 40% to about 95%, between at least about 40% to about 90%, between at least about 40% to about 85%, between at least about 40% to about 80%, between at least about 40% to about 75%, between at least about 40% to about 70%, between at least about 40% to about 60%, between at least about 40% to about 50%, between at least about 50% to about 100%, at least about 50% to about 99%, at least about 50% to about 95%, at least about 50% to about 90%, at least about 50% to about 85%, at least about 50% to about 80%, at least about 50% to about 75%, at least about 50% to about 70%, at least about 50% to about 60%, at least about 60% to about 100%, at least about 60% to about 99%, at least about 60% to about 95%, at least about 60% to about 90%, at least about 60% to about 85%, at least about 60% to about 80%, at least about 60% to about 75%, at least about 60% to about 70%, at least about 70% to about 100%, at least about 70% to about 99%, at least about 70% to about 95%, at least about 70% to about 90%, at least about 70% to about 85%, at least about 70% to about 80%, at least about 70% to about 75%, at least about 80% to about 100%, at least about 80% to about 99%, at least about 80% to about 95%, at least about 80% to about 90%, at least about 80% to about 85%, at least about 90% to about 100%, at least about 90% to about 99%, at least about 90% to about 95%, at least about 95% to about 99%, or at least about 95% to about 100%. In some embodiments, the specificity is at least about 30.0%, at least about 31.0%, at least about 32.0%, at least about 33.0%, at least about 34.0%, at least about 35.0%, at least about 36.0%, at least about 37.0%, at least about 38.0%, at least about 39.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 91.0%, at least about 92.0%, at least about 93.0%, at least about 94.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.0. For example, a sensitivity of at least about 99% and a specificity of at least about 75%, a sensitivity of at least about 99% and a specificity of at least about 99%, or a sensitivity of at least about 100% and a specificity of at least about 100%.

In some embodiments, the reference level of cardiac troponin I is from about 0.0100pg/mL to about 50.0pg/mL, from about 0.0200pg/mL to about 50.0pg/mL, from about 0.0300pg/mL to about 50.0pg/mL, from about 0.036pg/mL to about 50.0pg/mL, from about 0.0400pg/mL to about 50.0pg/mL, from about 0.0500pg/mL to about 50.0pg/mL, from about 0.1pg/mL to about 50.0pg/mL, from about 0.5pg/mL to about 50.0pg/mL, from about 1.0pg/mL to about 50.0pg/mL, from about 1.5pg/mL to about 50.0pg/mL, from about 2.0pg/mL to about 50.0pg/mL, from about 3.0pg/mL to about 50.0pg/mL, from about 3 mL, about 4.5pg/mL to about 50.0pg/mL, about 5.0pg/mL to about 50.0pg/mL, about 5.5pg/mL to about 50.0pg/mL, about 6.0pg/mL to about 50.0pg/mL, about 6.5pg/mL to about 50.0pg/mL, about 7.0pg/mL to about 50.0pg/mL, about 7.5pg/mL to about 50.0pg/mL, about 8.0pg/mL to about 50.0pg/mL, about 8.5pg/mL to about 50.0pg/mL, about 9.0pg/mL to about 50.0pg/mL, about 9.5pg/mL to about 50.0pg/mL, about 9.0pg/mL to about 50.0pg/mL, about 10.0pg/mL to about 50.0pg/mL, about 0pg/mL to about 40.0pg/mL, about 0100 mL to about 40.0 mL, about 40pg/mL to about 40.0/mL, about 40 mL to about 40.0/mL, about 0.0500pg/mL to about 40.0pg/mL, about 0.1pg/mL to about 40.0pg/mL, about 0.5pg/mL to about 40.0pg/mL, about 1.0pg/mL to about 40.0pg/mL, about 1.5pg/mL to about 40.0pg/mL, about 2.0pg/mL to about 40.0pg/mL, about 2.5pg/mL to about 40.0pg/mL, about 3.0pg/mL to about 40.0pg/mL, about 3.5pg/mL to about 40.0pg/mL, about 4.0pg/mL to about 40.0pg/mL, about 4.5pg/mL to about 40.0pg/mL, about 5.0pg/mL to about 40.0pg/mL, about 5pg/mL to about 40.0pg/mL, about 6.0pg/mL to about 40.0pg/mL, about 6.0pg/mL, about 8.0pg/mL to about 40.0pg/mL, about 8.5pg/mL to about 40.0pg/mL, about 9.0pg/mL to about 40.0pg/mL, about 9.5pg/mL to about 40.0pg/mL, about 10.0pg/mL to about 40.0pg/mL, about 0.0100pg/mL to about 35.0pg/mL, about 0.0200pg/mL to about 35.0pg/mL, about 0.0300pg/mL to about 35.0pg/mL, about 0.036pg/mL to about 35.0pg/mL, about 0.0400pg/mL to about 35.0pg/mL, about 0.0500pg/mL to about 35.0pg/mL, about 0.1pg/mL to about 35.0pg/mL, about 0pg/mL to about 35.0pg/mL, about 0.0500/mL to about 35.0/mL, about 0pg/mL to about 35.5 pg/mL, about 0.1pg/mL to about 2.5pg/mL, about 0.5pg/mL to about 2pg/mL, about 3.0pg/mL to about 35.0pg/mL, about 3.5pg/mL to about 35.0pg/mL, about 4.0pg/mL to about 35.0pg/mL, about 4.5pg/mL to about 35.0pg/mL, about 5.0pg/mL to about 35.0pg/mL, about 5.5pg/mL to about 35.0pg/mL, about 6.0pg/mL to about 35.0pg/mL, about 6.5pg/mL to about 35.0pg/mL, about 7.0pg/mL to about 35.0pg/mL, about 7.5pg/mL to about 35.0pg/mL, about 8.0pg/mL to about 35.0pg/mL, about 8.5pg/mL to about 35.0pg/mL, about 9.0pg/mL to about 30.0pg/mL, about 10 mL to about 0.0pg/mL, about 10.0pg/mL to about 0.0pg/mL, about 0.0300pg/mL to about 30.0pg/mL, about 0.036pg/mL to about 30.0pg/mL, about 0.0400pg/mL to about 30.0pg/mL, about 0.0500pg/mL to about 30.0pg/mL, about 0.1pg/mL to about 30.0pg/mL, about 0.5pg/mL to about 30.0pg/mL, about 1.0pg/mL to about 30.0pg/mL, about 1.5pg/mL to about 30.0pg/mL, about 2.0pg/mL to about 30.0pg/mL, about 2.5pg/mL to about 30.0pg/mL, about 3.0pg/mL to about 30.0pg/mL, about 3.5pg/mL to about 30.0pg/mL, about 4.0pg/mL to about 30.0pg/mL, about 0pg/mL to about 6.0pg/mL, about 0pg/mL to about 0pg/mL, about 6.5pg/mL to about 30.0pg/mL, about 7.0pg/mL to about 30.0pg/mL, about 7.5pg/mL to about 30.0pg/mL, about 8.0pg/mL to about 30.0pg/mL, about 8.5pg/mL to about 30.0pg/mL, about 9.0pg/mL to about 30.0pg/mL, about 9.5pg/mL to about 30.0pg/mL, about 9.0pg/mL to about 30.0pg/mL, about 10.0pg/mL to about 30.0pg/mL, about 0.0100pg/mL to about 25.0pg/mL, about 0.0200pg/mL to about 25.0pg/mL, about 0.0300pg/mL to about 25.0pg/mL, about 0.036pg/mL to about 25.0pg/mL, about 0pg/mL to about 0.0500pg/mL, about 0.0400/mL to about 0.0.0/mL, about 0/mL to about 25.0pg/mL, about 0/mL, about 1.5pg/mL to about 25.0pg/mL, about 2.0pg/mL to about 25.0pg/mL, about 2.5pg/mL to about 25.0pg/mL, about 3.0pg/mL to about 25.0pg/mL, about 3.5pg/mL to about 25.0pg/mL, about 4.0pg/mL to about 25.0pg/mL, about 4.5pg/mL to about 25.0pg/mL, about 5.0pg/mL to about 25.0pg/mL, about 5.5pg/mL to about 25.0pg/mL, about 6.0pg/mL to about 25.0pg/mL, about 6.5pg/mL to about 25.0pg/mL, about 7.0pg/mL to about 7.0pg/mL, about 7.0pg/mL to about 25.0pg/mL, about 7.0pg/mL to about 8.0pg/mL, about 25.0pg/mL to about 8.0pg/mL, about 10.0pg/mL to about 25.0pg/mL, about 0.0100pg/mL to about 24.0pg/mL, about 0.0200pg/mL to about 24.0pg/mL, about 0.0300pg/mL to about 24.0pg/mL, about 0.036pg/mL to about 24.0pg/mL, about 0.0400pg/mL to about 24.0pg/mL, about 0.0500pg/mL to about 24.0pg/mL, about 0.1pg/mL to about 24.0pg/mL, about 0.5pg/mL to about 24.0pg/mL, about 1.0pg/mL to about 24.0pg/mL, about 1.5pg/mL to about 24.0pg/mL, about 2.0pg/mL to about 24.0pg/mL, about 3.0pg/mL to about 3.0pg/mL, about 5.0pg/mL to about 24.0pg/mL, about 5.5pg/mL to about 24.0pg/mL, about 6.0pg/mL to about 24.0pg/mL, about 6.5pg/mL to about 24.0pg/mL, about 7.0pg/mL to about 24.0pg/mL, about 7.5pg/mL to about 24.0pg/mL, about 8.0pg/mL to about 24.0pg/mL, about 8.5pg/mL to about 24.0pg/mL, about 9.0pg/mL to about 24.0pg/mL, about 9.5pg/mL to about 24.0pg/mL, about 10.0pg/mL to about 24.0pg/mL, about 0pg/mL to about 24.0pg/mL, about 0.0pg/mL to about 23.0pg/mL, about 0pg/mL to about 0200.0 pg/mL, about 0pg/mL to about 23.0pg/mL, about 0pg/mL to about 20.0pg/mL, about 0300pg/mL, about 0pg/mL to about 23.0pg/mL, about 0pg/mL, about 0pg/mL to about 0400.23.0.0 g/mL, about 23.0.0.0 g/mL, about 23.0pg/mL, about 0pg, about 0.1pg/mL to about 23.0pg/mL, about 0.5pg/mL to about 23.0pg/mL, about 1.0pg/mL to about 23.0pg/mL, about 1.5pg/mL to about 23.0pg/mL, about 2.0pg/mL to about 23.0pg/mL, about 2.5pg/mL to about 23.0pg/mL, about 3.0pg/mL to about 23.0pg/mL, about 3.5pg/mL to about 23.0pg/mL, about 4.0pg/mL to about 23.0pg/mL, about 4.5pg/mL to about 23.0pg/mL, about 5.0pg/mL to about 23.0pg/mL, about 5.5pg/mL to about 23.0pg/mL, about 6.0pg/mL to about 23.0pg/mL, about 7.0pg/mL to about 7.0pg/mL, about 8.5pg/mL to about 23.0pg/mL, about 9.0pg/mL to about 23.0pg/mL, about 9.5pg/mL to about 23.0pg/mL, about 10.0pg/mL to about 23.0pg/mL, about 0.0100pg/mL to about 22.0pg/mL, about 0.0200pg/mL to about 22.0pg/mL, about 0.0300pg/mL to about 22.0pg/mL, about 0.036 0406 pg/mL to about 22.0pg/mL, about 0.0pg/mL to about 22.0pg/mL, about 0.0500pg/mL to about 22.0pg/mL, about 0.1pg/mL to about 22.0/mL, about 0.5pg/mL to about 22.0pg/mL, about 0.0pg/mL to about 22.0pg/mL, about 0pg/mL to about 2.0pg/mL, about 2 mL to about 22.0pg/mL, about 3.5pg/mL to about 22.0pg/mL, about 4.0pg/mL to about 22.0pg/mL, about 4.5pg/mL to about 22.0pg/mL, about 5.0pg/mL to about 22.0pg/mL, about 5.5pg/mL to about 22.0pg/mL, about 6.0pg/mL to about 22.0pg/mL, about 6.5pg/mL to about 22.0pg/mL, about 7.0pg/mL to about 22.0pg/mL, about 7.5pg/mL to about 22.0pg/mL, about 8.0pg/mL to about 22.0pg/mL, about 8.5pg/mL to about 22.0pg/mL, about 9.0pg/mL to about 9.0pg/mL, about 9.5pg/mL to about 10.0pg/mL, about 10pg/mL, about 21.0 mL to about 21.0pg/mL, about 21.0 mL, about 21.0pg/mL, about 0.036pg/mL to about 21.0pg/mL, about 0.0400pg/mL to about 21.0pg/mL, about 0.0500pg/mL to about 21.0pg/mL, about 0.1pg/mL to about 21.0pg/mL, about 0.5pg/mL to about 21.0pg/mL, about 1.0pg/mL to about 21.0pg/mL, about 1.5pg/mL to about 21.0pg/mL, about 2.0pg/mL to about 21.0pg/mL, about 2.5pg/mL to about 21.0pg/mL, about 3.0pg/mL to about 21.0pg/mL, about 3.5pg/mL to about 21.0pg/mL, about 4.0pg/mL to about 5.0pg/mL, about 4.0pg/mL to about 5pg/mL, about 5pg/mL to about 21.0pg/mL, about 6.0pg/mL, about 21.0pg/mL, about 7.0pg/mL to about 21.0pg/mL, about 7.5pg/mL to about 21.0pg/mL, about 8.0pg/mL to about 21.0pg/mL, about 8.5pg/mL to about 21.0pg/mL, about 9.0pg/mL to about 21.0pg/mL, about 9.5pg/mL to about 21.0pg/mL, about 10.0pg/mL to about 21.0pg/mL, about 0.0100pg/mL to about 20.0pg/mL, about 0.0200pg/mL to about 20.0pg/mL, about 0.0300pg/mL to about 20.0pg/mL, about 0.036pg/mL to about 20.0/mL, about 0.0400pg/mL to about 20.0pg/mL, about 0.0500/mL to about 1.0.0 pg/mL, about 2.0pg/mL to about 20.0pg/mL, about 2.5pg/mL to about 20.0pg/mL, about 3.0pg/mL to about 20.0pg/mL, about 3.5pg/mL to about 20.0pg/mL, about 4.0pg/mL to about 20.0pg/mL, about 4.5pg/mL to about 20.0pg/mL, about 5.0pg/mL to about 20.0pg/mL, about 5.5pg/mL to about 20.0pg/mL, about 6.0pg/mL to about 20.0pg/mL, about 6.5pg/mL to about 20.0pg/mL, about 7.0pg/mL to about 20.0pg/mL, about 7.5pg/mL to about 20.0pg/mL, about 8.0pg/mL to about 20.0pg/mL, about 10.0pg/mL, about 0.0100pg/mL to about 19.0pg/mL, about 0.0200pg/mL to about 19.0pg/mL, about 0.0300pg/mL to about 19.0pg/mL, about 0.036pg/mL to about 19.0pg/mL, about 0.0400pg/mL to about 19.0pg/mL, about 0.0500pg/mL to about 19.0pg/mL, about 0.1pg/mL to about 19.0pg/mL, about 0.5pg/mL to about 19.0pg/mL, about 1.0pg/mL to about 19.0pg/mL, about 1.5pg/mL to about 19.0pg/mL, about 2.0pg/mL to about 19.0pg/mL, about 2.5pg/mL to about 19.0pg/mL, about 3.0pg/mL to about 4pg/mL, about 0pg/mL to about 3.0pg/mL, about 5.5pg/mL to about 19.0pg/mL, about 6.0pg/mL to about 19.0pg/mL, about 6.5pg/mL to about 19.0pg/mL, about 7.0pg/mL to about 19.0pg/mL, about 7.5pg/mL to about 19.0pg/mL, about 8.0pg/mL to about 19.0pg/mL, about 8.5pg/mL to about 19.0pg/mL, about 9.0pg/mL to about 19.0pg/mL, about 9.5pg/mL to about 19.0pg/mL, about 10.0pg/mL to about 19.0pg/mL, about 0.0100pg/mL to about 18.0pg/mL, about 0.0200pg/mL to about 18.0pg/mL, about 0pg/mL to about 18.0.0 pg/mL, about 0pg/mL to about 18.0pg/mL, about 0pg/mL to about 18.030.0/mL, about 18.0pg/mL to about 18.0pg/mL, about 18.0 to about 18.0pg/mL, about 0pg/mL to about 18.0pg/mL, about 18.0 to about 0pg/mL to about 18., about 0.5pg/mL to about 18.0pg/mL, about 1.0pg/mL to about 18.0pg/mL, about 1.5pg/mL to about 18.0pg/mL, about 2.0pg/mL to about 18.0pg/mL, about 2.5pg/mL to about 18.0pg/mL, about 3.0pg/mL to about 18.0pg/mL, about 3.5pg/mL to about 18.0pg/mL, about 4.0pg/mL to about 18.0pg/mL, about 4.5pg/mL to about 18.0pg/mL, about 5.0pg/mL to about 18.0pg/mL, about 5.5pg/mL to about 18.0pg/mL, about 5pg/mL to about 18.0pg/mL, about 6.0pg/mL to about 18.0pg/mL, about 6.5pg/mL to about 6.0pg/mL, about 6.5pg/mL to about 18.0pg/mL, about 8.0pg/mL, about 8 mL, about 8.0pg/mL to about 18.0pg/mL, about 9.0pg/mL to about 18.0pg/mL, about 9.5pg/mL to about 18.0pg/mL, about 10.0pg/mL to about 18.0pg/mL, about 0.0100pg/mL to about 17.0pg/mL, about 0.0200pg/mL to about 17.0pg/mL, about 0.0300pg/mL to about 17.0pg/mL, about 0.036pg/mL to about 17.0pg/mL, about 0.0400pg/mL to about 17.0pg/mL, about 0.0500pg/mL to about 17.0pg/mL, about 0.1pg/mL to about 17.0pg/mL, about 0.5pg/mL to about 17.0pg/mL, about 1.0pg/mL to about 2.5pg/mL, about 1.0pg/mL to about 17.0pg/mL, about 2pg/mL to about 2.0pg/mL, about 2pg/mL, about 4.0pg/mL to about 17.0pg/mL, about 4.5pg/mL to about 17.0pg/mL, about 5.0pg/mL to about 17.0pg/mL, about 5.5pg/mL to about 17.0pg/mL, about 6.0pg/mL to about 17.0pg/mL, about 6.5pg/mL to about 17.0pg/mL, about 7.0pg/mL to about 17.0pg/mL, about 7.5pg/mL to about 17.0pg/mL, about 8.0pg/mL to about 17.0pg/mL, about pg 8.5pg/mL to about 17.0pg/mL, about 9.0pg/mL to about 17.0pg/mL, about 10.0pg/mL to about 15.0pg/mL, about 0100 mL, about 0 mL to about 16.0 mL, about 0 mL to about 0.0200 mL, about 16.0pg/mL to about 16.0 mL, about 0.0400pg/mL to about 16.0pg/mL, about 0.0500pg/mL to about 16.0pg/mL, about 0.1pg/mL to about 16.0pg/mL, about 0.5pg/mL to about 16.0pg/mL, about 1.0pg/mL to about 16.0pg/mL, about 1.5pg/mL to about 16.0pg/mL, about 2.0pg/mL to about 16.0pg/mL, about 2.5pg/mL to about 16.0pg/mL, about 3.0pg/mL to about 16.0pg/mL, about 3.5pg/mL to about 16.0pg/mL, about 4.0pg/mL to about 16.0pg/mL, about 4.5pg/mL to about 16.0pg/mL, about 5pg/mL to about 16.0pg/mL, about 6.0pg/mL to about 6.0pg/mL, about 7.5pg/mL to about 16.0pg/mL, about 8.0pg/mL to about 16.0pg/mL, about 8.5pg/mL to about 16.0pg/mL, about 9.0pg/mL to about 16.0pg/mL, about 9.5pg/mL to about 16.0pg/mL, about 10.0pg/mL to about 16.0pg/mL, about 0.0100pg/mL to about 15.0pg/mL, about 0.0200pg/mL to about 15.0pg/mL, about 0.0300pg/mL to about 15.0pg/mL, about 0.0500pg/mL to about 15.0pg/mL, about 0.036pg/mL to about 15.0pg/mL, about 0.0400/mL to about 15.0pg/mL, about 0.0500pg/mL to about 15.0pg/mL, about 0.030 pg/mL to about 15.0pg/mL, about 0.1.0/mL to about 15.0/mL, about 15.0pg/mL, about 0/mL to about 15.0.0/mL, about 2.5pg/mL to about 15.0pg/mL, about 3.0pg/mL to about 15.0pg/mL, about 3.5pg/mL to about 15.0pg/mL, about 4.0pg/mL to about 15.0pg/mL, about 4.5pg/mL to about 15.0pg/mL, about 5.0pg/mL to about 15.0pg/mL, about 5.5pg/mL to about 15.0pg/mL, about 6.0pg/mL to about 15.0pg/mL, about 6.5pg/mL to about 15.0pg/mL, about 7.0pg/mL to about 15.0pg/mL, about 7.5pg/mL to about 15.0pg/mL, about 8.0pg/mL to about 15.0pg/mL, about 8.5pg/mL to about 15.0pg/mL, about 10 mL, about 15.0pg/mL, about 15.0 to about 15.0pg/mL, the reference level of cardiac troponin I is about 1.0pg/mL, about 1.5pg/mL, about 2.0pg/mL, about 2.5pg/mL, about 3.0pg/mL, about 3.5pg/mL, about 4.0pg/mL, about 4.5pg/mL, about 5.0pg/mL, about 5.5pg/mL, about 5.6pg/mL, about 5.7pg/mL, about 5.8pg/mL, about 5.9pg/mL, about 6.0pg/mL, about 6.5pg/mL, about 7.0pg/mL, about 7.5pg/mL, about 8.0pg/mL, about 8.5pg/mL, about 9.0pg/mL, about 9.5pg/mL, or about 10.0 pg/mL.

In some embodiments, the reference age is less than 70 years of age, such as between at least about 35 and 70 years of age. In some embodiments, the reference age is an age of at least 35 years, an age of at least 40 years, an age of at least 41 years, an age of at least 42 years, an age of at least 43 years, an age of at least 44 years, an age of at least 45 years, an age of at least 46 years, an age of at least 47 years, an age of at least 48 years, an age of at least 49 years, an age of at least 50 years, an age of at least 55 years, an age of at least 60 years, an age of at least 65 years, or an age of at least 70 years.

In some embodiments, the method further comprises treating a human subject predicted to have an adverse outcome with a traumatic brain injury treatment as described below. In some embodiments, the method further comprises monitoring a human subject predicted to have an adverse outcome as described below. In some embodiments, the method further comprises requiring additional testing to obtain further clinical information regarding mild TBI in human subjects predicted to have adverse outcomes. In some embodiments, the method comprises treating a human subject predicted to have an adverse outcome with a cardioprotective treatment to protect the heart, as described below.

The nature of the assay employed in the methods described herein is not critical, and the assay may be any assay known in the art, for example, immunoassay, protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining, or spectroscopic methods, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assays are described in further detail in sections 11-13 herein. Also, the assay may be performed in a clinical chemistry format, such as would be known to one skilled in the art. For example, a clinical chemistry format may include assays involving one antibody or no antibody. Examples of analyzers that can be used in clinical chemistry formats are described in U.S. patent publication nos. 2016/0320422 and 2015/0112630.

9. Method for treating human subjects with traumatic brain injury using cardioprotective therapy

The present disclosure relates to, among other methods, a method for treating a human subject having or suspected of having a traumatic brain injury. Myocardial injury occurring during the acute phase of Traumatic Brain Injury (TBI) may be a contributing cause of poor TBI outcome. Thus, administration of one or more cardioprotective therapies or therapeutic agents (e.g., cardioimproving therapies) can improve TBI outcome and can be employed in the methods described herein. These cardioprotective therapies can be administered alone without any other therapeutic agent. Alternatively, these cardioprotective therapies can be administered in combination with other therapeutic agents administered to treat TBI, such as those described in section 10 below.

In particular, the disclosed methods involving one or more cardioprotective therapies or therapeutic agents include: a) performing an assay on a sample taken from a human subject within about 24 hours after an actual or suspected head injury to measure or detect a level of cardiac troponin I, wherein the sample is a biological sample; and b) providing a cardioprotective therapy or therapeutic agent to the subject if the level of cardiac troponin I in the sample is higher than the reference level of cardiac troponin I. In some embodiments, cardioprotective therapy optionally may include the administration of one or more beta-blockers, diuretics, Angiotensin Converting Enzyme (ACE) inhibitors, calcium channel blockers, lipid lowering therapies, statins (also known as 3-hydroxy-3-methylglutaryl coenzyme a (hmgcoa) reductase inhibitors), nitrates, antiplatelet agents, anticoagulants, and the like, as are known in the art. The nature, amount, and timing of administration of such cardioprotective therapies and therapeutic agents are well known in the art.

In some embodiments, the sample may be within about 0 minutes, within about 1 minute, within about 2 minutes, within about 3 minutes, within about 4 minutes, within about 5 minutes, within about 6 minutes, within about 7 minutes, within about 8 minutes, within about 9 minutes, within about 10 minutes, within about 11 minutes, within about 12 minutes, within about 13 minutes, within about 14 minutes, within about 15 minutes, within about 20 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours of a suspected head injury, Obtained or obtained from the subject within about 23 hours or within about 24 hours.

In general, the cTnI reference level may also be used as a benchmark to assess the results obtained in determining the cTnI of a test sample. In general, when making such a comparison, a reference level of cTnI is obtained by running a particular assay under appropriate conditions a sufficient number of times to correlate or correlate the presence, amount, or concentration of the analyte with a particular stage or endpoint of TBI or with a particular indication. Typically, the reference level of cTnI is obtained by an assay of a reference subject (or population of subjects). The measured cTnI may include fragments thereof, degradation products thereof, and/or enzymatic cleavage products thereof.

In some embodiments, the method may comprise obtaining a sample from a subject and contacting the sample with an antibody to cardiac troponin I such that a complex of the antibody and cardiac troponin I is formed. The method further comprises detecting the antibody-cardiac troponin I complex produced.

The nature of the assay employed in the methods described herein is not critical, and the assay may be any assay known in the art, for example, immunoassay, protein immunoprecipitation, immunoelectrophoresis, Western blot, or protein immunostaining, or spectroscopic methods, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Such assays are described in further detail in sections 11-13 herein. Also, the assay may be performed in a clinical chemistry format, such as would be known to one skilled in the art. For example, a clinical chemistry format may include assays involving one antibody or no antibody. Examples of analyzers that can be used in clinical chemistry formats are described in U.S. patent publication nos. 2016/0320422 and 2015/0112630.

10. Treatment and monitoring of subjects with traumatic brain injury

Subjects identified or assessed as having traumatic brain injury, e.g., mild traumatic brain injury or moderate, severe, or moderate to severe traumatic brain injury, in the above-described methods can be treated or monitored. In some embodiments, the method further comprises treating the human subject assessed as having traumatic brain injury with a traumatic brain injury treatment, e.g., any treatment known in the art. For example, treatment of traumatic brain injury can take various forms depending on the severity of the head injury. For example, for subjects with mild TBI, treatment may include one or more of: rest, avoid physical activity, e.g., exercise, protect from light or wear sunglasses under light), administering one or more therapeutic agents (e.g., drugs for relieving headache or migraine, anti-nausea drugs, etc.). Treatment of a patient with moderate, severe, or moderate to severe TBI may include administration of one or more appropriate therapeutic agents (e.g., diuretics, anticonvulsants, drugs that calm and induce drug-induced coma, or other pharmaceutical or biopharmaceutical type drugs) known to or developed in the future for the treatment of TBI, one or more surgical procedures to protect the airway (e.g., removal of hematomas, repair of skull fractures, decompression of boned flaps, etc.), and one or more therapies (e.g., one or more rehabilitations, physical therapies, occupational therapies, cognitive behavior therapies, anger management, psychological counseling, etc.). In some embodiments, the method further comprises monitoring a human subject assessed as having traumatic brain injury (e.g., mild or moderate, severe, or moderate to severe trauma). In some embodiments, subjects identified as having traumatic brain injury, e.g., mild traumatic brain injury or severe traumatic brain injury, can be monitored with CT scans or MRI. The treatments described herein for mild or moderate, severe, or moderate to severe TBI can be administered in combination with one or more cardioprotective therapies or agents described in section 9.

11. Method for measuring cTnI level

In the above methods, the cTnI level may be measured by any means, such as: antibody-dependent methods, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE, and Western blot analysis, immunostaining of proteins, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatography or spectroscopy, such as High Performance Liquid Chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). Also, the assay may be performed in clinical chemistry formats such as those that would be known to one skilled in the art.

In some embodiments, measuring the level of cTnI comprises contacting the sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the level of cTnI comprises contacting the sample simultaneously or sequentially in any order with: (1) a capture antibody (e.g., a cTnI capture antibody) that binds to an epitope on cTnI or a fragment of cTnI to form a capture antibody-cTnI antigen complex (e.g., a cTnI capture antibody-cTnI antigen complex), and (2) a detection antibody (e.g., a cTnI detection antibody) that comprises a detectable label and binds to an epitope on cTnI that is not bound by the capture antibody to form a cTnI antigen-detection antibody complex (e.g., a cTnI antigen-cTnI detection antibody complex), thereby forming a capture antibody-cTnI antigen-detection antibody complex (e.g., a cTnI-capture antibody-cTnI antigen-cTnI detection antibody complex), and measuring the amount or concentration of cTnI in the sample based on the signal generated by the detectable label in the capture antibody-cTnI antigen-detection antibody complex.

In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a cTnI antibody as described below.

In some embodiments, the sample is diluted or undiluted. The sample can be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliters, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, or about 25 microliters. In some embodiments, the sample is from about 1 to less than about 150 microliters, or from about 1 to less than about 25 microliters.

Some instruments other than bedside equipment (e.g., Abbott Laboratories instruments)And other core laboratory instruments) may be able to measure cTnI levels in a sample of about 0.032 μ g/L or less with a 10% CV.

Other detection methods include use on or may be adapted for use on a nanopore (nanopore) device or a nanopore (nanopore) device. Examples of nanopore devices are described in international patent publication No. wo 2016/161402, which is incorporated herein by reference in its entirety. Examples of nanopore devices are described in international patent publication No. wo 2016/161400, which is incorporated herein by reference in its entirety.

12. Cardiac troponin I antibodies

The methods described herein can use isolated antibodies, referred to as "cardiac troponin I antibodies," that specifically bind to cardiac troponin I, e.g., human cardiac troponin I (or a fragment thereof). The cardiac troponin I antibodies may be used to assess the condition of cardiac troponin I as a measure of traumatic brain injury, to detect the presence of cardiac troponin I in a biological sample, to quantify the amount of cardiac troponin I present in a biological sample, or to detect the presence of cardiac troponin I in a biological sample and to quantify the amount of cardiac troponin I.

a. Human cardiac troponin I (cTnI)

Human cardiac troponin i (ctni) and troponin t (tnt) and troponin c (tnc) are 3 subunits of the troponin complex forming striated muscle filaments. Cardiac troponin I is an inhibitory subunit; blocking actin-myosin interactions and thereby mediating striated muscle relaxation. The CTnI subfamily contains 3 genes: cTnI-skeletal muscle fast, cTnI-skeletal muscle slow, and cTnI-cardiac muscle. This gene encodes the cTnI-cardiac muscle protein and is expressed only in cardiac muscle tissue.

Human cardiac troponin I may have the following amino acid sequence:

human cardiac troponin I may be a fragment or variant of SEQ ID NO 1. Fragments of cardiac troponin I may be between 5 and 210 amino acids, between 10 and 210 amino acids, between 50 and 210 amino acids, between 60 and 210 amino acids, between 65 and 210 amino acids, between 100 and 210 amino acids, between 150 and 210 amino acids, between 100 and 210 amino acids, or between 175 and 210 amino acids in length. The fragment may comprise a contiguous number of amino acids from SEQ ID NO 1.

b. Cardiac troponin I recognition antibodies

The antibody is an antibody that binds to cardiac troponin I, a fragment thereof, an epitope of cardiac troponin I, or a variant thereof. The antibody may be a fragment of an anti-cardiac troponin I antibody or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, a fully human antibody or antibody fragment such as a Fab fragment, or a mixture thereof. Antibody fragments or derivatives may comprise F (ab')₂Fv or scFv fragment. Antibody derivatives can be generated by peptide mimetics. Furthermore, the techniques described for generating single chain antibodies may be adapted to generate single chain antibodies.

The anti-cardiac troponin I antibody may be a chimeric anti-cardiac troponin I or a humanized anti-cardiac troponin I antibody. In one embodiment, the humanized antibody and the chimeric antibody are monovalent. In one embodiment, both the humanized and chimeric antibodies comprise a single Fab region linked to an Fc region.

Human antibodies can be derived from phage display technology or from transgenic mice expressing human immunoglobulin genes. Human antibodies can be produced and isolated as a result of an immune response in a human. See, e.g., Funaro et al, bmcbio technology, 2008 (8): 85. thus, the antibody may be a product of a human rather than an animal repertoire. Because it is of human origin, the risk of reactivity to self-antigens can be minimized. Alternatively, human anti-cardiac troponin I antibodies may be selected and isolated using standard yeast display libraries and display techniques. For example, a natural human single chain variable fragment (scFv) library can be used to select human anti-cardiac troponin I antibodies. Transgenic animals can be used to express human antibodies.

A humanized antibody may be an antibody molecule from an antibody of a non-human species that binds an antigen of interest, having one or more Complementarity Determining Regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

The antibody differs from known antibodies in that it has a different biological function than antibodies known in the art.

(1) Epitope

The antibody can immunospecifically bind to human cardiac troponin I (SEQ ID NO:1), a fragment thereof, or a variant thereof. The antibody can immunospecifically recognize and bind at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, or at least ten amino acids within an epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least three consecutive amino acids, at least four consecutive amino acids, at least five consecutive amino acids, at least six consecutive amino acids, at least seven consecutive amino acids, at least eight consecutive amino acids, at least nine consecutive amino acids, or at least ten consecutive amino acids of the epitope region.

c. Antibody production/Generation

Antibodies can be prepared by any of a variety of techniques, including those well known to those skilled in the art. In general, antibodies can be produced by the following techniques: cell culture techniques, including the production of monoclonal antibodies by conventional techniques, or by transfection of antibody genes, heavy and/or light chains into a suitable bacterial or mammalian cell host, so as to allow antibody production, wherein the antibody may be recombinant. The term "transfection" in its various forms is intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although antibodies can be expressed in prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred, most preferably in mammalian host cells, because such eukaryotic cells (particularly mammalian cells) are more likely than prokaryotic cells to assemble and secrete a correctly folded and immunologically active antibody. ,

exemplary mammalian host cells for expression of recombinant antibodies include chinese hamster ovary (CHO cells) (including DHFR-CHO cells, described in Urlaub and Chasin, proc.natl.acad.sci.usa, 77: 4216-: 601-621 (1982); NS0 myeloma cells; COS cells; and SP2 cells. When a recombinant expression vector encoding a gene for an antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

Host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be appreciated that variations of the above procedure may be carried out. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of an antibody light and/or heavy chain. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light or heavy chains that is not necessary for binding to the antigen of interest. Antibodies also include molecules expressed from such truncated DNA molecules. Alternatively, bifunctional antibodies can be produced by cross-linking one antibody to a second antibody by standard chemical cross-linking methods, wherein one heavy and one light chain is an antibody (i.e., binds human troponin I) and the other heavy and light chains are specific for an antigen other than human cardiac troponin I.

In a preferred system for recombinant expression of the antibody, or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, antibody heavy and light chain genes are each operably linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows the use of methotrexate selection/amplification to select CHO cells that have been transfected with the vector. The selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover the antibodies from the culture medium. Further, the recombinant antibody may be synthesized by culturing the host cell in a suitable medium until the recombinant antibody is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.

Methods for producing monoclonal antibodies include the production of immortalized cell lines capable of producing antibodies with the desired specificity. Such cell lines can be generated from spleen cells obtained from the immunized animal. The animal may be immunized with cardiac troponin I or fragments and/or variants thereof. The peptide used to immunize an animal may comprise amino acids encoding a human Fc, e.g., a crystallizable fragment region or tail region of a human antibody. The spleen cells may then be immortalized by, for example, fusion with a myeloma cell fusion partner. A variety of fusion techniques may be employed. For example, spleen cells and myeloma cells can be combined with a non-ionic detergent for several minutes and then plated at low density on selective media that supports the growth of hybrid cells, but not myeloma cells. One such technique uses hypoxanthine, aminopterin, thymidine (HAT) selection. Another technique includes electrofusion. Colonies of hybrids are observed after a sufficient time, often about 1 to 2 weeks. Individual colonies were selected and their culture supernatants tested for binding activity to the polypeptide. Hybridomas having high reactivity and specificity can be used.

Monoclonal antibodies can be isolated from the supernatant of the growing hybridoma colonies. In addition, various techniques can be employed to increase yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibodies can then be harvested from ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used to purify antibodies.

The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (f (ab) fragments) each comprise a covalent heterodimer containing an intact antigen binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including F (ab')₂A fragment comprising two antigen binding sites.

Fv fragments can be produced by preferential proteolytic cleavage of IgM, and in individual cases IgG or IgA immunoglobulin molecules. Fv fragments can be generated using recombinant techniques. The Fv fragments include non-covalent VH' s.VL heterodimers, which contain antigen binding sites that retain many of the antigen recognition and binding capabilities of the native antibody molecule.

An antibody, antibody fragment or derivative can comprise sets of heavy and light chain complementarity determining regions ("CDRs") interposed between sets of heavy and light chain Frameworks (FRs), respectively, that provide support for the CDRs and define the spatial relationship of the CDRs to each other. The set of CDRs may contain three hypervariable regions of the heavy or light chain V regions.

Other suitable methods of generating or isolating antibodies with the necessary specificity can be used, including, but not limited to, methods of selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, phage, ribosome, oligonucleotide, RNA, cDNA, yeast, etc. display libraries) using methods known in the art; for example, they are available from various commercial suppliers, such as Cambridge Antibody Technologies (Cambridge, UK), Morphosys (Martinreid/Planegg, Del.), Biovariation (Aberdeen, Scotland, UK), BioInvent (Lund, Sweden). See U.S. Pat. Nos. 4,704,692; 5,723,323; 5,763,192, respectively; 5,814,476, respectively; 5,817,483, respectively; 5,824,514, respectively; 5,976,862. Alternative methods rely on immunization of transgenic animals capable of generating human antibody libraries (e.g., SCID mice, Nguyen et al, (1997) Microbiol. Immunol.41: 901-907; Sandhu et al, (1996) Crit. Rev. Biotechnol.16: 95-118; Eren et al, (1998) Immunol.93: 154-161), as known in the art and/or described herein. Such techniques include, but are not limited to, ribosome display (Hanes et al, (1997) Proc. Natl. Acad. Sci. USA, 94: 4937-; techniques for the production of single cell antibodies (e.g., the selected lymphocyte antibody method ("SLAM")) (U.S. Pat. No.5,627,052, Wen et al (1987) J.Immunol.17: 887-7848; Babcook et al (1996) Proc.Natl.Acad.Sci.USA 93: 7843-7848); gel microdroplets and flow cytometry (Powell et al (1990) Biotechnol.8: 333-; b cell selection (Steenbakkers et al, (1994) Molec. biol. reports 19: 125-134 (1994)).

Affinity matured antibodies can be produced by any of a number of procedures known in the art. See, for example, Marks et al, BioTechnology, 10: 779-783(1992) describes affinity maturation by VH and VL domain shuffling. Barbas et al, proc.nat.acad.sci.usa, 91: 3809-3813 (1994); schier et al, Gene, 169: 147-; yelton et al, j.immunol., 155: 1994-2004 (1995); jackson et al, j.immunol., 154 (7): 3310-3319 (1995); hawkins et al, j.mol.biol., 226: 889-896(1992) describes random mutagenesis of CDR and/or framework residues. Selective mutagenesis with activity-enhancing amino acid residues at selectively mutagenized positions as well as at contact or hypervariable positions is described in U.S. Pat. No.6,914,128B1.

Antibody variants can also be prepared by delivering a polynucleotide encoding an antibody to a suitable host such that a transgenic animal or mammal, e.g., goat, cow, horse, sheep, etc., is provided which produces such an antibody in its milk. Such methods are known in the art and are described, for example, in U.S. patent nos.5,827,690; 5,849,992, respectively; 4,873,316; 5,849,992, respectively; 5,994,616, respectively; 5,565,362, respectively; and 5,304,489.

Antibody variants can also be prepared by delivering polynucleotides to provide transgenic plants and cultured plant cells (such as, but not limited to, tobacco, maize, and duckweed) that produce such antibodies, specified portions, or variants in plant parts or from cultured cells thereof. For example, Cramer et al, (1999) curr. top. microbiol. immunol.240: 95-118 and references cited therein describe the production of transgenic tobacco leaves expressing large amounts of recombinant proteins, e.g., using inducible promoters. Transgenic maize has been used to express mammalian proteins at commercial production levels and has biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al, adv.exp.med.biol. (1999) 464: 127-147 and the references cited therein. Antibody variants have also been produced in large quantities from transgenic plant seeds, including tobacco seeds and potato tubers, containing antibody fragments, such as single chain antibodies (scFv'). See, e.g., Conrad et al (1998) Plant mol. biol.38: 101-109 and references cited therein. Thus, transgenic plants may also be used to produce antibodies according to known methods.

Antibody derivatives may be produced, for example, by modifying immunogenicity or by reducing, enhancing or altering binding, affinity, on-rate, off-rate, avidity, specificity, half-life or any other suitable property by adding exogenous sequences. Typically, part or all of the non-human or human CDR sequences are retained while the non-human sequences of the variable and constant regions are substituted with human or other amino acids.

A small antibody fragment can be a diabody with two antigen-binding sites, wherein the fragment comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH VL). See, for example, EP404,097; WO 93/11161; and Hollinger et al, (1993) proc.natl.acad.sci.usa 90: 6444-6448. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. See also, U.S. patent No.6,632,926 to Chen et al, which is incorporated herein by reference in its entirety, discloses antibody variants having one or more amino acids inserted into a hypervariable region of a parent antibody and having at least about two-fold greater binding affinity for a target antigen than the parent antibody.

The antibody may be a linear antibody. Procedures for the manufacture of linear antibodies are known in the art and are described in Zapata et al, (1995) Protein eng.8 (10): 1057-. Briefly, these antibodies comprise a pair of Fd segments (VH-CH1-VH-CH1) in tandem, which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

The antibodies can be recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein a purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

It may be useful to detectably label the antibody. Methods of conjugating antibodies to these agents are known in the art. For illustrative purposes only, the antibody may be labeled with a detectable moiety, such as a radioactive atom, chromophore, fluorophore, or the like. Such labeled antibodies are useful in diagnostic techniques in vivo or in isolated test samples. They may be linked to a cytokine, to a ligand, to another antibody. Suitable agents conjugated to antibodies to achieve an anti-tumor effect include: cytokines such as interleukin 2(IL-2) and Tumor Necrosis Factor (TNF); photosensitizers for photodynamic therapy including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrins and phthalocyanines; radionuclides such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99 m (99mTc), rhenium-186 (186Re), and rhenium-188 (188 Re); antibiotics such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostain, and carboplatin; bacterial, plant and other toxins, such as diphtheria toxin, pseudomonas exotoxin a, staphylococcal enterotoxin a, abrin a toxin, ricin a (deglycosylated ricin a and native ricin a), TGF-alpha toxin, cytotoxin of chinese cobra (naja naja atra), and poliumvariegate (phytotoxin); ribosome-inactivating proteins from plants, bacteria and fungi, such as restrictocin (ribosome-inactivating protein produced by Aspergillus restrictus), saporin (ribosome-inactivating protein from Saponaria officinalis) and RNase, tyrosine kinase inhibitors, ly207702 (difluoropurine nucleosides), liposomes containing anticystic agents (e.g., antisense oligonucleotides, toxin-encoding plasmids, methotrexate, etc.), and other antibodies or antibody fragments, such as F (ab).

Antibody production by using hybridoma technology, the Selected Lymphocyte Antibody Method (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.

(1) Anti-cardiac troponin I monoclonal antibodies using hybridoma technology

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and taught, for example, in the following references: harlow et al, antibodies: a Laboratory Manual, second edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); hammerling et al, "in monoclonal Antibodies and T-Cell Hybridomas" (Inmonoclonal Antibodies and T-Cell hybrids), (Elsevier, N.Y., 1981). It should also be noted that the term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a unicellular clone, including any eukaryotic or prokaryotic cell or phage clone, rather than the method by which it is produced.

Methods of producing monoclonal antibodies and antibodies produced by the methods can include culturing hybridoma cells that secrete antibodies of the disclosure, wherein, preferably, the hybridomas are produced by: spleen cells isolated from an animal immunized with cardiac troponin I, such as a rat or mouse, are fused with myeloma cells, and hybridoma clones secreting an antibody capable of binding to a polypeptide of the present disclosure are selected from the hybridomas produced by the fusion. Briefly, rats can be immunized with the cardiac troponin I antigen. In a preferred embodiment, the cardiac troponin I antigen is administered together with an adjuvant to stimulate an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immune stimulating complex). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering the polypeptide in a localized deposit, or they may contain agents that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if the polypeptide is being administered, the immunization program will include two or more administrations of the polypeptide over up to several weeks; however, a single administration of the polypeptide may also be used.

After immunization of an animal with the cardiac troponin I antigen, antibodies and/or antibody-producing cells can be obtained from the animal. Serum containing anti-cardiac troponin I antibodies was obtained from the animals by exsanguinating or sacrificing the animals. The serum may be used as it is obtained from animals, an immunoglobulin fraction may be obtained from the serum, or an anti-cardiac troponin I antibody may be purified from the serum. The serum or immunoglobulins obtained in this way are polyclonal and therefore have a heterogeneous array of properties.

Once an immune response is detected, e.g., an antibody specific for cardiac troponin I is detected in rat serum, rat spleens are harvested and splenocytes isolated. The spleen cells are then fused by well-known techniques with any suitable myeloma cells, such as cells of the SP20 cell line available from the American Type Culture Collection (ATCC, Manassas, Va., US). Hybridomas were selected and cloned by limiting dilution. The hybridoma clones are then assayed for cells secreting antibodies capable of binding troponin I by methods known in the art. Ascites fluid, which usually contains high levels of antibodies, can be produced by immunizing rats with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas can be prepared from immunized animals. Following immunization, the animals are sacrificed and spleen B cells are fused with immortalized myeloma cells as is well known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secretory cell lines). Following fusion and antibiotic selection, the hybridomas are screened using troponin I or a portion thereof or cells expressing troponin I. In a preferred embodiment, the initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA), preferably an ELISA. An example of an ELISA screen is provided in PCT publication No. wo 00/37504.

Hybridomas producing anti-cardiac troponin I antibodies are selected, cloned, and further screened for desirable properties, including robust hybridoma growth, high antibody production, and desirable antibody properties. Hybridomas can be cultured and expanded in vivo in syngeneic animals, in animals lacking the immune system, such as nude mice, or in vitro in cell culture. Methods for selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art.

In a preferred embodiment, the hybridoma is a rat hybridoma. In another embodiment, the hybridoma is produced in a non-human, non-rat species, such as mouse, sheep, pig, goat, cow, or horse. In yet another preferred embodiment, the hybridoma is a human hybridoma in which a human non-secretory myeloma is fused to a human cell expressing an anti-cardiac troponin I antibody.

Antibody fragments that recognize a particular epitope can be generated by known techniques. For example, Fab and F (ab')₂Fragments can be generated by using e.g. papain (producing two identical Fab fragments) or pepsin (producing F (ab')₂Fragments) are produced by proteolytic cleavage of the immunoglobulin molecule. IgG fractionSubsidiary F (ab')₂The fragment retains two antigen binding sites of a larger ("parent") IgG molecule, including the two light chains (containing the variable light chain region and the constant light chain region), the CH1 domain of the heavy chain, and the hinge region that forms disulfide bonds of the parent IgG molecule. Thus, F (ab')₂The fragments are still able to cross-link the antigenic molecule like the parent IgG molecule.

(2) Anti-cardiac troponin I monoclonal antibody using SLAM

In another aspect of the present disclosure, as disclosed in U.S. Pat. Nos.5,627,052; PCT publication nos. wo 92/02551; and Babcook et al, proc.natl.acad.sci.usa, 93: 7843-7848(1996), recombinant antibodies were generated from individual isolated lymphocytes using a procedure known in the art as the Selected Lymphocyte Antibody Method (SLAM). In this method, antigen-specific hemolytic plaque assays are used to screen single cells secreting antibodies of interest, e.g. lymphocytes derived from any one of the immunized animals, wherein the antigen cardiac troponin I, a subunit of cardiac troponin I or a fragment thereof is coupled to sheep erythrocytes using a linker, e.g. biotin, and used to identify single cells secreting antibodies specific for cardiac troponin I. After the antibody-secreting cells of interest are identified, the heavy and light chain variable region cdnas are rescued from the cells by reverse transcriptase-PCR (RT-PCR), and these variable regions can then be expressed in mammalian host cells, such as COS or CHO cells, in the context of appropriate immunoglobulin constant regions (e.g., human constant regions). Host cells transfected with amplified immunoglobulin sequences derived from lymphocytes selected in vivo can then undergo further in vitro analysis and selection, e.g., panning of transfected cells to isolate cells expressing antibodies to cardiac troponin I. The amplified immunoglobulin sequences may be further manipulated in vitro, for example by in vitro affinity maturation methods. See, for example, PCT publication No. wo 97/29131 and PCT publication No. wo 00/56772.

(3) Anti-cardiac troponin I monoclonal antibody using transgenic animal

In another embodiment of the disclosure, some or all of the human immunizations are included by priming with a cardiac troponin I antigenNon-human animals immunized at the globin locus are used to generate antibodies. In one embodiment, the non-human animal is

Transgenic mice, an engineered mouse strain, comprising a large fragment of a human immunoglobulin locus and lacking mouse antibody production. See, e.g., Green et al, Nature Genetics, 7: 13-21(1994) and U.S. Pat. Nos.5,916,771; 5,939,598; 5,985,615, respectively; 5,998,209, respectively; 6,075,181; 6,091,001, respectively; 6,114,598, respectively; and6,130,364. SeealsoPCTPublications Nos. WO91/10741; WO 94/02602; WO 96/34096; WO 96/33735; WO 98/16654; WO 98/24893; WO 98/50433; WO 99/45031; WO 99/53049; WO 00/09560; and WO 00/37504.The transgenic mice produce an adult-like human repertoire of fully human antibodies and produce antigen-specific human monoclonal antibodies. By introducing YAC fragments of megabase size and germ line configuration of human heavy chain locus and x light chain locus,

the transgenic mice contained about 80% of the human antibody repertoire. See Mendez et al, Nature Genetics, 15: 146-: 483-495(1998), the disclosure of which is incorporated herein by reference.

(4) Anti-cardiac troponin I monoclonal antibodies using recombinant antibody libraries

In vitro methods can also be used to make antibodies of the disclosure, wherein antibody libraries are screened to identify antibodies with the desired cardiac troponin I binding specificity. Such methods of screening recombinant antibody libraries are well known in the art and include those described, for example, in the following references: U.S. Pat. No.5,223,409(Ladner et al); PCT publication No. WO 92/18619(Kang et al); PCT publication No. WO 91/17271(Dower et al); PCT publication No. WO 92/20791(Winter et al); PCT publication No. WO 92/15679(Markland et al); PCT publication No. WO 93/01288(Breitling et al); PCT publication No. WO 92/01047(McCafferty et al); PCT publication No. wo 92/09690 (garrrad et al); fuchs et al, Bio/Technology, 9: 1369-; hay et al, hum. 81-85 (1992); hue et al, Science, 246: 1275-1281 (1989); McCafferty et al, Nature, 348: 552 (1990); griffiths et al, EMBO j., 12: 725 + 734 (1993); hawkins et al, j.mol.biol., 226: 889-896 (1992); clackson et al, Nature, 352: 624-628 (1991); gram et al, proc.natl.acad.sci.usa, 89: 3576-3580 (1992); garrrard et al, Bio/Technology, 9: 1373-1377 (1991); hoogenboom et al, nucleic acids res, 19: 4133 4137 (1991); barbas et al, proc.natl.acad.sci.usa, 88: 7978-7982 (1991); U.S. patent application publication nos. 2003/0186374; and PCT publication No. wo 97/29131, the contents of each of which are incorporated herein by reference.

The recombinant antibody library may be from a subject immunized with cardiac troponin I or a portion of cardiac troponin I. Alternatively, the recombinant antibody library may be from a non-immunized subject, i.e. a subject not immunized with cardiac troponin I, e.g. a human antibody library from a human subject not immunized with human cardiac troponin I. Antibodies of the present disclosure are selected by screening a recombinant antibody library with a peptide comprising human cardiac troponin I, thereby selecting those antibodies that recognize troponin I. Methods for performing such screening and selection are well known in the art, for example as described in the references in the preceding paragraph. To select antibodies with a specific binding affinity for cardiac troponin I, e.g.I with a specific K_offAntibodies with rate constants that dissociate from human cardiac troponin can be selected to have a desired K using surface plasmon resonance methods known in the art_offAntibody with rate constant. To select antibodies of the disclosure having specific neutralizing activity against cardiac troponin I, e.g., having a specific IC₅₀Can be used using standard methods known in the art for assessing inhibition of cardiac troponin I activity.

In one aspect, the invention relates to an isolated antibody, or antigen-binding portion thereof, that binds human cardiac troponin I. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

For example, antibodies can also be produced using various phage display methods known in the art. In the phage display method, functional antibody domains are displayed on the surface of phage particles with polynucleotide sequences encoding them. Such phage can be used to display antigen binding domains expressed from libraries or combinatorial antibody libraries (e.g., human or murine). The phage expressing the antigen binding domain that binds to the antigen of interest can be selected or identified with the antigen, e.g., using a labeled antigen or an antigen bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage comprising fd and M13 binding domains expressed from the phage and Fab, Fv, or disulfide-bond stabilized Fv antibody domains recombinantly fused to phage gene III or gene VIII proteins. Examples of phage display methods that can be used to prepare antibodies include those disclosed in the following references: brinkmann et al, j.immunol.methods, 182: 41-50 (1995); ames et al, j.immunol.methods, 184: 177-186 (1995); ketleborough et al, eur.j.immunol., 24: 952 and 958 (1994); persic et al, Gene, 187: 9-18 (1997); burton et al, Advances in Immunology, 57: 191-280 (1994); PCT publication nos. wo 92/01047; PCT published nos. wo 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753, respectively; 5,821,047, respectively; 5,571,698; 5,427,908; 5,516,637; 5,780,225, respectively; 5,658,727, respectively; 5,733,743, respectively; and 5,969,108.

As described in the above references, after phage selection, the antibody coding regions can be isolated from the phage and used to generate whole antibodies, including human antibodies or any other antigen binding fragment of interest, and expressed in any target host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described in detail below. For example, the use of the present invention may be adoptedRecombinant production of Fab, Fab 'and F (ab')₂Techniques for fragmentation, such as those disclosed in the following documents: PCT publication nos. wo 92/22324; mullinax et al, BioTechniques, 12 (6): 864-869 (1992); sawai et al, am.j.reprod.immunol., 34: 26-34 (1995); and Better et al, Science, 240: 1041-1043(1988). Examples of techniques that can be used to produce single chain Fv's and antibodies include those described in the following references: U.S. Pat. Nos. 4,946,778 and 5,258,498; huston et al, Methods in Enzymology, 203: 46-88 (1991); shu et al, proc.natl.acad.sci.usa, 90: 7995-7999 (1993); and Skerra et al, Science, 240: 1038-1041(1988).

Alternative approaches to screening recombinant antibody libraries by phage display, other methodologies known in the art for screening large combinatorial libraries, can be applied to identify antibodies of the present disclosure. One type of alternative expression system is one in which the recombinant antibody library is expressed as RNA-protein fusions, such as PCT publication No. wo 98/31700(Szostak and Roberts), and Roberts and Szostak, proc.natl.acad.sci.usa, 94: 12297, 12302 (1997). In this system, a covalent fusion is made between the mRNA and the peptide or protein it encodes by in vitro translation of a synthetic mRNA carrying the peptidyl receptor antibiotic, puromycin, at the 3' end. Thus, specific mrnas can be enriched from complex mRNA mixtures (e.g., combinatorial libraries) based on the properties of the encoded peptide or protein, e.g., antibody or portion thereof, e.g., binding of the antibody or portion thereof to a dual specific antigen. The nucleic acid sequences encoding the antibodies or portions thereof recovered from screening such libraries may be expressed by recombinant means as described above (e.g., in mammalian host cells), and further affinity maturation may be carried out by screening for mRNA-peptide fusions in which mutations have been introduced into the originally selected sequence, or by other methods for in vitro affinity maturation of recombinant antibodies as described above. . One preferred example of this methodology is the PROfusion exhibition technique.

In another approach, antibodies can also be produced using yeast display methods known in the art. In the yeast display method, using genetic methods to bind antibody domains to yeast cell walls, and display it on the yeast surface. In particular, such yeast can be used to display from the library or combinatorial antibody library (e.g., human or mouse) expression of antigen binding domain. Examples of yeast display methods that can be used to prepare antibodies are included in U.S. Pat. No.6,699,658(Wittrup et al), which is incorporated herein by reference.

d. Production of recombinant cardiac troponin I antibodies

Antibodies can be produced by any of a number of techniques known in the art. For example, expression from a host cell into which expression vectors encoding the heavy and light chains are transfected by standard techniques. The term "transfection" in its various forms is intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the antibodies of the present disclosure may be expressed in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells is preferred, most preferably in mammalian host cells, because such eukaryotic cells (particularly mammalian cells) are more likely than prokaryotic cells to assemble and secrete a correctly folded and immunologically active antibody.

Exemplary mammalian host cells for expressing recombinant antibodies of the present disclosure include chinese hamster ovary (CHO cells) (including DHFR-CHO cells, described in Urlaub and Chasin, proc.natl.acad.sci.usa, 77: 4216-: 601-621 (1982); NS0 myeloma cells; COS cells; and SP2 cells. When a recombinant expression vector encoding a gene for an antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

Host cells may also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be appreciated that variations of the above procedure may be carried out. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of the light and/or heavy chain of an antibody of the disclosure. Recombinant DNA techniques can also be used to remove some or all of the DNA encoding one or both of the light or heavy chains that is not necessary for binding to the antigen of interest. The antibodies of the present disclosure also encompass molecules expressed from such truncated DNA molecules. In addition, bifunctional antibodies can be produced by crosslinking an antibody of the disclosure with a second antibody by standard chemical crosslinking methods, wherein one heavy and one light chain is an antibody of the disclosure (i.e., binds to human troponin I) and the other heavy and light chains are specific for an antigen other than human cardiac troponin I.

In a preferred system for recombinant expression of the antibodies or antigen-binding portions thereof of the present disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, antibody heavy and light chain genes are each operably linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows the use of methotrexate selection/amplification for the selection of CHO cells that have been transfected with the vector. The selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover the antibodies from the culture medium. Still further, the present disclosure provides methods of synthesizing recombinant antibodies by culturing a host cell of the present disclosure in a suitable culture medium until a recombinant antibody of the present disclosure is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.

(1) Humanized antibodies

The humanized antibody may be an antibody or a variant, derivative, analog or portion thereof that immunospecifically binds to an antigen of interest and comprises Framework (FR) regions having substantially the amino acid sequence of a human antibody and Complementarity Determining Regions (CDRs) having substantially the amino acid sequence of a non-human antibody. A humanized antibody may be an antibody from a non-human species that binds an antigen of interest, having one or more Complementarity Determining Regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

As used herein, the term "substantially" in the context of a CDR means that the amino acid sequence of the CDR is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a CDR of a human antibody. Humanized antibodies comprise at least one, and usually two, variable domains (Fab, Fab ', F (ab')₂FabC, Fv), all or substantially all of the CDR regions in the variable domain correspond to CDR regions of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are framework regions of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. In some embodiments, the humanized antibody contains both the light chain and at least the variable domain of the heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only humanized heavy chains. In a specific embodiment, the humanized antibody contains only humanized variable domains of the light and/or heavy chain.

Humanized antibodies may be selected from any class of immunoglobulin including IgM, IgG, IgD, IgA, and IgE, as well as any isotype including, but not limited to, IgG1, IgG2, IgG3, and IgG 4. Humanized antibodies may comprise sequences from more than one class or isotype, and specific constant domains may be selected to optimize the desired effector function using techniques well known in the art.

The framework regions and CDR regions of the humanized antibody need not correspond exactly to the parent sequence, e.g., the donor antibody CDR or consensus framework can be mutagenized by substituting, inserting, and/or deleting at least one amino acid residue such that the CDR or framework residue at that position does not correspond to the donor antibody or consensus framework. In one embodiment, such mutations, however, are not extensive. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "consensus framework" refers to framework regions in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In the immunoglobulin family, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in the family. If the two amino acids occur equally frequently, either one of the two can be included in the consensus sequence.

Humanized antibodies can be designed to minimize unwanted immune responses to rodent anti-human antibodies, which limits the duration and effectiveness of therapeutic applications of these moieties in human recipients. A humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human residues are often referred to as "import" (import) residues, which are typically taken from the variable domain. Humanization may be performed by substituting a hypervariable region sequence for the corresponding sequence of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies in which significantly less than the entire human variable domain has been replaced by the corresponding sequence from a non-human species. See, for example, U.S. Pat. No.4,816,567, the contents of which are incorporated herein by reference. The humanized antibody may be a human antibody in which some hypervariable region residues, and possibly some FR residues, are substituted by residues from analogous sites in rodent antibodies. Humanization or engineering of antibodies of the present disclosure can be performed using any known method, such as, but not limited to, the methods described in the following references: U.S. Pat. Nos.5,723,323; 5,976,862, respectively; 5,824,514, respectively; 5,817,483, respectively; 5,814,476, respectively; 5,763,192, respectively; 5,723,323; 5,766,886; 5,714,352, respectively; 6,204,023, respectively; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

The humanized antibodies can retain high affinity for cardiac troponin I as well as other advantageous biological properties. Humanized antibodies can be made by methods that use three-dimensional models of the parent and humanized sequences to analyze the parent sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are commonly available. Computer programs can be used to illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from the recipient and import sequences to achieve desired antibody properties, such as increased affinity for cardiac troponin I. In general, hypervariable region residues are likely to be directly and most significantly involved in affecting antigen binding.

As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") may be produced. For example, human antibodies can be isolated from libraries by PROfusion and/or yeast related techniques. Transgenic animals (e.g., mice) can also be generated that are capable of producing a complete human antibody repertoire without the production of endogenous immunoglobulins following immunization. For example, the junction region of the antibody heavy chain in chimeric and germline mutant mice (J) _H) Homozygous deletion of the gene results in complete suppression of endogenous antibody production. Delivery of human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies upon antigen challenge. Humanized or fully human antibodies can be prepared according to the methods described in the following references: U.S. patent nos. 5,770,429; 5,833,985, respectively; 5,837,243, respectively; 5,922,845, respectively; 6,017,517, respectively; 6,096,311, respectively; 6,111,166, respectively; 6,270,765, respectively; 6,303,755, respectively; 6,365,116, respectively; 6,410,690, respectively; 6,682,928, respectively; and 6,984,720, the contents of each of which are incorporated herein by reference.

e. Anti-cardiac troponin I antibodies

Anti-cardiac troponin I antibodies may be produced using the techniques described above as well as using conventional techniques known in the art. In some embodiments, the anti-cardiac troponin I antibody may be unconjugated myocardiumTroponin I antibodies, for example, cardiac troponin I antibodies, obtainable from: abcam (e.g., anti-cardiac troponin I antibody (ab47003)), Thermofish (e.g., cardiac troponin I monoclonal antibody (12F10), cardiac troponin I polyclonal antibody, cardiac troponin I antibody (1HCLC), ABFINITY^TMRabbit oligoclonal cardiac troponin I antibody (1H11L19), ABFINITY^TMRabbit monoclonal), Santa Cruz (e.g. cardiac troponin I antibody (C-4) (catalog No. sc-133117), cardiac troponin I antibody (4) (catalog No. sc-130351), cardiac troponin I antibody (12) (catalog No. sc-130350), cardiac troponin I antibody (H-170) (catalog No. sc-15368), cardiac troponin I antibody (C-19) (catalog No. sc-8118), cardiac troponin I-C antibody (G-11) (catalog No. sc-376662), cardiac troponin I-C antibody (M46) (catalog No. sc-52277), cardiac troponin I-C antibody (10B11) (catalog No. sc-52266), and hytest (monoclonal mouse anti-cardiac troponin I (catalog No. 4T 21).

13. Variants of the method

The disclosed methods of determining the presence or amount of an analyte of interest (e.g., cTnI) present in a sample can be as described herein. The method may also be adapted with reference to other methods for analyte analysis. Examples of well known variants include, but are not limited to: immunoassays, such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including enzyme assays (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays) (ELISA), competitive inhibition immunoassays (e.g., forward and reverse), enzyme-multiplied immunoassay techniques (EMIT), competitive binding assays, Bioluminescence Resonance Energy Transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, instant capture assays (capture on the same assay), and the like.

a. Immunoassay method

The analyte of interest, and/or a peptide of a fragment thereof (e.g., cTnI and/or a peptide or fragment thereof, i.e., a cTnI fragment), can be analyzed in an immunoassay using a cTnI antibody. The presence or amount of an analyte (e.g., cTnI) can be determined using an antibody and detecting specific binding to the analyte (e.g., cTnI). For example, an antibody or antibody fragment thereof can specifically bind to an analyte (e.g., cTnI). If desired, one or more of the antibodies can be used in combination with one or more commercially available monoclonal/polyclonal antibodies. Such antibodies can be obtained, for example, from the following companies: r & D Systems, Inc. (Minneapolis, MN) and Enzo Life Sciences International, Inc. (Plymouth Meeting, Pa.).

Using immunoassays such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassays (RIA)) and enzyme detection (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA) (e.g., Quantikine ELISA assay, R)&D Systems, Minneapolis, MN)) can readily determine the presence or amount of an analyte (e.g., cTnI) present in a body sample. An example of a bedside device that may be used is

(Abbott, Laboratories, Abbott Park, IL). Other methods which may be used include chemiluminescent particle immunoassays, particularly those employingThis immunoassay is performed by an automated analyzer (Abbott laboratories, Abbott Park, IL). Other methods include, for example, mass spectrometry, and immunohistochemistry (e.g., with sections from tissue biopsies) using anti-analyte (e.g., anti-cTnI) antibodies (monoclonal, polyclonal, chimeric, humanized, human antibodies, etc.) or antibody fragments thereof against the analyte (e.g., cTnI). Other detection methods include those described in, for example, the following documents: U.S. patent nos. 6,143,576; 6,113,855; 6,019,944, respectively; 5,985,579, respectively; 5,947,124, respectively; 5,939,272, respectively; 5,922,615, respectively; 5,885,527, respectively; 5,851,776, respectively; 5,824,799, respectively; 5,679,526, respectively; 5,525,524, respectively; and 5,480,792, each of which is incorporated herein by reference in its entirety. Specific immunological binding of an antibody to an analyte (e.g., cTnI) can be achieved by direct labeling of the antibody with a fluorescent or luminescent tag, a metal or antibody radionuclide, or by an indirect label such as alkaline phosphatase or horseradish peroxidaseAnd (6) detecting.

The use of immobilized antibodies or antibody fragments thereof may be incorporated in immunoassays. The antibodies can be immobilized on various supports such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter well), pieces of solid matrix material, and the like. The assay strip may be prepared by coating the antibody or antibodies in an array on a solid support. Such strips can then be immersed in a test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a stain.

Homogeneous forms may be used. For example, after obtaining a test sample from a subject, a mixture is prepared. The mixture contains a test sample of the analyte to be assessed (e.g., cTnI) and a specific binding partner. The order of addition of the test sample and specific binding partner to form the mixture is not critical. The test sample is immediately contacted with the specific binding partner. In some embodiments, the specific binding partner and any cTnI contained in the test sample may form a specific binding partner-analyte (e.g., cTnI) -antigen complex. The specific binding partner can be an anti-analyte antibody (e.g., an anti-cTnI antibody that binds to an epitope having an amino acid sequence comprising at least three (3) contiguous amino acids of SEQ ID NO: 1). In addition, the specific binding partner may be labeled with or contain a detectable label as described above.

A multiphase form may be used. For example, after obtaining a test sample from a subject, a first mixture is prepared. The mixture comprises a test sample of an analyte to be assessed (e.g., cTnI) and a first specific binding partner, wherein the first specific binding partner and any cTnI contained in the test sample form a first specific binding partner-analyte (e.g., cTnI) -antigen complex. The first specific binding partner can be an anti-analyte antibody (e.g., an anti-cTnI antibody that binds to an epitope having an amino acid sequence comprising at least three (3) consecutive amino acids of seq id NO: 1). The order in which the test sample and the first specific binding partner are added to form the mixture is not critical.

The first specific binding partner may be immobilized on a solid phase. ImmunoassayThe solid phase (for the specific binding partner) used in the assay may be any solid phase known in the art, such as, but not limited to, magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, membranes, filter papers, discs, and chips. In those embodiments where the solid phase is a bead, the bead may be a magnetic bead or a magnetic particle. The magnetic beads/particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or magnetofluidic. Typical ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO₂MnAs, MnBi, EuO, and NiO/Fe. Examples of ferromagnetic materials include NiFe₂O₄、CoFe₂O₄、Fe₃O₄(or FeO. Fe₂O₃). The beads may have a solid core portion that is magnetic and surrounded by one or more non-magnetic layers. Alternatively, the magnetic portion may be a layer surrounding the non-magnetic core. The solid support to which the first specific binding member is immobilised may be stored in dry form or in liquid form. The magnetic beads may be subjected to a magnetic field either before or after the sample is contacted with the magnetic beads having the first specific binding member immobilized thereon.

After the mixture containing the first specific binding partner-analyte (e.g., cTnI) antigen complex is formed, any unbound analyte (e.g., cTnI) is removed from the complex using any technique known in the art. For example, unbound analyte (e.g., cTnI) can be removed by washing. However, it is desirable that the first specific binding partner be present in excess of any analyte (e.g., cTnI) present in the test sample, such that all analyte (e.g., cTnI) present in the test sample is bound by the first specific binding partner.

After removal of any unbound analyte (e.g., cTnI), a second specific binding partner is added to the mixture to form a first specific binding partner-analyte of interest (e.g., cTnI) -second specific binding partner complex. The second specific binding partner may be an anti-analyte antibody (e.g., an anti-cTnI antibody that binds to an epitope having an amino acid sequence comprising at least three (3) consecutive amino acids of SEQ ID NO: 1). In addition, the second specific binding partner may be labeled with or contain a detectable label as described above.

The use of immobilized antibodies or antibody fragments thereof may be incorporated in immunoassays. The antibodies can be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles (e.g., magnetic beads), latex particles or surface-modified latex particles, polymer or polymer membranes, plastic or plastic films, planar substrates, surfaces of assay plates (e.g., microtiter wells), pieces of solid substrate material, and the like. The assay strip may be prepared by coating the antibody or antibodies in an array on a solid support. Such strips can then be immersed in a test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a stain.

(1) Sandwich immunoassay

Sandwich immunoassays measure the amount of antigen between two layers of antibody (i.e., at least one capture antibody) and detection antibody (i.e., at least one detection antibody). The capture antibody and the detection antibody bind to different epitopes on an antigen, e.g., an analyte of interest (e.g., cTnI). Ideally, binding of the capture antibody to the epitope does not interfere with binding of the detection antibody to the epitope. Monoclonal or polyclonal antibodies can be used as capture and detection antibodies in sandwich immunoassays.

Typically, at least two antibodies are employed to isolate and quantify the analyte (e.g., cTnI) in the test sample. More specifically, the at least two antibodies bind to certain epitopes of the analyte (e.g., cTnI) forming an immune complex known as a "sandwich (sandwich)". One or more antibodies can be used to capture an analyte (e.g., cTnI) in a test sample (these antibodies are often referred to as "capture" antibodies), and one or more antibodies are used to bind a detectable (i.e., quantifiable) label on a sandwich (these antibodies are often referred to as "detection" antibodies). In a sandwich assay, it is desirable that the binding of an antibody to its epitope is not impaired by the binding of any other antibody in the assay to its respective epitope. The antibodies are selected such that the one or more first antibodies contacted with a test sample suspected of containing an analyte (e.g., cTnI) do not bind all or a portion of the epitope recognized by the second or subsequent antibody, thereby interfering with the ability of the one or more second detection antibodies to bind to the analyte (e.g., cTnI).

The antibody may be used as a primary antibody in the immunoassay. The antibodies immunospecifically bind to an epitope on the analyte (e.g., cTnI). In addition to the antibodies of the present disclosure, the immunoassay may further comprise a second antibody that immunospecifically binds to an epitope that is not recognized or bound by the first antibody.

A test sample suspected of containing an analyte (e.g., cTnI) can be contacted with at least one first capture antibody and at least one second detection antibody simultaneously or sequentially. In a sandwich assay format, a test sample suspected of containing an analyte (e.g., cTnI) is first contacted with the at least one first capture antibody that specifically binds to a particular epitope under conditions that allow formation of a first antibody-analyte (e.g., cTnI) antigen complex. If more than one capture antibody is used, a first plurality of capture antibody-cTnI antigen complexes is formed. In a sandwich assay, the antibody, preferably the at least one capture antibody, is used in molar excess of the maximum amount of analyte (e.g., cTnI) expected in the test sample. For example, about 5. mu.g/mL to about 1mg/mL of antibody can be used per mL of microparticle coating buffer.

i. anti-cTnI capture antibodies

Optionally, the at least one first capture antibody can be bound to a solid support prior to contacting the test sample with the at least one first capture antibody, which facilitates separation of the first antibody-analyte (e.g., cTnI) complex from the test sample. Any solid support known in the art may be used, including but not limited to solid supports in the form of wells, tubes, or beads (e.g., microparticles) made of polymeric material. The antibody can be bound to the solid support by adsorption, by covalent bonding using a chemical coupling agent, or by other means known in the art, provided that such binding does not interfere with the ability of the antibody to bind the analyte (e.g., cTnI). In addition, if necessary, the solid support can be derivatized to allow reaction with various functional groups on the antibody. Such derivatization requires the use of certain coupling agents, such as, but not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

After incubation of the test sample suspected of containing the analyte (e.g., cTnI), a first capture antibody (or antibodies) -analyte (e.g., cTnI) complex is allowed to form. The incubation can be performed at a pH of about 4.5 to about 10.0, at a temperature of about 2 ℃ to about 45 ℃, and for at least about one (1) minute to about eighteen (18) hours, about 2-6 minutes, about 7-12 minutes, about 5-15 minutes, or about 3-4 minutes.

Detecting the antibody

After the first/multiple capture antibody-analyte (e.g., cTnI) complex is formed, the complex is then contacted with at least one second detection antibody (under conditions that allow formation of the first/multiple antibody-analyte (e.g., cTnI) antigen-second antibody complex). In some embodiments, the test sample is contacted with the detection antibody simultaneously with the capture antibody. If a first antibody-analyte (e.g., cTnI) complex is contacted with more than one detection antibody, a first/multiple capture antibody-analyte (e.g., cTnI) -multiple detection antibody complex is formed. As with the first antibody, when the at least second (and subsequent) antibodies are contacted with the first antibody-analyte (e.g., cTnI) complexes, incubation for a period of time under conditions similar to those described above is required to form the first/plurality of antibody-analyte (e.g., cTnI) -second/plurality of antibody complexes. Preferably, at least one of the second antibodies contains a detectable label. The detectable label may be bound to the at least one second antibody prior to, simultaneously with, or after formation of the first/plurality of antibody-analyte (e.g., cTnI) -second/plurality of antibody complexes. Any detectable label known in the art may be used.

Chemiluminescence assays can be performed according to Adamczyk et al, anal. chim. acta 579 (1): 61-67 (2006). Microplate chemophotometers (Mithras LB-940, Berthold Technologies u.s.a., LLC, Oak Ridge, TN) are capable of rapidly assaying multiple small volume samples, although any suitable assay format may be used. The chemophotometer may be equipped with multiple reagent syringes using a 96-well black polystyrene microplate (Costar # 3792). Each sample may be added to a separate well and then the other reagents added simultaneously/sequentially as determined by the type of assay employed. It is desirable to avoid the use of acridinium aryl esters to form pseudobases in neutral or alkaline solutions, for example by acidification. The chemiluminescent response was then recorded well by well. In this regard, the time at which the chemiluminescent response is recorded will depend in part on the delay between addition of the reagent used and the particular acridinium.

The order of addition of the test sample and the first specific binding partner to form a mixture for use in a chemiluminescent assay is not critical. If the first specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-antigen (e.g., cTnI) complex is formed. Alternatively, if a second specific binding partner is used, and the second specific binding partner is detectably labeled with an acridinium compound, a detectably labeled first specific binding partner-analyte (e.g., cTnI) -second specific binding partner complex is formed. Any unbound specific binding partner, whether labeled or unlabeled, may be removed from the mixture using any technique known in the art, such as washing.

The hydrogen peroxide can be generated in situ in the mixture or supplied to the mixture prior to, simultaneously with, or after addition of the acridinium compound described above. The hydrogen peroxide may be generated in situ in a number of ways, as will be apparent to those skilled in the art.

Alternatively, a source of hydrogen peroxide may be simply added to the mixture. For example, the source of hydrogen peroxide may be one or more buffers or other solutions known to contain hydrogen peroxide. In this respect, the hydrogen peroxide solution may be added alone.

Upon the simultaneous or subsequent addition of at least one basic solution to the sample, a detectable signal, i.e., a chemiluminescent signal, is produced, indicating the presence of the analyte (e.g., cTnI). The alkaline solution comprises at least one base and has a pH greater than or equal to 10, preferably greater than or equal to 12. Examples of alkaline solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of alkaline solution added to the sample depends on the concentration of the alkaline solution. The amount of alkaline solution to be added to the sample can be readily determined by one skilled in the art based on the concentration of alkaline solution used. Other labels than chemiluminescent labels may be used. For example, enzymatic labels (including but not limited to alkaline phosphatase) may be used.

The generated chemiluminescent or other signal may be detected using conventional techniques known to those skilled in the art. Based on the intensity of the generated signal, the amount of the analyte of interest (e.g., cTnI) in the sample can be quantified. Specifically, the amount of analyte (e.g., cTnI) in the sample is directly proportional to the intensity of the signal generated. The amount of analyte (e.g., cTnI) can be quantified by comparing the amount of light generated to a standard curve of analyte (e.g., cTnI) or to a reference standard. Standard curves can be generated by mass spectrometry, gravimetric analysis, and other techniques known in the art using serial dilutions or solutions of known analyte (e.g., cTnI) concentrations.

(2) Forward competitive inhibition assay

In the forward competition format, aliquots of a known concentration of a labeled analyte of interest (e.g., cTnI) having a fluorescent label, a tag with a cleavable linker, etc., are used to compete with the analyte of interest (e.g., cTnI) in the test sample for binding to an antibody to the analyte of interest (e.g., an antibody to cTnI).

In a forward competition assay, an immobilized specific binding partner (e.g., an antibody) may be contacted with a test sample and a labeled analyte of interest, analyte fragment of interest, or analyte variant of interest, either sequentially or simultaneously. The analyte peptide, analyte fragment of interest, or analyte variant of interest can be labeled with any detectable label, including detectable labels comprised of a label with a cleavable linker. In this assay, the antibody may be immobilized on a solid support. Alternatively, the antibody may be conjugated to an antibody, e.g. an anti-species (anti-pepties) antibody, which has been immobilized on a solid support, e.g. a particulate or planar substrate.

The labeled analyte of interest, test sample and antibody are incubated under conditions similar to those described above for the sandwich assay format. Two different species of antibody-analyte complexes of interest can then be generated. Specifically, one of the antibody-analyte of interest complexes generated contains a detectable label (e.g., a fluorescent label, etc.), while the other antibody-analyte of interest complex does not contain a detectable label. The antibody-analyte of interest complex may, but need not, be separated from the remainder of the test sample prior to quantifying the detectable label. Regardless of whether the antibody-analyte of interest complex separates from the remainder of the test sample, the amount of detectable label in the antibody-analyte of interest complex is then quantified. The concentration of the analyte of interest (e.g., membrane-bound analyte of interest, soluble analyte of interest, a fragment of soluble analyte of interest, a variant of the analyte of interest (membrane-bound or soluble analyte of interest), or any combination thereof) in the test sample is then determined as described above.

(3) Reverse competition inhibition assay

In a reverse competition assay, an immobilized analyte of interest (e.g., cTnI) can be contacted with a test sample and at least one labeled antibody sequentially or simultaneously.

The analyte of interest may be bound to a solid support, such as those discussed above with respect to the sandwich assay format.

The immobilized analyte of interest, the test sample and the at least one labeled antibody are incubated under conditions similar to those described above for the sandwich assay format. Two different species of analyte-antibody complexes of interest are then generated. Specifically, one of the generated analyte-antibody complexes of interest is immobilized and contains a detectable label (e.g., a fluorescent label, etc.), while the other analyte-antibody complex of interest is not immobilized and contains a detectable label. The non-immobilized analyte-antibody complexes of interest and the remainder of the test sample are removed from the presence of immobilized analyte-antibody complexes of interest by techniques known in the art, such as washing. Once the non-immobilized analyte-antibody complex of interest is removed, the amount of detectable label in the immobilized analyte-antibody complex of interest is then quantified after the label is cleaved off. The concentration of the analyte of interest in the test sample can then be determined by comparing the amount of detectable label as described above.

(4) One-step immunoassay or "instant capture" assay

In a ready-to-capture immunoassay, a solid substrate is pre-coated with a fixative. The capture agent, analyte (e.g., cTnI), and detection agent are added together to the solid matrix, and then a washing step is performed prior to detection. The capture agent can bind to an analyte (e.g., cTnI) and comprises a ligand for the immobilization agent. The capture and detection agents may be antibodies or any other moiety capable of capture or detection as described herein or known in the art. The ligand may comprise a peptide tag and the immobilisation agent may comprise an anti-peptide tag antibody. Alternatively, the ligand and the immobilizing agent can be any pair of reagents (e.g., a specific binding pair, and others such as are known in the art) that can be bound together for use in a real-time capture assay. More than one analyte may be measured. In some embodiments, the solid substrate may be coated with an antigen and the analyte to be analyzed is an antibody.

In certain other embodiments, in a one-step immunoassay or "instant capture," a solid support (e.g., a microparticle) pre-coated with a fixative (e.g., biotin, streptavidin, etc.) and at least a specific binding member and a second specific binding member (which function as capture and detection reagents, respectively) is used. The first specific binding member comprises a ligand for an immobilization agent (e.g., if the immobilization agent on the solid support is streptavidin, the ligand on the first specific binding member may be biotin) and also binds to an analyte of interest (e.g., cTnI). The second specific binding member comprises a detectable label and binds to the analyte of interest (e.g., cTnI). The solid support and the first and second specific binding members may be added to the test sample (sequentially or simultaneously). The ligand on the first specific binding member binds to the immobilisation agent on the solid support to form a solid support/first specific binding member complex. Any analyte of interest present in the sample binds to the solid support/first specific binding member complex to form a solid support/first specific binding member/analyte complex. The second specific binding member binds to the solid support/first specific binding member/analyte complex and the detectable label is detected. An optional washing step may be employed prior to detection. In certain embodiments, more than one analyte may be measured in a one-step assay. In certain other embodiments, more than two specific binding members may be employed. In certain other embodiments, multiple detectable labels may be added. In certain other embodiments, multiple analytes of interest may be detected, or their amount, level, or concentration measured, determined, or assessed.

The use of a point-of-care capture assay can be performed in a variety of formats as described herein and known in the art. For example, the format may be a sandwich assay as described above, but may also be a competition assay, a single specific binding member may be employed, or other variants, such as known variants, may be used.

14. Other factors

The methods of diagnosis, prognosis and/or assessment described above may also include diagnosis, prognosis and assessment using other factors. In some embodiments, traumatic brain injury can be diagnosed using the glasgow coma scale or the extended Glasgow Outcome Scale (GOSE). Other tests, scales or indicators may also be used alone or in combination with the glasgow coma scale. An example is the Ranchos Los Amigos scale. The Ranchos Los Amigos scale measures the level of awareness, cognition, behavior, and interaction with the environment. The Ranchos Los Amigos scale includes: stage I: no reaction is carried out; II stage: a Generalized reaction (Generalized Response); grade III: local reaction (Localized Response); stage IV: confusion-manic reaction (convosed-aged); and V stage: confusion-inappropriate reaction (condensed-inappropriately); stage VI: confusion-appropriate reaction (convosed-autopropriate); and VII, stage: auto-appropriate reaction (Automatic-autopropropriate); and VIII: purpose- -appropriate reaction (purposedul-aprypropriate).

15. Sample (I)

In some embodiments, the sample is obtained after the human subject has suffered head injury from physical shock, blunt impact of external mechanical or other forces resulting in closed or open head trauma, one or more drops, explosions or explosions, or other types of blunt force trauma. In some embodiments, the sample is obtained after ingestion or exposure to a chemical, toxin, or combination of chemical and toxin by a human subject. Examples of such chemicals or toxins are fire, mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (e.g., carbon monoxide, hydrogen sulfide and cyanide), organic metals (e.g., methyl mercury, tetraethyl lead and organic tin), and/or one or more drugs of abuse. In some embodiments, the sample is obtained from a subject having an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or a combination thereof.

In yet another embodiment, the sample used in the methods described herein can also be used to determine the level of cTnI in a subject by using an anti-cTnI antibody or antibody fragment thereof as described below for determining whether the subject has or is at risk of developing mild traumatic brain injury. Thus, in particular embodiments, the present disclosure also provides a method for determining whether a subject suffering from or at risk of traumatic brain injury as discussed herein and known in the art is a candidate for therapy or treatment. In general, as described herein, the subject is at least one of the following: (i) (ii) experiencing head injury; (ii) ingestion and/or exposure to one or more chemicals and/or toxins; (iii) suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or any combination thereof; or (iv) any combination of (i) - (iii); alternatively, a concentration or amount that has been actually diagnosed as having or at risk of TBI (e.g., a subject having an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, one or more viruses, meningitis, hydrocephalus, or a combination thereof), and/or exhibiting an adverse (i.e., clinically undesirable) of cTnI or a segment of cTnI.

a. Testing or biological samples

As used herein, "sample," "test sample," "biological sample" refers to a fluid sample containing or suspected of containing cTnI. The sample may be obtained from any suitable source. In some cases, the sample may comprise a liquid, a fluidized particulate solid, or a fluid suspension of solid particles. In some cases, the sample may be processed prior to the analysis described herein. For example, a sample may be isolated or purified from its source prior to analysis; however, in certain embodiments, untreated cTnI-containing samples can be assayed directly. In a particular example, the source containing cTnI is a human substance (e.g., bodily fluid, blood such as whole blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, feces, tissue, organ, etc.). The tissue may include, but is not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, cardiac muscle tissue, brain tissue, bone marrow, cervical tissue, skin, and the like. The sample may be a liquid sample or a liquid extract of a solid sample. In some cases, the source of the sample may be an organ or tissue that can be lysed by tissue disintegration/cell lysis, such as a biopsy sample.

A wide variety of volumes of fluid sample may be analyzed. In some exemplary embodiments, the sample volume can be about 0.5nL, about 1nL, about 3nL, about 0.01 μ L, about 0.1 μ L, about 1 μ L, about 5 μ L, about 10 μ L, about 100 μ L, about 1mL, about 5mL, about 10mL, and the like. In some cases, the volume of the fluid sample is between about 0.01 μ L and about 10mL, between about 0.01 μ L and about 1mL, between about 0.01 μ L and about 100 μ L, or between about 0.1 μ L and about 10 μ L.

In some cases, the fluid sample may be diluted prior to use in the assay. For example, in embodiments where the source containing cTnI is a human bodily fluid (e.g., blood, serum), the fluid may be diluted with a suitable solvent (e.g., a buffer such as PBS buffer). Prior to use, the fluid sample may be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more. In other cases, the fluid sample is not diluted prior to use in the assay.

In some cases, the sample may undergo pre-analytical processing. Pretreatment for analysis may provide additional functions such as removal of non-specific proteins and/or efficient and inexpensive implementation of mixing functions. General methods of pretreatment for analysis may include the use of electrokinetic trapping, electrokinetic, surface acoustic wave, isotachophoresis, dielectrophoresis, electrophoresis, or other preconcentration techniques known in the art. In some cases, the fluid sample may be concentrated prior to use in the assay. For example, in embodiments where the source containing cTnI is a human bodily fluid (e.g., blood, serum), the can be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. Prior to use, the fluid sample may be concentrated by a factor of about 1, about 2, about 3, about 4, about 5, about 6, about 10, about 100, or more.

b. Control

It may be desirable to include a control sample. The control sample may be analyzed simultaneously with the sample from the subject as described above. The results obtained from the subject sample can be compared to the results obtained from the control sample. A standard curve may be provided whereby the results of the measurements of the samples may be compared. If a fluorescent label is used, such a standard curve provides a level of the marker that is a function of the unit of measurement, i.e., the intensity of the fluorescent signal. Using samples taken from multiple donors, standard curves can be provided for reference levels of cTnI in normal healthy tissue and "risk" levels of cTnI in tissue taken from donors that may have one or more of the above characteristics.

Thus, in view of the foregoing, a method of determining the presence, amount, or concentration of cTnI in a test sample is provided. The method comprises determining cTnI in the test sample by immunoassay, for example, using at least one capture antibody that binds to an epitope on cTnI and at least one detection antibody that binds to an epitope on cTnI that is different from the epitope of the capture antibody and optionally comprises a detectable label, and comparing the signal generated by the detectable label as a direct or indirect indicator of the presence, amount or concentration of cTnI in the test sample with the signal generated as a direct or indirect indicator of the presence, amount or concentration of cTnI in the calibrator. The calibrator is optional and is preferably part of a series of calibrators, wherein each calibrator differs in the concentration of cTnI from the other calibrators in the series.

16. Reagent kit

Provided herein are kits that can be used to determine or assess the cTnI and/or cTnI fragments of a test sample. The kit comprises at least one component for determining cTnI in a test sample and instructions for determining cTnI in a test sample. For example, the kit can comprise instructions for determining cTnI in a test sample by an immunoassay, such as a chemiluminescent microparticle immunoassay. The instructions contained in the kit may be affixed to the packaging material or may be included as packaging instructions. Although the description is generally of written or printed material, they are not limited thereto. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tape, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "specification" may include the address of the Internet site that provides the specification.

The at least one component can include at least one composition comprising one or more isolated antibodies or antibody fragments thereof that specifically bind to cTnI. The antibody may be a cTnI detection antibody and/or a capture antibody.

Alternatively, or in addition, the kit may comprise a calibrator or control, such as purified and optionally lyophilized cTnI, and/or at least one container for performing the assay (e.g., a test tube, microtiter plate or strip, which may have been coated with an anti-cTnI antibody), and/or a buffer, such as an assay buffer or wash buffer, any of which may be provided as a concentrated solution, a substrate solution for a detectable label (e.g., an enzymatic label), or a stop solution. Preferably, the kit comprises all components necessary for performing the assay, i.e. reagents, standards, buffers, diluents, etc. The instructions may also include instructions for generating a standard curve.

The kit may further comprise a reference standard for quantifying cTnI. Reference standards can be used to establish standard curves for interpolating and/or extrapolating the concentration of cTnI. In some embodiments, the reference standard for CTnI may correspond to the 99 th percentile derived from a healthy reference population. Such reference standards may be determined using conventional techniques known in the art.

Any antibody provided in the kit, e.g., a recombinant antibody specific for cTnI, can incorporate a detectable label, e.g., a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, etc., or the kit can include a reagent for labeling the antibody or a reagent for antibody detection (e.g., a detection antibody) and/or a reagent for labeling the analyte (e.g., cTnI) or a reagent for detecting the analyte (e.g., cTnI). The antibodies, calibrator and/or control may be provided in separate containers or pre-dispensed into an appropriate assay format, such as a microtiter plate.

Optionally, the kit includes quality control components (e.g., sensitivity panels, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described on the insert for various immunodiagnostic products. Members of the sensitivity group are optionally used to establish assay performance characteristics, and are also optionally useful indicators of the integrity of immunoassay kit reagents and assay standardization.

The kit may also optionally include other reagents required to perform diagnostic assays or to facilitate quality control assessments, such as buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, and the like. Other components, such as buffers and solutions used to separate and/or process the test sample (e.g., pretreatment reagents), may also be included in the kit. The kit may also include one or more other controls. One or more components of the kit may be lyophilized, in which case the kit may further comprise reagents suitable for reconstitution of the lyophilized components.

The various components of the kit are optionally provided in suitable containers, such as microtiter plates, as desired. The kit may also include a container for holding or storing a sample (e.g., a container or a cassette for a urine, whole blood, plasma, or serum sample). The kit optionally may also contain, where appropriate, reaction vessels, mixing vessels, and other components that facilitate preparation of reagents or test samples. The kit may also include one or more instruments for assisting in obtaining the test sample, such as syringes, pipettes, forceps, measuring spoons, and the like.

If the detectable label is at least one acridinium compound, the kit can comprise at least one acridinium-9-carboxamide, at least one aryl acridinium 9-carboxylate ester, or any combination thereof. If the detectable label is at least one acridinium compound, the kit can comprise a source of hydrogen peroxide, such as a buffer, a solution, and/or at least one basic solution. If desired, the kit may contain a solid phase, such as magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, membranes, filter paper, discs or chips.

If desired, the kit may further comprise one or more components, alone or in combination with instructions, for determining other analytes in a test sample, which may be biomarkers, for example, biomarkers of traumatic brain injury or disorder.

a. Modification of kits and methods

The kits (or components thereof), and methods of assessing or determining the concentration of cTnI in a test sample by immunoassay as described herein, can be adapted for use in a variety of automated and semi-automated systems (including systems in which the solid phase comprises microparticles) as described, for example, in U.S. patent No.5,063,081, U.S. patent application publication nos. 2003/0170881, 2004/0018577, 2005/0054078, and 2006/0160164, and as described, for example, by Abbott laboratories (Abbott Park, IL) as Abbott Point of Care (c) ((r))

or i-STATAlinity，Abbott Laboratories), and those described in U.S. Pat. Nos. 5,089,424 and 5,006,309 and referenced by, for example, Abbott Laboratories (Abbott Park, IL) asOr those commercially sold by the Abbott alliance family of devices.

Some differences between automated or semi-automated systems as compared to non-automated systems (e.g., ELISA) include the substrate to which the first specific binding partner (e.g., analyte antibody or capture antibody) is attached (which may affect sandwich formation and analyte reactivity), as well as the length and timing of the capture, detection, and/or any optional washing steps. Non-automated formats such as ELISA may require relatively long incubation times of the sample and capture reagents (e.g., about 2 hours), while automated or semi-automated formats (e.g.,and any subsequent platform, Abbott Laboratories) may be relatively short (e.g.,

about 18 minutes). Similarly, non-automated formats such as ELISA may allow relatively long incubation times (e.g., about 2 hours) for incubating detection antibodies, such as conjugate reagents, while automated or semi-automated formats (e.g.,

and any subsequent platform, Abbott Laboratories) may be relatively short (e.g.,

and any subsequent plateaus for approximately 4 minutes).

Other platforms available from Abbott Laboratories include, but are not limited to

(see, e.g., U.S. Pat. No.5,294,404, which is incorporated herein by reference in its entirety),

EIA (beads) and Quantum^TMII, and other platforms. In addition, the assays, kits, and kit components can be used in other forms, for example, on an electrochemical assay system or other handheld or bedside assay systems. As previously mentioned, the present disclosure, for example, can be applied to a commercial Abbott Point of Care (C) (Abbott Laboratories) electrochemical immunoassay system. Immunosensors, methods of making the same, and methods of operating in disposable test devices are described, for example, in U.S. patent No.5,063,081, U.S. patent application publication nos. 2003/0170881, 2004/0018577, 2005/0054078, and 2006/0160164, the teachings of which are incorporated herein by reference in their entirety.

In particular, with respect to targeting assays toThe system was modified, and the following configuration was preferred. Micromachined silicon chips were fabricated using a pair of gold ampere working electrodes and a silver-silver chloride-silver reference electrode. On one working electrode, polystyrene beads (0.2 mm diameter) with immobilized capture antibody were adhered to the patterned polymer coating of polyvinyl alcohol on the electrode. Assembling the chip into a fluid form suitable for immunoassay

In the cylinder core. On a part of the silicon chip, there is a specific binding partner for cTnI, such as one or more cTnI antibodies, one or more monoclonal/polyclonal antibodies or fragments thereof, variants thereof, or variant fragments thereof that can bind to cTnI) or one or more anti-cTnI DVD-Ig (or fragments thereof, variants thereof, or variant fragments thereof that can bind to cTnI), any of which can be detectably labeled. Fluid bag in the cartridgeIs an aqueous reagent comprising a p-aminophenol phosphate ester.

In operation, a sample from a subject suspected of having TBI is added to the storage chamber of a test cartridge and the cartridge is insertedIn the reader. A pump element in the cartridge pushes the sample into the conduit containing the chip. The sample is contacted with the sensor to dissolve the enzyme conjugate into the sample. The sample is shaken across the sensor to facilitate sandwich formation for about 2-12 minutes. In the penultimate step of the assay, the sample is pushed into a waste compartment and excess enzyme conjugate and sample are washed from the sensor chip with a wash solution containing the alkaline phosphatase substrate. In the last step of the assay, the alkaline phosphatase label reacts with the p-aminophenol phosphate to cleave the phosphate group and allow the released p-aminophenol to be electrochemically oxidized at the working electrode. Based on the measured current, the reader can calculate the amount of cTnI in the sample through a built-in algorithm and a factory-determined calibration curve.

The methods and kits described herein must include other reagents and methods for performing immunoassays. For example, various buffers are included, such as those known in the art and/or that can be readily prepared or optimized to be used, such as for washing, as a conjugate diluent, and/or as a calibrator diluent. Exemplary conjugate diluents are those used in certain kits (Abbott Laboratories, Abbott Park, IL)

A conjugate diluent comprising 2- (N-morpholino) ethanesulfonic acid (MES), a salt, a protein blocker, an antimicrobial agent, and a detergent. An exemplary calibrator diluent is used in certain kits (Abbott Laboratories, Abbott Park, IL)

Human calibrator diluent comprising MES-containing buffer, other salts, protein blockers and antibacterialsAnd (3) preparing. In addition, as described in U.S. patent application No.61/142,048 filed on 31/12/2008, a nucleic acid sequence linked to a signal antibody can be used as a signal amplifier, for example, in

Improved signal generation is obtained in the cartridge format.

While certain embodiments herein are advantageous for use in assessing diseases, such as traumatic brain injury, the assays and kits can also optionally be used to assess cTnI in other diseases, disorders, and conditions, as appropriate.

The assay methods can also be used to identify compounds that ameliorate diseases, such as traumatic brain injury. For example, a cell expressing cTnI can be contacted with a candidate compound. Using the assay methods described herein, the level of cTnI expression in cells contacted with a compound can be compared to control cells.

The present disclosure has several aspects, which are illustrated by the following non-limiting examples.

17. Examples of the embodiments

It will be apparent to those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and appreciated, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, it will be more clearly understood by reference to the following examples, which are intended merely to illustrate some aspects and embodiments of the present disclosure, and should not be taken as limiting the scope of the disclosure. All journal references, U.S. patents, and publications mentioned herein are incorporated by reference in their entirety.

The present disclosure has several aspects, which are illustrated by the following non-limiting examples.