阅读说明：本技术 通过生物标记物联检体外评估类风湿性关节炎是否存在的方法 (The method that whether there is by biomarker joint inspection evaluating in vitro rheumatoid arthritis ) 是由 饶星 廖智星 刘宇卉 李临 其他发明人请求不公开姓名 于 2018-07-23 设计创作，主要内容包括：本发明涉及一种用于通过生物化学标记物体外评估类风湿性关节炎(RA)是否存在的方法。该方法通过联合检测血清14-3-3η蛋白、anti-CCP抗体和抗Carp抗体中至少2种的水平,并将检测结果与RA相关联,可以显著提高RA炎性关节病变患者的RA阳性的检测准确度。(The present invention relates to a kind of methods for whether there is by biochemical markers evaluating in vitro rheumatoid arthritis (RA).This method will test that result is associated with RA by least two kinds of levels in joint-detection serum 14-3-3 η albumen, anti-CCP antibody and anti-Carp antibody, can significantly improve the accuracy in detection of the RA positive of RA inflamed joints lesion patient.)

1. each biomarker is dense in utilization homogeneous immunodetection detection rheumatoid arthritis (RA) biomarker group The purposes in the reagent that preparation whether there is by biochemical markers evaluating in vitro rheumatoid arthritis (RA) is spent, Include:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combination of each biomarker in biomarker group Concentration value；With

C) the combined concentration value obtained in step b) whether there is with RA it is associated, wherein corresponding to being measured from reference group Biomarker group in each marker truncation combined concentration value compared to increased combined value instruction RA presence；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few.

2. purposes according to claim 1, which is characterized in that by the combined concentration value of step b) and derived from except the RA positive The cutoff value of reference group except patient is compared, and the reference group is comprising obvious healthy person and is selected from osteoarthritis (OA) patient of patient and other autoimmune disease patients.

3. each biomarker is dense in utilization homogeneous immunodetection detection rheumatoid arthritis (RA) biomarker group The purposes in reagent of the preparation by the severity of biochemical markers evaluating in vitro rheumatoid arthritis (RA) is spent, Include:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) the combined concentration value obtained in step b) is associated with the severity of RA, wherein with the phase that is measured from reference group In the biomarker group answered the truncation combined concentration value of each marker compared to increased combined value instruction patient in RA it is serious Degree；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few.

4. each biomarker is dense in utilization homogeneous immunodetection detection rheumatoid arthritis (RA) biomarker group Spend the system for distinguishing rheumatoid arthritis (RA) and other autoimmune diseases in vitro by biochemical markers in preparation Purposes in agent comprising:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) RA and other autoimmune diseases are distinguished from the combined concentration value obtained in step b), wherein with from referring to group The truncation combined concentration value of each marker is compared to increased combined value instruction RA's in the corresponding biomarker group of bulk measurement In the presence of；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few；It is preferred that other autoimmune diseases include other joint diseases；It is further preferably described other Joint disease is osteoarthritis (OA).

5. rheumatoid arthritis (RA) biomarker group is used to prepare assesses rheumatoid joint in sample to be tested in vitro The purposes in reagent that scorching (RA) whether there is, wherein using homogeneous immunodetection for rheumatoid arthritis (RA) biology The combined concentration value of each biomarker measurement and the corresponding biomarker for being measured from reference group in marker group The truncation combined concentration value of each marker is compared to the presence for increasing instruction RA in object group；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few.

6. purposes described in any one of -5 according to claim 1, which is characterized in that the biomarker group includes At least two kinds of and other biomarkers object in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody, preferably 2 Kind and other biomarkers object；It is preferred that the other biomarkers object is RA.

7. purposes described in any one of -6 according to claim 1, which is characterized in that the biomarker group includes Anti-CCP antibody and 14-3-3eta albumen.

8. purposes according to claim 7, which is characterized in that the step further include measurement 14-3-3eta albumen or its The content for the immune complex that segment or the 14-3-3eta albumen or its segment and at least one antibody are formed.

9. purposes according to claim 8, which is characterized in that determined based on 14-3-3eta protein standard working curve The content of 14-3-3eta albumen in sample to be tested.

10. purposes according to claim 8, which is characterized in that the step further includes by measured 14-3-3eta egg The content for the immune complex that white or its segment or the 14-3-3eta albumen or its segment and at least one antibody are formed, with 14-3- described in sample before normal reference sample, rheumatoid arthritis control sample or treatment from same subject The immune complex that 3eta albumen or its segment or the 14-3-3eta albumen or its segment are formed at least one antibody contains Amount is compared.

11. purposes according to claim 8, which is characterized in that the step include by the sample with comprising can be with At least one specific epitopes of 14-3-3eta albumen or its segment specifically bind the antibody to form immune complex.

12. the purposes according to any one of claim 8-11, which is characterized in that the antibody includes can be with 14- First antibody that first epitope specificity of 3-3eta albumen combines and can be special with the second epitope of 14-3-3eta albumen Property combine secondary antibody, wherein second epitope and the first epitope non-overlapping.

13. purposes according to claim 12, which is characterized in that the first antibody is described by physical efficiency in conjunction with receptor Enough react with singlet oxygen generates detectable chemiluminescence signal.

14. purposes according to claim 13, which is characterized in that the receptor includes olefin(e) compound and metal-chelating Object is non-particulate forms, and solvable in water-bearing media；And/or the receptor is filled with luminophor and group of the lanthanides member The high molecular particle of element.

15. purposes according to claim 12, which is characterized in that the first antibody and secondary antibody are separately selected From monoclonal antibody and/or polyclonal antibody, preferably monoclonal antibody.

16. purposes according to claim 8, which is characterized in that the amino acid sequence of the 14-3-3eta albumen or its segment Column are as shown in SEQUENCE NO.1.

17. purposes according to claim 16, which is characterized in that it is 14-3-3eta that the epitope, which is selected from amino acid fragment, The relative specificity segment of the sequence of albumen: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa.

18. one kind passes through biochemistry mark based on the concentration of each biomarker in rheumatic arthritis (RA) biomarker group The reagent set that note object evaluating in vitro rheumatoid arthritis (RA) whether there is comprising for utilizing homogeneous immunodetection Detect the reagent of each biomarker concentration in rheumatoid arthritis (RA) biomarker group, wherein the biomarker Object group contain it is at least two kinds of in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody, preferably 2 kinds.

19. one kind passes through biochemistry mark based on the concentration of each biomarker in rheumatic arthritis (RA) biomarker group Remember the reagent set of the severity of object evaluating in vitro rheumatoid arthritis (RA) comprising for utilizing homogeneous immune detection Method detects the reagent of each biomarker concentration in rheumatoid arthritis (RA) biomarker group, wherein the biology mark It is at least two kinds of in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody to remember that object group contains, preferably 2 kinds.

20. one kind passes through biochemistry mark based on the concentration of each biomarker in rheumatic arthritis (RA) biomarker group Remember the reagent set of object outskirt classification rheumatic arthritis (RA) and other autoimmune diseases comprising for using Phase immunodetection detects the reagent of each biomarker concentration in rheumatoid arthritis (RA) biomarker group, wherein The biomarker group contain it is at least two kinds of in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody, preferably It is 2 kinds；It is preferred that other autoimmune diseases include other joint diseases；Further preferred other joint diseases are Osteoarthritis (OA).

21. based on rheumatoid arthritis (RA) in rheumatoid arthritis (RA) biomarker group evaluating in vitro sample to be tested The reagent set that whether there is, wherein using homogeneous immunodetection in rheumatoid arthritis (RA) biomarker group The combined concentration value of each biomarker measurement with for respectively being marked in the corresponding biomarker group that reference group measures Remember the truncation combined concentration value of object compared to the presence for increasing instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few.

22. reagent set described in any one of 8-21 according to claim 1, which is characterized in that the biomarker group Comprising at least two kinds of and other biomarkers object in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody, preferably For 2 kinds and other biomarkers object；It is preferred that the other biomarkers object is RA.

23. reagent set described in any one of 8-21 according to claim 1, which is characterized in that the reagent set includes Using the reagent of Anti-CCP antibody and 14-3-3eta protein concentration in homogeneous immunodetection detection biomarker group.

24. reagent set according to claim 23, which is characterized in that for detecting the homogeneous immune of Anti-CCP antibody Detection reagent includes:

Component a1, it includes the first antigens that can be specifically bound with the epitope binding site of anti-CCP antibody；

Component b1, it includes anti-immunity complex antibody, the anti-immunity complex antibody can specific recognition and combine with The anti-CCP of the anti-CCP antibody in the first immune complex that first antigen is formed, antigen that nonrecognition dissociates, unbonded is anti- Body.

25. reagent set according to claim 24, which is characterized in that first antigen or the anti-immunity compound Antibody is combined with receptor, and the receptor can be reacted with singlet oxygen generates detectable chemiluminescence signal；Preferably, institute Stating receptor includes olefin(e) compound and metallo-chelate, is non-particulate forms, and solvable in water-bearing media；And/or it is described Receptor is the high molecular particle filled with luminophor and lanthanide series.

26. the reagent set according to claim 24 or 25, which is characterized in that the reagent set also includes component c1, It includes can be in the donor of excited state generation singlet oxygen；It is preferred that one in the donor and specific binding pair member Member combines, and another member in specific binding pair member and first antigen or the anti-immunity complex antibody knot It closes；It is further preferred that the donor is in conjunction with Streptavidin, correspondingly the first antigen or the anti-immunity complex antibody In conjunction with biotin.

27. reagent set according to claim 25, which is characterized in that for detecting homogeneously exempting from for 14-3-3eta albumen Epidemic disease detection reagent includes:

Component a2, it includes the receptor and first antibody in combination for generating detectable signal can be reacted with singlet oxygen Or its binding fragment, the first antibody or its binding fragment can be in conjunction with the first epitope specificities of 14-3-3eta albumen；

Component b2, it includes can be with the secondary antibody or its bonding pad in conjunction with the second epitope specificity of 14-3-3eta albumen Section, second epitope and the first epitope non-overlapping；

Component c2 comprising can be in the donor of excited state generation singlet oxygen.

28. reagent set according to claim 27, which is characterized in that the reagent further includes the 14- as calibration object 3-3eta albumen sterling, the calibration object be calibrated product dilution proportionally gradient dilution at various concentration working calibration product Solution.

29. the reagent set according to claim 27 or 28, which is characterized in that the secondary antibody or its binding fragment with A member in specific binding pair member combines, and another Yuan combination in the donor and specific binding pair member； It is preferred that the secondary antibody or its binding fragment be in conjunction with biotin, and the donor is in conjunction with Streptavidin.

30. one kind passes through bioid for the concentration based on each biomarker in rheumatic arthritis (RA) biomarker group The kit that marker evaluating in vitro rheumatoid arthritis (RA) whether there is is learned, it includes any in claim 18-29 Reagent set described in one.

31. one kind passes through bioid for the concentration based on each biomarker in rheumatic arthritis (RA) biomarker group The kit for learning the severity of marker evaluating in vitro rheumatoid arthritis (RA), it includes appoint in claim 18-29 Reagent set described in meaning one.

32. one kind passes through bioid for the concentration based on each biomarker in rheumatic arthritis (RA) biomarker group The kit that label object distinguishes rheumatoid arthritis (RA) and other autoimmune diseases in vitro is learned, it includes rights to want Seek reagent set described in any one of 18-29.

33. based on rheumatoid arthritis (RA) in rheumatoid arthritis (RA) biomarker group evaluating in vitro sample to be tested The kit that whether there is, it includes the reagent sets described in any one of claim 18-29.

34. the method that one kind whether there is by biochemical markers evaluating in vitro rheumatoid arthritis (RA) comprising Using the reagent set as described in any one of claim 18-29 or using such as any one of claim 30-33 institute The kit stated detects the concentration of each biomarker in rheumatic arthritis (RA) biomarker group and passes through bioid Learning marker evaluating in vitro rheumatoid arthritis (RA) whether there is.

35. according to the method for claim 34, which is characterized in that this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combination of each biomarker in biomarker group Concentration value；With

C) the combined concentration value obtained in step b) whether there is with RA it is associated, wherein corresponding to being measured from reference group Biomarker group in each marker truncation combined concentration value compared to increased combined value instruction RA presence；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few.

36. according to the method for claim 35, which is characterized in that by the combined concentration value of step b) and derived from except RA sun Property patient except the cutoff value of reference group be compared, the reference group includes obvious healthy person and is selected from osteoarthritis (OA) patient of patient and other autoimmune disease patients.

37. one kind passes through the method for the severity of biochemical markers evaluating in vitro rheumatoid arthritis (RA), packet It includes using the reagent set as described in any one of claim 18-29 or using such as any one of claim 30-33 The kit detects the concentration of each biomarker in rheumatic arthritis (RA) biomarker group and passes through biology Chemical labeling beyond the region of objective existence assesses the severity of rheumatoid arthritis (RA).

38. according to the method for claim 37, which is characterized in that this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) the combined concentration value obtained in step b) is associated with the severity of RA, wherein with the phase that is measured from reference group In the biomarker group answered the truncation combined concentration value of each marker compared to increased combined value instruction patient in RA it is serious Degree；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few.

39. one kind distinguishes rheumatoid arthritis (RA) and other autoimmune diseases by biochemical markers in vitro Method comprising using the reagent set as described in any one of claim 18-29 or using as in claim 30-33 Kit described in any one detects the concentration of each biomarker in rheumatic arthritis (RA) biomarker group simultaneously Distinguish rheumatoid arthritis (RA) and other autoimmune diseases in vitro by biochemical markers.

40. according to the method for claim 39, which is characterized in that this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) RA and other autoimmune diseases are distinguished from the combined concentration value obtained in step b), wherein with from referring to group The truncation combined concentration value of each marker is compared to increased combined value instruction RA's in the corresponding biomarker group of bulk measurement In the presence of；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few；It is preferred that other autoimmune diseases include other joint diseases；It is further preferably described other Joint disease is osteoarthritis (OA).

41. using the reagent set as described in any one of claim 18-29 or using any in such as claim 30-33 The method that kit described in one whether there is for rheumatoid arthritis (RA) in assessment sample to be tested in vitro, wherein The combined concentration value of biomarker each in rheumatoid arthritis (RA) biomarker group measurement is joined with for coming from The truncation combined concentration value of each marker in the corresponding biomarker group of group's measurement is examined compared to the presence for increasing instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody extremely It is 2 kinds, preferably 2 kinds few.

42. the method according to any one of claim 34-41, which is characterized in that the biomarker group includes At least two kinds of and other biomarkers object in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody, preferably 2 Kind and other biomarkers object；It is preferred that the other biomarkers object is RF.

43. the method according to any one of claim 34-42, which is characterized in that appoint using in claim 24-26 Reagent set described in one anticipate to detect the concentration of Anti-CCP by homogeneous immune detection comprising:

Sample to be tested is mixed with component a1, obtains the first mixture by step R1；

First mixture is mixed with component b1, obtains the second mixture by step R2；

Second mixture is mixed with component c1, obtains the third mixture for producing detectable chemiluminescence signal by step R3；

Step R4, the intensity of chemiluminescence signal described in detecting step R3, so that it is determined that the content of Anti-CCP antibody.

44. according to the method for claim 43, which is characterized in that the method also includes the production before step R1 The step of Anti-CCP antibody standard working curve.

45. the method according to claim 43 or 44, which is characterized in that in step R4, change described in detecting step R3 The intensity of luminous signal is learned, and determines Anti-CCP antibody in sample to be tested based on Anti-CCP antibody standard working curve Content.

46. the method according to any one of claim 34-42, which is characterized in that appoint using in claim 27-29 Reagent set described in one anticipate to detect the concentration of 14-3-3eta albumen by homogeneous immunodetection comprising:

Step R1 by sample to be tested and component a2 and combines b2 and mixes, obtains third mixture；

Third mixture is mixed with component c2, obtains the 4th mixture by step R2；

Step R3 contacts energy or reactive compound with the 4th mixture, and the donor is excited to generate singlet oxygen, The receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence and/or intensity of chemiluminescence signal described in detecting step R3, thus judge survey sample to be tested in be It is no that there are the contents of 14-3-3eta albumen and/or determining 14-3-3eta albumen.

47. according to the method for claim 46, which is characterized in that the method also includes the production before step R1 The step of 14-3-3eta protein standard working curve.

48. according to the method for claim 47, which is characterized in that in step R4, the hair of chemistry described in detecting step R3 The intensity of optical signal, and determine based on 14-3-3eta protein standard working curve containing for 14-3-3eta albumen in sample to be tested Amount.

49. one kind is for detecting the chemiluminescence immunoassay of each biomarker in rheumatoid arthritis (RA) biomarker group Detection system comprising using the reagent set as described in any one of claim 18-29 or using such as claim 30- Kit described in any one of 33 detects sample to be tested using method described in any one of claim 34-48 In with the presence or absence of each biomarker in rheumatoid arthritis (RA) biomarker group and/or determine rheumatoid arthritis (RA) in biomarker group each biomarker content.

50. detection system according to claim 49, which is characterized in that the system comprises:

Reaction unit is used for reagent set or claim described in any one of sample to be tested and claim 18-29 Reagent in kit described in any one of 30-33 chemically reacts；

Excitation and reading plotter generate active oxygen, receptor microballoon using the excitation donor microballoon of 600-700nm wavelength The transmitting light that 520-620nm is generated with the reactive oxygen species received, records the optical signal of above-mentioned transmitting light；

Processor, being surveyed in sample to be tested according to the presence of the optical signal of the transmitting light recorded and/or intensity judgement is It is no that there are the contents of object to be measured molecule and/or determining object to be measured molecule.

51. detection system according to claim 50, which is characterized in that the processor is fitted using cubic spline interpolation It is fitted, directly gives the concentration value of object to be measured molecule in sample to be tested.

52. it is a kind of for detecting the homogeneous immunologic detection method of Anti-CCP, use any one of claim 49-51 institute The detection system stated and the reagent set as described in any one of claim 24-26 are detected by homogeneous immune detection The concentration of Anti-CCP comprising:

Sample to be tested is mixed with component a1, obtains the first mixture by step R1；

First mixture is mixed with component b1, obtains the second mixture by step R2；

Second mixture is mixed with component c1, obtains the third mixture for producing detectable chemiluminescence signal by step R3；

Step R4, the intensity of chemiluminescence signal described in detecting step R3, so that it is determined that the content of Anti-CCP antibody.

53. method according to claim 52, which is characterized in that the method also includes the production before step R1 The step of Anti-CCP antibody standard working curve.

54. the method according to claim 52 or 53, which is characterized in that in step R4, change described in detecting step R3 The intensity of luminous signal is learned, and determines Anti-CCP antibody in sample to be tested based on Anti-CCP antibody standard working curve Content.

55. it is a kind of for detecting the homogeneous immunologic detection method of Anti-CCP, use any one of claim 49-51 institute The detection system stated and the reagent set as described in any one of claim 27-29 are examined by homogeneous immunodetection Survey the concentration of 14-3-3eta albumen comprising:

Step R1 by sample to be tested and component a2 and combines b2 and mixes, obtains third mixture；

Third mixture is mixed with component c2, obtains the 4th mixture by step R2；

Step R3 contacts energy or reactive compound with the 4th mixture, and the donor is excited to generate singlet oxygen, The receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence and/or intensity of chemiluminescence signal described in detecting step R3, thus judge survey sample to be tested in be It is no that there are the contents of 14-3-3eta albumen and/or determining 14-3-3eta albumen.

56. method according to claim 55, which is characterized in that the method also includes the production before step R1 The step of 14-3-3eta protein standard working curve.

57. method according to claim 56, which is characterized in that in step R4, the hair of chemistry described in detecting step R3 The intensity of optical signal, and determine based on 14-3-3eta protein standard working curve containing for 14-3-3eta albumen in sample to be tested Amount.

Technical field

The invention belongs to technical field of immunoassay, and in particular to one kind passes through biomarker joint inspection evaluating in vitro class wind Kit and preparation method that wet arthritis whether there is, application method.

Background technique

Biomarker (Biomarker) refers to can be with tagging system, organ, tissue, cell and subcellular structure or function The biochemical indicator of change or the change that may occur of energy, has very extensive purposes.

Rheumatoid arthritis (RA) is one kind using arthrosynovitis as main feature, and clinical manifestation is with chronic multiple joint The systemic autoimmune disease that finally can lead to joint deformity based on inflammation.Using biomarker to the course of disease of RA patient Comprehensive descision is made with characteristics such as the state of an illness, genetic background, epigenetics, the precision diagnosis and treatment of RA may be implemented, improves RA The life quality of patient.Therefore, how to improve the accuracy in detection of the RA positive of RA inflamed joints lesion patient is one urgent The technical issues of it is urgently to be resolved.

Summary of the invention

In order to solve the above technical problems, the present invention provides one kind for passing through biomarker joint inspection evaluating in vitro class wind The method that wet arthritis whether there is.This method is by joint-detection serum 14-3-3 η albumen, anti-CCP antibody and resists At least two kinds of level in Carp antibody, and it is associated with RA to will test result, can significantly improve RA inflamed joints lesion patient The RA positive accuracy in detection.

For this purpose, first aspect present invention provides a kind of utilization homogeneous immunodetection detection rheumatoid arthritis (RA) The concentration of each biomarker passes through biochemical markers evaluating in vitro rheumatoid joint in preparation in biomarker group The purposes in reagent that scorching (RA) whether there is comprising:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a) obtains each biomarker in biomarker group Combined concentration value；With

C) the combined concentration value obtained in step b) whether there is with RA it is associated, wherein with being measured from reference group Presence of the truncation combined concentration value of each marker in corresponding biomarker group compared to increased combined value instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some embodiments of the invention, by the combined concentration value of step b) and derived from addition to RA positive patient The cutoff value of reference group is compared, and the reference group is comprising obvious healthy person and selected from osteoarthritis (OA) patient and its The patient of his autoimmune disease patient.

Second aspect of the present invention provides a kind of biological using homogeneous immunodetection detection rheumatoid arthritis (RA) The concentration of each biomarker passes through biochemical markers evaluating in vitro rheumatoid arthritis in preparation in marker group (RA) purposes in the reagent of severity, comprising:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) the combined concentration value obtained in step b) is associated with the severity of RA, wherein being measured with from reference group Corresponding biomarker group in each marker truncation combined concentration value compared to RA in increased combined value instruction patient Severity；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

Third aspect present invention provides a kind of biological using homogeneous immunodetection detection rheumatoid arthritis (RA) The concentration of each biomarker distinguishes rheumatoid arthritis by biochemical markers in preparation in vitro in marker group (RA) with the preparation of other autoimmune diseases in purposes comprising:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) RA and other autoimmune diseases are distinguished from the combined concentration value obtained in the step b), wherein with from ginseng It is indicated according to the truncation combined concentration value of each marker in the corresponding biomarker group of group's measurement compared to increased combined value The presence of RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some embodiments of the invention, other autoimmune diseases include other joint diseases；It is described its Its joint disease is osteoarthritis (OA).

Fourth aspect present invention provides a kind of rheumatoid arthritis (RA) biomarker group and is used to prepare in vitro The purposes in reagent that rheumatoid arthritis (RA) whether there is in assessment sample to be tested, wherein utilizing homogeneous immunodetection The combined concentration value of biomarker each in rheumatoid arthritis (RA) biomarker group measurement is joined with for coming from The truncation combined concentration value of each marker in the corresponding biomarker group of group's measurement is examined compared to the presence for increasing instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In the present invention, the sample to be tested is selected from blood, blood derivatives, serum, blood plasma, urine, celiolymph, essence Liquid, saliva, synovial fluid, pulmonary emphysema hydrops and tissue.

In some preferred embodiments of the invention, the biomarker group includes Anti-CCP antibody, 14-3- At least two kinds of and other biomarkers object in 3eta albumen and Anti-carp antibody, preferably 2 kinds and other biomarkers object； It is preferred that the other biomarkers object is RA.

Some preferred embodiments according to the present invention, the biomarker group include Anti-CCP antibody and 14- 3-3eta albumen.

In some embodiments of the invention, the step further includes measurement 14-3-3eta albumen or its segment or described The content for the immune complex that 14-3-3eta albumen or its segment and at least one antibody are formed.

In some embodiments of the invention, it is determined in sample to be tested based on 14-3-3eta protein standard working curve The content of 14-3-3eta albumen.

According to the present invention, the step further includes by measured 14-3-3eta albumen or its segment or the 14-3- The content for the immune complex that 3eta albumen or its segment and at least one antibody are formed, with normal reference sample, rheumatoid 14-3-3eta albumen or its segment or described described in sample before arthritis control sample or treatment from same subject 14-3-3eta albumen or its segment are compared with the content for the immune complex that at least one antibody is formed.

In some embodiments of the invention, the step include by the sample with comprising can be with 14-3-3eta egg White or its segment at least one specific epitopes specifically bind the antibody to form immune complex.

In some embodiments of the invention, the antibody include can be special with the first epitope of 14-3-3eta albumen Property the first antibody that combines and can be with the secondary antibody in conjunction with the second epitope specificity of 14-3-3eta albumen, wherein institute State the second epitope and the first epitope non-overlapping.

In the present invention, for the first antibody in conjunction with receptor, it is detectable that the receptor can react generation with singlet oxygen Chemiluminescence signal.

In some embodiments of the invention, the receptor includes olefin(e) compound and metallo-chelate, is non-particle Form, and it is solvable in water-bearing media；And/or the receptor is that the macromolecule filled with luminophor and lanthanide series is micro- Grain.

In the present invention, the first antibody and secondary antibody are separately selected from monoclonal antibody and/or Anti-TNF-α Body, preferably monoclonal antibody.

In the present invention, the amino acid sequence of the 14-3-3eta albumen or its segment is as shown in SEQUENCE NO.1.

In some embodiments of the invention, the epitope is selected from the sequence that amino acid fragment is 14-3-3eta albumen Relative specificity segment: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa.

Fifth aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker passes through the reagent set that biochemical markers evaluating in vitro rheumatoid arthritis (RA) whether there is, Including for dense using each biomarker in homogeneous immunodetection detection rheumatoid arthritis (RA) biomarker group The reagent of degree, wherein the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

Sixth aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker passes through the reagent set of the severity of biochemical markers evaluating in vitro rheumatoid arthritis (RA), It includes for utilizing each biomarker in homogeneous immunodetection detection rheumatoid arthritis (RA) biomarker group The reagent of concentration, wherein the biomarker group contains Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody In it is at least two kinds of, preferably 2 kinds.

Seventh aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker distinguishes rheumatoid arthritis (RA) and other autoimmune diseases by biochemical markers in vitro Reagent set comprising for using each in homogeneous immunodetection detection rheumatoid arthritis (RA) biomarker group The reagent of biomarker concentration, wherein the biomarker group contain Anti-CCP antibody, 14-3-3eta albumen and It is at least two kinds of in Anti-carp antibody, preferably 2 kinds.

Eighth aspect present invention, which is provided, waits for test sample based on rheumatoid arthritis (RA) biomarker group evaluating in vitro The reagent set that rheumatoid arthritis (RA) whether there is in product, wherein being closed using homogeneous immunodetection for rheumatoid Save the combined concentration value and the phase for measuring from reference group of each biomarker measurement in scorching (RA) biomarker group The truncation combined concentration value of each marker is compared to the presence for increasing instruction RA in the biomarker group answered；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some preferred embodiments of the invention, the biomarker group includes Anti-CCP antibody, 14-3- It is at least two kinds of in 3eta albumen and Anti-carp antibody, preferably 2 kinds and other biomarkers object, the preferably described other biological Marker is RA.

In the present invention, the sample to be tested is selected from blood, blood derivatives, serum, blood plasma, urine, celiolymph, essence Liquid, saliva, synovial fluid, pulmonary emphysema hydrops and tissue.

Some preferred embodiments according to the present invention, the reagent set include being detected using homogeneous immunodetection The reagent of Anti-CCP antibody and 14-3-3eta protein concentration in biomarker group.

In some specific preferred embodiments of the invention, the homogeneous immune detection for detecting Anti-CCP antibody is tried Agent includes:

Component a1, it includes the first antigens that can be specifically bound with the epitope binding site of anti-CCP antibody；

Component b1, it includes anti-immunity complex antibody, the anti-immunity complex antibody being capable of specific recognition and knot Close the anti-CCP antibody in the first immune complex formed with the first antigen, nonrecognition it is free, unbonded antigen it is anti- CCP antibody.

In the present invention, first antigen or the anti-immunity complex antibody are combined with receptor, and the receptor can It is reacted with singlet oxygen and generates detectable chemiluminescence signal；Preferably, the receptor includes olefin(e) compound and metal chelating Object is closed, is non-particulate forms, and solvable in water-bearing media；And/or the receptor is filled with luminophor and group of the lanthanides The high molecular particle of element.

In some preferred embodiments of the invention, the reagent set also includes component c1, and it includes can swash The donor of hair-like state generation singlet oxygen；It is preferred that the donor is in conjunction with a member in specific binding pair member, and it is special Property in conjunction with pairing member in another member in conjunction with first antigen or the anti-immunity complex antibody；Further preferably Ground, the donor is in conjunction with Streptavidin, and correspondingly the first antigen or the anti-immunity complex antibody are in conjunction with biotin.

In some specific preferred embodiments of the invention, for detecting the homogeneous immune detection of 14-3-3eta albumen Reagent includes:

Component a2, it includes the receptor and in combination first for generating detectable signal can be reacted with singlet oxygen Antibody or its binding fragment, the first antibody or its binding fragment can be with the first epitope specificities of 14-3-3eta albumen In conjunction with；

Component b2, it includes can be with the secondary antibody or its knot in conjunction with the second epitope specificity of 14-3-3eta albumen Close segment, second epitope and the first epitope non-overlapping；

Component c2 comprising can be in the donor of excited state generation singlet oxygen.

In some embodiments of the invention, the reagent further includes the 14-3-3eta albumen sterling as calibration object, institute State calibration object be calibrated product dilution proportionally gradient dilution at various concentration working calibration product solution.

In some preferred embodiments of the invention, the secondary antibody or its binding fragment and specific binding pair A member in member combines, and another Yuan combination in the donor and specific binding pair member；It is preferred that described second is anti- Body or its binding fragment are in conjunction with biotin, and the donor is in conjunction with Streptavidin.

Ninth aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker passes through the kit that biochemical markers evaluating in vitro rheumatoid arthritis (RA) whether there is, packet Containing reagent set described in the 5th-eight aspect of the present invention.

Tenth aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker passes through the kit of the severity of biochemical markers body evaluating in vitro rheumatoid arthritis (RA), It includes reagent sets described in the 5th-eight aspect of the present invention.

Tenth one side of the invention provides a kind of for based on each life in rheumatic arthritis (RA) biomarker group The concentration of substance markers object distinguishes rheumatoid arthritis (RA) and other autoimmune diseases by biochemical markers in vitro The kit of disease, it includes reagent sets described in the 5th-eight aspect of the present invention.

The twelfth aspect of the present invention provides to be measured based on rheumatoid arthritis (RA) biomarker group evaluating in vitro The kit that rheumatoid arthritis (RA) whether there is in sample, it includes reagent sets described in the 5th-eight aspect of the present invention Dress.

The 13rd aspect of the present invention provides a kind of by biochemical markers evaluating in vitro rheumatoid arthritis (RA) method that whether there is comprising using the reagent set as described in terms of the present invention the 5th-eight or using such as the present invention Kit described in 9th-ten two aspect detects the dense of each biomarker in rheumatic arthritis (RA) biomarker group Spending and passing through biochemical markers evaluating in vitro rheumatoid arthritis (RA) whether there is.

In some embodiments of the invention, this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a) obtains each biomarker in biomarker group Combined concentration value；With

C) the combined concentration value obtained in step b) whether there is with RA it is associated, wherein with being measured from reference group Presence of the truncation combined concentration value of each marker in corresponding biomarker group compared to increased combined value instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some further embodiments of the invention, by the combined concentration value of step b) and derived from except RA positive trouble The cutoff value of reference group except person is compared, and the reference group includes obvious healthy person and is selected from osteoarthritis (OA) The patient of patient and other autoimmune disease patients.

Fourteenth aspect of the present invention provides a kind of by assessing rheumatoid arthritis (RA) outside biochemical markers Severity method comprising using the reagent set as described in the 5th-eight aspect of the present invention or use such as of the invention the Kit described in nine-ten two aspects detects the concentration of each biomarker in rheumatic arthritis (RA) biomarker group And the severity by assessing rheumatoid arthritis (RA) outside biochemical markers.

In some embodiments of the invention, this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) the combined concentration value obtained in step b) is associated with the severity of RA, wherein being measured with from reference group Corresponding biomarker group in each marker truncation combined concentration value compared to RA in increased combined value instruction patient Severity；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of.

The fifteenth aspect of the present invention provides one kind and distinguishes rheumatoid arthritis in vitro by biochemical markers (RA) with the method for other autoimmune diseases comprising using as described in terms of the of the invention 5th-eight reagent set or Each life in rheumatic arthritis (RA) biomarker group is detected using the kit as described in terms of the present invention the 9th-ten two The concentration of substance markers object simultaneously distinguishes rheumatoid arthritis (RA) and other autoimmune by biochemical markers in vitro Disease.

In some embodiments of the invention, this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) RA and other autoimmune diseases are distinguished from the combined concentration value obtained in the step b), wherein with from ginseng It is indicated according to the truncation combined concentration value of each marker in the corresponding biomarker group of group's measurement compared to increased combined value The presence of RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of.

In some further embodiments of the invention, other autoimmune diseases include other joint diseases Disease；Other joint diseases are osteoarthritis (OA).

The 16th aspect of the present invention provides a kind of reagent set using as described in terms of the of the invention 5th-eight or use Kit as described in terms of the present invention the 9th-ten two is used to assess rheumatoid arthritis (RA) in sample to be tested in vitro No existing method, wherein dense for the combination of biomarker each in rheumatoid arthritis (RA) biomarker group measurement Angle value is compared with for the truncation combined concentration value of each marker in the corresponding biomarker group that reference group measures Increase the presence of instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of.

In the present invention, the sample to be tested is selected from blood, blood derivatives, serum, blood plasma, urine, celiolymph, essence Liquid, saliva, synovial fluid, pulmonary emphysema hydrops and tissue.

In some preferred embodiments of the invention, the biomarker group includes Anti-CCP antibody, 14-3- At least two kinds of and other biomarkers object in 3eta albumen and Anti-carp antibody, preferably 2 kinds and other biomarkers object； It is preferred that the other biomarkers object is RF.

According to method described in the 13rd to 16 aspect, Anti-CCP antibody and 14-3-3eta egg are detected using containing White homogeneous immunological detection reagent suit detects the concentration of Anti-CCP by homogeneous immune detection comprising:

Sample to be tested is mixed with component a1, obtains the first mixture by step R1；

First mixture is mixed with component b1, obtains the second mixture by step R2；

Step R3 mixes the second mixture with component c1, and the third for obtaining producing detectable chemiluminescence signal is mixed Close object；

Step R4, the intensity of chemiluminescence signal described in detecting step R3, so that it is determined that the content of Anti-CCP antibody.

In some preferred embodiments of the invention, the method also includes the production Anti-CCP before step R1 The step of antibody standard working curve.

In some further preferred embodiments of the invention, in step R4, the hair of chemistry described in detecting step R3 The intensity of optical signal, and determine based on Anti-CCP antibody standard working curve containing for Anti-CCP antibody in sample to be tested Amount.

According to method described in the 13rd to 16 aspect, Anti-CCP antibody and 14-3-3eta egg are detected using containing White homogeneous immunological detection reagent suit detects the concentration of 14-3-3eta albumen by homogeneous immunodetection comprising:

Step R1 by sample to be tested and component a2 and combines b2 and mixes, obtains third mixture；

Third mixture is mixed with component c2, obtains the 4th mixture by step R2；

Step R3 contacts energy or reactive compound with the 4th mixture, and the donor is excited to generate single line State oxygen, the receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence and/or intensity of chemiluminescence signal described in detecting step R3, to judge to survey sample to be tested In with the presence or absence of 14-3-3eta albumen and/or determine 14-3-3eta albumen content.

In some preferred embodiments of the invention, the method also includes the production 14-3- before step R1 The step of 3eta protein standard working curve.

In some further preferred embodiments of the invention, in step R4, the hair of chemistry described in detecting step R3 The intensity of optical signal, and determine based on 14-3-3eta protein standard working curve containing for 14-3-3eta albumen in sample to be tested Amount.

The 17th aspect of the present invention provides a kind of each in rheumatoid arthritis (RA) biomarker group for detecting The chemiluminescence immune detection system of biomarker comprising using reagent set as described in respect of the second aspect of the invention or make Method with kit as described in the third aspect of the present invention or as described in the method as described in terms of the present invention the 13 to the 16th To detect in sample to be tested with the presence or absence of each biomarker and/or determination in rheumatoid arthritis (RA) biomarker group The content of each biomarker in rheumatoid arthritis (RA) biomarker group.

According to certain embodiments of the present invention, the system comprises:

Reaction unit is used for reagent set or third aspect present invention described in sample to be tested and second aspect of the present invention Reagent in the kit chemically reacts；

Excitation and reading plotter generate active oxygen, receptor using the excitation donor microballoon of 600-700nm wavelength Microballoon generates the transmitting light of 520-620nm with the reactive oxygen species received, records the optical signal of above-mentioned transmitting light；

Processor surveys sample to be tested according to the presence of the optical signal of the transmitting light recorded and/or intensity judgement In with the presence or absence of object to be measured molecule and/or determine object to be measured molecule content.

In some embodiments of the invention, the processor is fitted using cubic spline interpolation fitting, directly to Out in sample to be tested object to be measured molecule concentration value.

The 18th aspect of the present invention provides a kind of for detecting the homogeneous immunologic detection method of Anti-CCP, uses this It invents detection system described in the 17th aspect and passes through as described in the present invention for detecting the reagent set of Anti-CCP Homogeneous immune detection detects the concentration of Anti-CCP comprising:

Sample to be tested is mixed with component a1, obtains the first mixture by step R1；

First mixture is mixed with component b1, obtains the second mixture by step R2；

Step R3 mixes the second mixture with component c1, and the third for obtaining producing detectable chemiluminescence signal is mixed Close object；

Step R4, the intensity of chemiluminescence signal described in detecting step R3, so that it is determined that the content of Anti-CCP antibody.

In some embodiments of the invention, the method also includes the production Anti-CCP antibody marks before step R1 The step of quasi- working curve.

In some further embodiments of the invention, in step R4, the letter of chemiluminescence described in detecting step R3 Number intensity, and determine based on Anti-CCP antibody standard working curve the content of Anti-CCP antibody in sample to be tested.

The 19th aspect of the present invention provides a kind of for detecting the homogeneous immunologic detection method of Anti-CCP, uses it Using detection system described in the 17th aspect of the present invention and as described in the present invention for detecting the reagent of 14-3-3eta albumen It is set with to detect the concentration of 14-3-3eta albumen by homogeneous immunodetection comprising:

Step R1 by sample to be tested and component a2 and combines b2 and mixes, obtains third mixture；

Third mixture is mixed with component c2, obtains the 4th mixture by step R2；

Step R3 contacts energy or reactive compound with the 4th mixture, and the donor is excited to generate single line State oxygen, the receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence and/or intensity of chemiluminescence signal described in detecting step R3, to judge to survey sample to be tested In with the presence or absence of 14-3-3eta albumen and/or determine 14-3-3eta albumen content.

In some embodiments of the invention, the method also includes the production 14-3-3eta albumen before step R1 The step of standard working curve.

In some further embodiments of the invention, in step R4, the letter of chemiluminescence described in detecting step R3 Number intensity, and determine based on 14-3-3eta protein standard working curve the content of 14-3-3eta albumen in sample to be tested.

It is provided by the present invention to whether there is by biochemical markers evaluating in vitro rheumatoid arthritis (RA) Method passes through level at least two kinds of in joint-detection serum 14-3-3 η albumen, anti-CCP antibody and anti-Carp antibody, and will Testing result is associated with RA, can significantly improve the accuracy in detection of the RA positive of RA inflamed joints lesion patient.

Specific embodiment

To be readily appreciated that the present invention, the present invention is described more detail below.But before describing the present invention in detail, it should be understood that The present invention is not limited to the specific embodiments of description.It is also understood that term used herein is only for description specific implementation Mode, and be not offered as restrictive.

In the case where providing numberical range, it should be understood that in the upper and lower bound of the range and the prescribed limit Any other regulation or between two parties each of between numerical value numerical value is encompassed by the present invention between two parties.These small range of upper limits It can independently be included in lesser range with lower limit, and be also covered by the present invention, obey any clear in prescribed limit The limit of exclusion.Defined range include one or two limit in the case where, exclude any of the limit that those include or The range of the two is also included in the present invention.

Unless otherwise defined, all terms used herein and those skilled in the art's is usual Understand meaning having the same.Although similar or equivalent any method and material can also with method described herein and material To use in implementation or test of the invention, but preferred method and material will now be described.

I, term

" main body to be measured ", " subject " and " patient " are used interchangeably, in the case where being not particularly illustrated or limiting, Refer to mammal, such as people and non-human primates and rabbit, rat, mouse, goat, pig and other food in one's mouth animal species.

English corresponding to term " homogeneous " of the present invention is defined as " homogeneous ", and referring to need not be to combination Antigen antibody complex and remaining free antigen or antibody separated and can both be detected.

Term " sample to be tested " of the present invention, which refers to, to include containing a kind of mixture of analyte, analyte But it is not limited to protein, hormone, antibody or antigen.The typical sample to be tested that can be used in method disclosed by the invention includes Body fluid and tissue, such as blood, blood derivatives, serum, blood plasma, urine, celiolymph, sperm, saliva, synovial fluid, pulmonary emphysema Hydrops and tissue etc..

Heretofore described term " citrulling peptide " refers to the specific antigen that can have positive reaction with RA serum: silk egg collection White tiles section, preceding filaggrin precursor, synthesis polypeptide and recombinant polypeptide, coupling have peptide fragment after the various modifications such as polypeptide of marker, Its main feature is that containing citrulling, citrulline residue is the substrate essential component of antiCCP antibody identification.

Term " citrulling epitope " of the present invention refers to that antigenic surface can be tied by cyclic citrullinated peptid specificity The region of conjunction, including citrulline residue and its locating surrounding's amino acid sequence.

Term " the epitope recognition site of cyclic citrullinated peptid " of the present invention is also known as " identification epitope Site " refer to identification possessed by cyclic citrullinated peptid and combine " citrulling epitope " region, for example, anti-cyclic citrulline The first recognition site of epitope of peptide antibody and epitope the second recognition site non-overlapping of cyclic citrullinated peptid, That is the two belongs to the epitope recognition site of the different location with identical combination characteristic.

Term " the citrulling peptide fragment mixture " of the present invention refers to is mixed by the single peptide fragment containing citrulling of at least two The mixture formed after conjunction is also possible to wherein the peptide fragment containing citrulling can be the ring-like peptide fragment containing citrulling containing melon The line style peptide fragment of propylhomoserin.

Term " 14-3-3 " of the present invention and " 14-3-3 albumen " are used interchangeably, and refer to the universal table in eukaryocyte What is reached guards at least one member of the 14-3-3 family intracellular for adjusting molecule.14-3-3 albumen, which has, combines many Various Functions Signal transducer, the ability including kinases, phosphatase and transmembrane receptor.In fact, more than 100 kinds of signal transducers It is reported as the ligand of 14-3-3.14-3-3 albumen can be considered as being evolved into for Tetratrico peptide repeated fragment superfamily Member.They usually have 9 or 10 α spirals, generally along its amino terminal spiralization homodimer and/or heterodimer phase Interaction.These albumen include multiple known structure domains, and the structural domain includes for bivalent cation interaction, phosphoric acid Change the region of acetylation and proteolytic cleavage and other.The known 14- for expressing seven kinds of different genetic codings in mammals 3-3 protein isoform, every kind of isotype include 242-255 amino acid.Seven kinds of 14-3-3 protein isoforms are named as 14-3-3 α/β(alpha/beta)、14-3-3δ/ξ(delta/zeta)、14-3-3ε(epsilon)、14-3-3γ(gamma)、14-3-3 η (eta), 14-3-3 τ/θ (tau/theta) and 14-3-3 σ (sigma/stratifin).14-3-3 albumen has high level Sequence similarity, it is known that experience translation post-processing such as, phosphorylation, it is citrullinated, etc..Referring to such as, Megidish etc. (1998) J.Biol.Chem.273:21834-45.Therefore, anti-14-3-3 autoantibody can specifically bind and/or identify and is more than one 14-3-3 protein isoform, or can specifically bind and/or identify a kind of only isotype (e.g., 14-3-3 η).In addition, anti-14-3- 3 antibody are combinable and/or identification is by such as, the 14-3-3- albumen of natural (e.g., after translation) or chemical method modification.

Heretofore described term " relative specificity segment " refers to 7 isotype 14-3-3 eggs for 14-3-3 family It is white, through research, the inventor has found that 14-3-3eta albumen or the amino acid sequence of its segment as shown in SEQUENCE NO.1 Middle segment 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa are the spy for only belonging to 14-3-3 η (eta) albumen Anisotropic epitope is intersected with the amino acid sequence of other 6 isotype 14-3-3 albumen of 14-3-3 family without any, by its production Raw monoclonal antibody, only identifies or combines 14-3-3 η (eta) albumen, nonrecognition or combine other 6 of 14-3-3 family it is same Kind type 14-3-3 albumen.

Heretofore described " arthritis " is used interchangeably with " arthritis illness " and " arthralgia ", in addition to what is indicated sentences Outside, the inflammatory disease of human synovial is typically referred to.Pain, swelling, it is stiff and be difficult to movement it is usually related with arthritis illness. Arthritis more than 100 kinds of different situations by forming.These are damaged it may is that any situation from relatively slight form to serious Harmful system form.Arthritis illness can be caused by any reason of many reasons, including infection, wound, degenerative disease, generation Thank disorder or interference or other unknown etiologies.Arthritis illness can be more specifically described according to hypotype, for example, rheumatoid joint Inflammation, mixed connective tissue disease (MCTD), crystal-induced arthritis, adjuvant arthritis, spondyloarthropathy, osteoarthritis, class meat Tumor disease, palindromic rheumatism, post-traumatic arthritis, the relevant arthritis of malignant tumour, septic arthritis, Lyme arthritis, Osteoarthritis, bacterial infectivity arthritis, etc..Arthritis can also be with the disease of other identifications, including gout, tatanic ridge Column inflammation, systemic loupus erythematosus, inflammatory bowel disease, psoriasis, etc..Clearly defined arthritis illness, which refers to, to be known about pass The scorching type of section and its stage, e.g., break out, alleviate, recurring, etc..

Term " antibody " of the present invention and " immunoglobulin " are used with most wide meaning, the antibody including any isotype Or immunoglobulin, retain the antibody fragment of the specific binding to antigen；Including but not limited to Fab, Fv, scFv, Fd segment, Chimeric antibody, humanized antibody, single-chain antibody, bispecific antibody, and antigen-binding portion thereof and non-antibody comprising antibody The fusion protein of albumen.In the case where any need, antibody can further with other parts, such as specific binding pair Member, such as biotin or Streptavidin (a member in biotin-Streptavidin specific binding pair member) etc. are sewed It closes.

Term " monoclonal antibody " of the present invention refers to the immunoglobulin secreted by the bone-marrow-derived lymphocyte of monoclonal, It can be prepared by method known in those skilled in the art.

Term " polyclonal antibody " of the present invention refers to the immune globulin generated by more than one bone-marrow-derived lymphocyte clone White set can be prepared by method known in those skilled in the art.

Term " antigen " of the present invention is to refer to stimulation body to generate immune response, and can resist with immune response product Body and sensitized lymphocyte combine in vivo and in vitro, and the substance of immunological effect occurs.

Term " in conjunction with " of the present invention refer to due to for example covalently, the interaction such as electrostatic, hydrophobic, ion and/or hydrogen bond, Including but not limited to two intermolecular direct joints caused by such as salt bridge and the interaction of water bridge.

Term " specific binding " of the present invention or " specific bond " refer to mutual discrimination and choosing between two kinds of substances Selecting property association reaction, said from stereochemical structure angle be exactly conformation between corresponding reactant correspondence.

Term " specific binding pair member " of the present invention refers to molecule a pair of of in this way, they can be mutually specific In conjunction with for example, enzyme-substrate, Ag-Ab, ligand-receptor.The example of one specific specific binding pair member couple is Biotin-Streptavidin system, wherein " biotin " is widely present in animal vegetable tissue, there are two cyclic annular knots on molecule Structure, respectively imidazolone ring and thiphene ring, wherein imidazolone ring is the main portions in conjunction with Streptavidin.The biology of activation Element can under the mediation of protein cross agent, with known almost all creatures macromolecular be coupled, including protein, nucleic acid, Polysaccharide and lipid etc.；And " Streptavidin " is a kind of protein secreted by streptomycete, molecular weight 65kD." strepto- is affine Molecule is made of element " 4 identical peptide chains, wherein every peptide chain can combine a biotin.Therefore each antigen or antibody It can be coupled multiple biotin molecules simultaneously, so that generating " tentacle effect " improves sensitivity for analysis.In the case where any need, Any reagent used in the present invention, including antigen, antibody, receptor or donor, can biotin-conjugated-strepto- according to actual needs Any member in Avidin specific binding pair member.

Term " donor " of the present invention can generate after referring to the activation by energy or reactive compound and receptor The sensitizer of the reactive intermediate of such as singlet oxygen of reaction.Donor can be (such as dyestuff and aromatic compound) of photoactivation Or (such as enzyme, metal salt) of chemical activation.In particular embodiments of the invention, the donor is photosensitizer, described Photosensitizer can be photosensitizer known in the art, preferably stable with respect to the light and not compound with singlet oxygen effecting reaction, Its non-limiting example includes Asia disclosed in such as United States Patent (USP) US5709994 (patent document is hereby incorporated by reference) The compounds such as methyl blue, rose-red, porphyrin, phthalocyanine and chlorophyll and these compounds have 1-50 replacing group Derivative, the substituent group is used for so that these compounds are with more lipophilicity or with more hydrophily, and/or as connection To the linking group of specific binding pair member.The example of other photosensitizers well known by persons skilled in the art can also be at this It is used in invention, such as the content recorded in United States Patent (USP) US6406913, which is hereby expressly incorporated by reference.At this It invents in other specific embodiments, the donor is other sensitizers of chemical activation, and non-limiting example is certain Compound, their catalyzing hydrogen peroxides are converted into singlet oxygen and water.The example of some other donor includes: 1,4- dicarboxyl second Base-Isosorbide-5-Nitrae-naphthalene endoperoxides object, 9,10- diphenylanthrancene -9,10- endoperoxides object etc. heat these compounds or these changes Conjunction object, which directly absorbs light, can discharge singlet oxygen.

Term " receptor " of the present invention is to refer to react the compound that can produce detectable signal with singlet oxygen. For donor by energy or reactive compound induced activation and the singlet oxygen for discharging upper state, the singlet oxygen of the upper state is close The receptor of distance is captured, to transmit energy to activate the receptor.In some embodiments of the invention, the receptor It is such substance, the chemical reaction of experience and singlet oxygen is to form unstable metastable state intermediate, the metastable state Intermediate can decompose, and subsequently or simultaneously shine.The typical example of these substances includes but is not limited to: enol ether, enamine, 9- alkane Pitch xanthan gum, 9- alkylidene-N- alkyl Acridane, aromatic ethylene ether, bicyclic ethylene oxide, thioxene, armaticity imidazoles or gloss Essence.In other specific embodiments of the invention, the receptor can be reacted with singlet oxygen to be formed and can be resolved into The olefines of the hydroperoxides or dioxy cyclobutane of ketone or carboxylic acid derivates；Two can be stablized by what the effect of light was decomposed Oxygen cyclobutane；It can be reacted with singlet oxygen to form the acetylene class of diones；Azo-compound or azo carbonyl can be formed The hydrazone class or hydrazides of compound, such as luminol；With the aromatic compounds that can form endoperoxides species.It can be according to this Specific, the non-limiting example for the receptor that the invention disclosed and claimed utilizes are recorded in U.S. Patent number US5340716 (patent document is hereby incorporated by reference).In other specific embodiments of the invention, the receptor includes olefinic compound The case where object and metallo-chelate are non-particlizeds and solvable in water-bearing media, this receptor can be found in patent PCT/US2010/025433 (patent document is hereby incorporated by reference).

" donor " can be to be coated on matrix by functional group and be formed filled with sensitized in the present invention The high molecular particle for closing object can generate singlet oxygen under light excitation；And/or " receptor " can be through function base Group is coated on the high molecular particle for being formed on matrix and being filled with luminophor and lanthanide series.

" matrix " of the present invention is microballoon or particle known in those skilled in the art, can be any size , it can be organic or inorganic, can be inflatable or nondistensible, can be porous or non-porous , with any density, but preferably have and the close density of water, can preferably float in water, and by transparent, partially transparent Or opaque material is constituted.Described matrix can be with or without charge, when having charge, preferably negative electrical charge.The base It is (such as hydrocarbon that body can be solid (such as polymer, metal, glass, organic and inorganic matter such as mineral, salt and diatom), small oil droplet Compound, fluorocarbon, silicic fluid), vesica is (such as such as phosphatide or natural such as cell and organelle of synthesis Official).Matrix can be latex particle or other particles, lipid bilayer such as liposome, phosphatide containing organic or inorganic polymer Vesica, small oil droplet, silicon particle, metal-sol, cell and crystallite dyestuff.Matrix usually has multifunctionality, or can pass through Special or non-specific covalently or non-covalently interaction and be integrated on donor or receptor.Be available there are many functional group or Person is merged in.Typical functional group includes carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, sulfydryl etc..It is suitable for One unrestricted example of matrix of the invention is carboxy-modified latex particle.The details of this matrix can be found in United States Patent (USP) US5709994 and US5780646 (this two patents document is hereby incorporated by reference).

Term " epitope " of the present invention is any egg for referring to specific binding immunoglobulin or T cell receptor White determinant.In some embodiments of the invention, epitope is that antigenic surface can be by the region of antibody specificity set. Epitopic determinants usually may include the chemically active surface group of molecule, such as, but not limited to: amino acid, carbohydrate side chain, phosphinylidyne Base and/or sulfonyl.In some other specific embodiment of the invention, epitope can specific specific three-valued structures feature and Specific charge feature.

Term " homogeneous immunological detection reagent suit " of the present invention refers to the whole that homogeneous immune detection must use The combination of reagent or medicament.

Heretofore described " concentration of each biomarker in detection rheumatoid arthritis (RA) biomarker group " Refer to " biomarker joint inspection ".

II, embodiment

As previously described, because RA performance multiplicity, part atypical early stage and (or) serum show negative patient and are often missed It examines, fail to pinpoint a disease in diagnosis.In order to improve the existing Diagnostic Strategy of RA, diagnostic level is improved, the present inventor has carried out largely Ra diagnostic method Research.

The present inventor is the study found that 14-3-3 η albumen significantly increases in RA serum and knuckle synovia, and can raise a variety of The expression of inflammatory factor relevant to RA prompts it that may participate in the generation of RA disease.The present inventor further passes through detection RA, non- In RA inflamed joints lesion patient and same period physical examination of healthy population serum 14-3-3 η albumen, anti-CCP antibody and anti-Carp antibody At least two kinds of levels, and analyze it and compare, and is associated with RA, discovery joint-detection serum 14-3-3 η albumen, Anti-CCP antibody and at least two kinds of level of anti-Carp antibody, and it is associated with RA to will test result, can significantly improve RA inflammation The accuracy in detection of the RA positive of property arthropathy patient.The present invention is based on what above-mentioned discovery was made.

Therefore, first aspect present invention is related to each biology in a kind of detection rheumatoid arthritis (RA) biomarker group The reagent that the concentration of marker whether there is in preparation by biochemical markers evaluating in vitro rheumatoid arthritis (RA) In purposes comprising:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a) obtains each biomarker in biomarker group Combined concentration value；With

C) the combined concentration value obtained in step b) whether there is with RA it is associated, wherein with being measured from reference group Presence of the truncation combined concentration value of each marker in corresponding biomarker group compared to increased combined value instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some embodiments of the invention, by the combined concentration value of step b) and derived from addition to RA positive patient The cutoff value of reference group is compared, and the reference group is comprising obvious healthy person and selected from osteoarthritis (OA) patient and its The patient of his autoimmune disease patient.

Second aspect of the present invention is related to each biomarker in a kind of detection rheumatoid arthritis (RA) biomarker group The concentration of object is in the reagent that preparation passes through the severity for assessing rheumatoid arthritis (RA) outside biochemical markers Purposes, comprising:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) the combined concentration value obtained in step b) is associated with the severity of RA, wherein being measured with from reference group Corresponding biomarker group in each marker truncation combined concentration value compared to RA in increased combined value instruction patient Severity；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

Third aspect present invention is related to each biomarker in a kind of detection rheumatoid arthritis (RA) biomarker group The concentration of object distinguishes rheumatoid arthritis (RA) and other autoimmune diseases by biochemical markers in preparation in vitro Purposes in the preparation of disease comprising:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) RA and other autoimmune diseases are distinguished from the combined concentration value obtained in the step b), wherein with from ginseng It is indicated according to the truncation combined concentration value of each marker in the corresponding biomarker group of group's measurement compared to increased combined value The presence of RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some embodiments of the invention, other autoimmune diseases include other joint diseases；It is described its Its joint disease is osteoarthritis (OA).

Fourth aspect present invention is related to a kind of rheumatoid arthritis (RA) biomarker group and is used to prepare to comment in vitro The purposes in the reagent that rheumatoid arthritis in sample to be tested (RA) whether there is is estimated, wherein for rheumatoid arthritis (RA) the combined concentration value of each biomarker measurement is corresponding with for measuring from reference group in biomarker group The truncation combined concentration value of each marker is compared to the presence for increasing instruction RA in biomarker group；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In the present invention, the sample to be tested is selected from blood, blood derivatives, serum, blood plasma, urine, celiolymph, essence Liquid, saliva, synovial fluid, pulmonary emphysema hydrops and tissue, the preferably described sample to be tested are selected from blood, blood plasma, serum, synovial fluid and group It knits, the further preferred sample to be tested is selected from blood, blood plasma and serum, and the still more preferably described sample to be tested is serum.

In the present invention, Anti-CCP antibody is captured by one or more CCP as antigen；And/or anti-Anti-carp Antibody is captured by one or more carp as antigen；And/or the 14-3-3eta albumen is by one or more 14-3- The antibody capture of 3eta albumen.

In some preferred embodiments of the invention, the biomarker group includes Anti-CCP antibody, 14-3- At least two kinds of and other biomarkers object in 3eta albumen and Anti-carp antibody, preferably 2 kinds and other biomarkers object.

In some specific preferred embodiments of the invention, the other biomarkers object is RA.

In some specific embodiments of the invention, the concentration of each biomarker is using homogeneous in biomarker group Immunodetection is detected.

Some preferred embodiments according to the present invention, the biomarker group include Anti-CCP antibody and 14- 3-3eta albumen.

In some embodiments of the invention, the step further includes measurement 14-3-3eta albumen or its segment or described The content for the immune complex that 14-3-3eta albumen or its segment and at least one antibody are formed.

In some embodiments of the invention, it is determined in sample to be tested based on 14-3-3eta protein standard working curve The content of 14-3-3eta albumen.

According to the present invention, the step further includes by measured 14-3-3eta albumen or its segment or the 14-3- The content for the immune complex that 3eta albumen or its segment and at least one antibody are formed, with normal reference sample, rheumatoid 14-3-3eta albumen or its segment or described described in sample before arthritis control sample or treatment from same subject 14-3-3eta albumen or its segment are compared with the content for the immune complex that at least one antibody is formed.

In some embodiments of the invention, the step include by the sample with comprising can be with 14-3-3eta egg White or its segment at least one specific epitopes specifically bind the antibody to form immune complex.

In some embodiments of the invention, the antibody include can be special with the first epitope of 14-3-3eta albumen Property the first antibody that combines and can be with the secondary antibody in conjunction with the second epitope specificity of 14-3-3eta albumen, wherein institute State the second epitope and the first epitope non-overlapping.

In the present invention, for the first antibody in conjunction with receptor, it is detectable that the receptor can react generation with singlet oxygen Chemiluminescence signal.

In some embodiments of the invention, the receptor includes olefin(e) compound and metallo-chelate, is non-particle Form, and it is solvable in water-bearing media；And/or the receptor is that the macromolecule filled with luminophor and lanthanide series is micro- Grain.

In the present invention, the first antibody and secondary antibody are separately selected from monoclonal antibody and/or Anti-TNF-α Body, preferably monoclonal antibody.

In the present invention, the amino acid sequence of the 14-3-3eta albumen or its segment is as shown in SEQUENCE NO.1.

In some embodiments of the invention, the epitope is selected from the sequence that amino acid fragment is 14-3-3eta albumen Relative specificity segment: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa.

Hereinafter the 5th to the 19th aspect further provides realization specific embodiments of the present invention.

Fifth aspect present invention is related to a kind of for based on biology mark each in rheumatic arthritis (RA) biomarker group The concentration of note object passes through the reagent set that biochemical markers evaluating in vitro rheumatoid arthritis (RA) whether there is, packet Include the reagent for detecting each biomarker concentration in rheumatoid arthritis (RA) biomarker group, wherein the life Substance markers object group contain it is at least two kinds of in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody, preferably 2 kinds.

Sixth aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker passes through the reagent set of the severity of biochemical markers evaluating in vitro rheumatoid arthritis (RA), It includes for utilizing each biomarker in homogeneous immunodetection detection rheumatoid arthritis (RA) biomarker group The reagent of concentration, wherein the biomarker group contains Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody In it is at least two kinds of, preferably 2 kinds.

Seventh aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker distinguishes rheumatoid arthritis (RA) and other autoimmune diseases by biochemical markers in vitro Reagent set comprising for using each in homogeneous immunodetection detection rheumatoid arthritis (RA) biomarker group The reagent of biomarker concentration, wherein the biomarker group contain Anti-CCP antibody, 14-3-3eta albumen and It is at least two kinds of in Anti-carp antibody, preferably 2 kinds.

Eighth aspect present invention, which is provided, waits for test sample based on rheumatoid arthritis (RA) biomarker group evaluating in vitro The reagent set that rheumatoid arthritis (RA) whether there is in product, wherein being closed using homogeneous immunodetection for rheumatoid Save the combined concentration value and the phase for measuring from reference group of each biomarker measurement in scorching (RA) biomarker group The truncation combined concentration value of each marker is compared to the presence for increasing instruction RA in the biomarker group answered；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In the present invention, Anti-CCP antibody is captured by one or more CCP as antigen；And/or anti-Anti-carp Antibody is captured by one or more carp as antigen；And/or the 14-3-3eta albumen is by one or more 14-3- The antibody capture of 3eta albumen.

In some preferred embodiments of the invention, the biomarker group includes Anti-CCP antibody, 14-3- It is at least two kinds of in 3eta albumen and Anti-carp antibody, preferably 2 kinds and other biomarkers object.

In some specific preferred embodiments of the invention, the other biomarkers object is RA.

In the present invention, the sample to be tested is selected from blood, blood derivatives, serum, blood plasma, urine, celiolymph, essence Liquid, saliva, synovial fluid, pulmonary emphysema hydrops and tissue, the preferably described sample to be tested are selected from blood, blood plasma, serum, synovial fluid and group It knits, the further preferred sample to be tested is selected from blood, blood plasma and serum, and the still more preferably described sample to be tested is serum.

Ninth aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker passes through the kit that biochemical markers evaluating in vitro rheumatoid arthritis (RA) whether there is, packet Containing reagent set described in the 5th-eight aspect of the present invention.

Tenth aspect present invention provides a kind of for based on each biology in rheumatic arthritis (RA) biomarker group The concentration of marker passes through the kit of the severity of biochemical markers body evaluating in vitro rheumatoid arthritis (RA), It includes reagent sets described in the 5th-eight aspect of the present invention.

Tenth one side of the invention provides a kind of for based on each life in rheumatic arthritis (RA) biomarker group The concentration of substance markers object distinguishes rheumatoid arthritis (RA) and other autoimmune diseases by biochemical markers in vitro The kit of disease, it includes reagent sets described in the 5th-eight aspect of the present invention.

The twelfth aspect of the present invention provides to be measured based on rheumatoid arthritis (RA) biomarker group evaluating in vitro The kit that rheumatoid arthritis (RA) whether there is in sample, it includes reagent sets described in the 5th-eight aspect of the present invention Dress.

The 13rd aspect of the present invention provides a kind of by biochemical markers evaluating in vitro rheumatoid arthritis (RA) method that whether there is comprising using the reagent set as described in terms of the present invention the 5th-eight or using such as the present invention Kit described in 9th-ten two aspect detects the dense of each biomarker in rheumatic arthritis (RA) biomarker group Spending and passing through biochemical markers evaluating in vitro rheumatoid arthritis (RA) whether there is.

In some embodiments of the invention, this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a) obtains each biomarker in biomarker group Combined concentration value；With

C) the combined concentration value obtained in step b) whether there is with RA it is associated, wherein with being measured from reference group Presence of the truncation combined concentration value of each marker in corresponding biomarker group compared to increased combined value instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some further embodiments of the invention, by the combined concentration value of step b) and derived from except RA positive trouble The cutoff value of reference group except person is compared, and the reference group includes obvious healthy person and is selected from osteoarthritis (OA) The patient of patient and other autoimmune disease patients.

Fourteenth aspect of the present invention provides a kind of by assessing rheumatoid arthritis (RA) outside biochemical markers Severity method comprising using the reagent set as described in the 5th-eight aspect of the present invention or use such as of the invention the Kit described in nine-ten two aspects detects the concentration of each biomarker in rheumatic arthritis (RA) biomarker group And the severity by assessing rheumatoid arthritis (RA) outside biochemical markers.

In some embodiments of the invention, this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) the combined concentration value obtained in step b) is associated with the severity of RA, wherein being measured with from reference group Corresponding biomarker group in each marker truncation combined concentration value compared to RA in increased combined value instruction patient Severity；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

The fifteenth aspect of the present invention provides one kind and distinguishes rheumatoid arthritis in vitro by biochemical markers (RA) with the method for other autoimmune diseases comprising using as described in terms of the of the invention 5th-eight reagent set or Each life in rheumatic arthritis (RA) biomarker group is detected using the kit as described in terms of the present invention the 9th-ten two The concentration of substance markers object simultaneously distinguishes rheumatoid arthritis (RA) and other autoimmune by biochemical markers in vitro Disease.

In some embodiments of the invention, this method comprises:

A) concentration of each biomarker in biomarker group is detected in sample to be tested respectively；

B) concentration value of each biomarker measured by combination a), obtains the combined concentration value of each biomarker；With

C) RA and other autoimmune diseases are distinguished from the combined concentration value obtained in the step b), wherein with from ginseng It is indicated according to the truncation combined concentration value of each marker in the corresponding biomarker group of group's measurement compared to increased combined value The presence of RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some further embodiments of the invention, other autoimmune diseases include other joint diseases Disease；Other joint diseases are osteoarthritis (OA).

The 16th aspect of the present invention provides a kind of reagent set using as described in terms of the of the invention 5th-eight or use Kit as described in terms of the present invention the 9th-ten two is used to assess rheumatoid arthritis (RA) in sample to be tested in vitro No existing method, wherein dense for the combination of biomarker each in rheumatoid arthritis (RA) biomarker group measurement Angle value is compared with for the truncation combined concentration value of each marker in the corresponding biomarker group that reference group measures Increase the presence of instruction RA；

Wherein, the biomarker group contains in Anti-CCP antibody, 14-3-3eta albumen and Anti-carp antibody It is at least two kinds of, preferably 2 kinds.

In some preferred embodiments of the invention, the biomarker group includes Anti-CCP antibody, 14-3- At least two kinds of and other biomarkers object in 3eta albumen and Anti-carp antibody, preferably 2 kinds and other biomarkers object.

In some specific preferred embodiments of the invention, the other biomarkers object is RA.

In the present invention, the sample to be tested is selected from blood, blood derivatives, serum, blood plasma, urine, celiolymph, essence Liquid, saliva, synovial fluid, pulmonary emphysema hydrops and tissue, the preferably described sample to be tested are selected from blood, blood plasma, serum, synovial fluid and group It knits, the further preferred sample to be tested is selected from blood, blood plasma and serum, and the still more preferably described sample to be tested is serum.

Some preferred embodiment modes according to the present invention, the reagent set include being examined using homogeneous immunodetection Survey the reagent of Anti-CCP antibody and 14-3-3eta protein concentration in biomarker group.

In some specific preferred embodiments of the invention, for detecting the homogeneous immune inspection of cyclic citrullinated peptid Test agent includes:

Component a1, it includes the first antigens that can be specifically bound with the epitope binding site of anti-CCP antibody；

Component b1, it includes anti-immunity complex antibody, the anti-immunity complex antibody being capable of specific recognition and knot Close the anti-CCP antibody in the first immune complex formed with the first antigen, nonrecognition it is free, unbonded antigen it is anti- CCP antibody.

In some embodiments of the invention, first antigen or the anti-immunity complex antibody are mutually tied with receptor It closes, the receptor can be reacted with singlet oxygen generates detectable chemiluminescence signal.

In other embodiments of the invention, it is non-grain that the receptor, which includes olefin(e) compound and metallo-chelate, Sub- form, and it is solvable in water-bearing media；And/or the receptor is the macromolecule filled with luminophor and lanthanide series Particle.

According to the present invention, the reagent set also includes component c1, and it includes can generate singlet oxygen in excited state Donor；It is preferred that the donor is in conjunction with a member in specific binding pair member, and in specific binding pair member Another member is in conjunction with first antigen or the anti-immunity complex antibody；It is further preferred that the donor and strepto- parent It is combined with element, correspondingly the first antigen or the anti-immunity complex antibody are in conjunction with biotin.

In the present invention, it is non-particulate forms, and aqueous that the donor, which is photoactivation or chemical activation sensitizer, It is solvable in medium；And/or the donor is the high molecular particle filled with Photoactive compounds, can generate list under light excitation Line state oxygen.

In the present invention, the anti-immunity complex antibody is anti-by the anti-CCP in identification epitope and the first immune complex Body combines, and the identification epitope is comformational epitope and/or linear epitope.

It will be understood by a person skilled in the art that the anti-immunity complex antibody identifies anti-CCP in the first immune complex The constant region of antibody.The light chain portion of anti-CCP antibody in the first immune complex of the anti-immunity complex antibody nonrecognition Point.The Fc section of anti-CCP antibody in the first immune complex of the anti-immunity complex antibody specific recognition.

In the present invention, the anti-immunity complex antibody is polyclonal antibody and/or monoclonal antibody；Preferably, described Anti-immunity complex antibody is monoclonal antibody.

In some embodiments, the preparation method of the polyclonal antibody includes: that employment immune complex carries out animal It is immune, obtain the animal blood serum for containing the polyclonal antibody；The animal blood serum obtains specific knowledge through affinitive layer purification The polyclonal antibody of others' immune complex.

In some embodiments, the preparation method of the monoclonal antibody include: will be after people's immune complex be immune The spleen cell of mouse is cultivated after merging with murine myeloma cell, is detected to cell culture supernatant, and sun is retained Property cell strain.

According to the present invention, first antigen is citrullinated antigens.

In some preferred embodiments of the invention, first antigen be selected from synthesis the ring-like peptide containing citrulling, Line style peptide containing citrulling is synthesized the polypeptide formed on a peptide chain by the single peptide fragment containing citrulling of at least two, containing extremely The citrulling peptide fragment mixture and citrullinated albumen of few 2 single peptide fragments containing citrulling.

In some particularly preferred embodiments of the invention, first antigen is selected from the ring containing cyclic citrulline of synthesis Type peptide, the line style peptide containing citrulling, synthesized by the single peptide fragment containing citrulling of at least two the polypeptide formed on a peptide chain and Citrulling peptide fragment mixture containing the single peptide fragment containing citrulling of at least two.

In some further particularly preferred embodiments of the invention, first antigen is to contain citrulling by 2-4 The peptide fragment polypeptide that is formed on a peptide chain of synthesis or citrulling peptide fragment mixing containing the 2-4 single peptide fragments containing citrulling Object；It is preferred that the peptide fragment containing citrulling is selected from SEQ ID No.2-5.

Table 1

Sequence number	Sequence
		SEQ ID No.2	Ring-(HQCHQEST-Cit-GRSRGRCGRSGS)
SEQ ID No.3	ARGGSRERARGRGRG-Cit-GEKR
		SEQ ID No.4	GGSKTSLYNLR-Cit-GTALAIPQ
SEQ ID No.5	APPPISGGGY-cit-A-cit-PAKAAAT

In some preferred embodiments, first antigen is combined by intermediate with receptor, and the intermediate is Hydrophilic macromolecule substance.

Preferably, the intermediate is protein, is preferably selected from hemocyanin, ovalbumin, bovine serum albumin(BSA) or ox Thyroglobulin.

Preferably, the intermediate is selected from dendritic macromole, polycarboxylate, poly- sulfydryl and polyethylene glycol.

In some embodiments, the total concentration of the receptor and the first antigen in combination is 10-200 μ g/mL, preferably 20-150 μ g/mL, more preferable 25-100 μ g/mL；And/or the anti-immunity complex antibody and specificity in combination are tied The total concentration for closing a member in pairing member is 0.1-10 μ g/mL, preferably 0.5-5 μ g/mL, more preferable 1-3 μ g/mL.

In some further specific preferred embodiments of the invention, Anti-CCP antibody and 14- are detected using containing The homogeneous immunological detection reagent suit of 3-3eta albumen detects the concentration of Anti-CCP by homogeneous immune detection comprising:

Sample to be tested is mixed with component a1, obtains the first mixture by step R1；

First mixture is mixed with component b1, obtains the second mixture by step R2；

Step R3 mixes the second mixture with component c1, and the third for obtaining producing detectable chemiluminescence signal is mixed Close object；

Step R4, the intensity of chemiluminescence signal described in detecting step R3, so that it is determined that the content of Anti-CCP antibody.

In some preferred embodiments of the invention, the method also includes the production Anti-CCP before step R1 The step of antibody standard working curve.

In some further preferred embodiments of the invention, in step R4, the hair of chemistry described in detecting step R3 The intensity of optical signal, and determine based on Anti-CCP antibody standard working curve containing for Anti-CCP antibody in sample to be tested Amount.

It is above-mentioned to be passed through using the homogeneous immunological detection reagent suit containing detection Anti-CCP antibody and 14-3-3eta albumen Homogeneous immune detection can be understood as come the method for detecting the concentration of Anti-CCP the following steps are included:

S1 is formed by the first antigen in conjunction with the anti-CCP antibody in sample to be tested by the first antigen-anti-CCP antibody structure At the first immune complex；

S2, by anti-immunity complex antibody in conjunction with first immune complex, formation is resisted by the first antigen-anti-CCP The second immune complex that body-anti-immunity complex antibody is constituted；

S3, the second immune complex of detection whether there is；If the second immune complex exists, show in sample to be tested There are anti-CCP antibodies.

In some embodiments of the invention, detecting the second immune complex by chemiluminescent method in step S3 is No presence.

In the present invention, for first immune complex through the first antigen in conjunction with donor, corresponding described second is immune For compound through anti-immunity complex antibody in conjunction with receptor, the receptor can be reacted with singlet oxygen generates detectableization Luminous signal is learned, the donor can generate singlet oxygen in excited state.

In the present invention, for first immune complex through the first antigen in conjunction with receptor, corresponding described second is immune For compound through anti-immunity complex antibody in conjunction with donor, the receptor can be reacted with singlet oxygen generates detectableization Luminous signal is learned, the donor can generate singlet oxygen in excited state.

It is above-mentioned to use containing detection Anti-CCP antibody and 14-3-3eta albumen in other embodiments of the invention The homogeneous immunological detection reagent suit method of concentration that Anti-CCP is detected by homogeneous immune detection can be understood as wrapping Include following steps:

T1, by the first antigen in conjunction with biotin with the anti-CCP antibody in sample to be examined in conjunction with, formed by biotin- The third immune complex that first antigen-anti-CCP antibody is constituted；

T2, by the anti-immunity compound of the anti-CCP antibody in the specific recognition third immune complex in conjunction with receptor Antibody with third immune complex combine, formed by the-the first antigen of biotin-anti-CCP antibody-anti-immunity complex antibody-by The 4th immune complex that body is constituted；

T3 is formed affine by donor-strepto- by the donor in conjunction with Streptavidin in conjunction with the 4th immune complex The 5th immune complex that element-the-the first antigen of biotin-anti-CCP antibody-anti-immunity complex antibody-receptor is constituted；

T4, the 5th immune complex of detection whether there is；If the 5th immune complex exists, exist in sample to be tested Anti-CCP antibody；

Alternatively,

R1, by the first antigen in conjunction with receptor with the anti-CCP antibody in sample to be examined in conjunction with, formed by receptor-the first The 6th immune complex that antigen-anti-CCP antibody is constituted；

R2, the anti-immunity of the anti-CCP antibody in the 6th immune complex of specific recognition in conjunction with biotin is compound Object antibody is combined with the 6th immune complex, is formed by the-the first antigen of receptor-anti-CCP antibody-anti-immunity complex antibody-life The 7th immune complex that object element is constituted；

R3 is formed in conjunction with the biotin in same 7th immune complex of donor in conjunction with Streptavidin by receptor- First antigen-anti-CCP antibody-anti-immunity complex antibody-biotin-Streptavidin-donor is constituted the 8th immune multiple Close object；

R4, the 8th immune complex of detection whether there is；If the 8th immune complex exists, exist in sample to be tested Anti-CCP antibody；

Wherein, it in the presence of the 5th immune complex or the 8th immune complex, is excited with energy or reactive compound Donor generates singlet oxygen, and the receptor is reacted with singlet oxygen generates detectable chemiluminescence signal.

In some embodiments, the method also includes anti-CCP antibody standard work is made before step T1 or step R1 The step of making curve.

In some further embodiments, in step T4 or step R4, the intensity of the chemiluminescence signal is detected, And the content of anti-CCP antibody in sample to be tested is determined based on anti-CCP antibody standard working curve.

In some further specific embodiments, it is immunized using the exciting light irradiation the 5th of 600-700nm wavelength compound Object or the 8th immune complex, excited donor generate singlet oxygen, and receptor is reacted with the singlet oxygen touched generates 520- The transmitting light of 620nm, the signal value of detection transmitting light, thus judge to survey in sample to be tested with the presence or absence of anti-CCP antibody and/or The concentration of anti-CCP antibody.

In some specific preferred embodiments of the invention, for detecting the homogeneous immune detection of 14-3-3eta albumen Reagent includes:

Component a2, it includes the receptor and in combination first for generating detectable signal can be reacted with singlet oxygen Antibody or its binding fragment, the first antibody or its binding fragment can be with the first epitope specificities of 14-3-3eta albumen In conjunction with；

Component b2, it includes can be with the secondary antibody or its knot in conjunction with the second epitope specificity of 14-3-3eta albumen Close segment, second epitope and the first epitope non-overlapping；

Component c2 comprising can be in the donor of excited state generation singlet oxygen.

In some embodiments of the invention, the amino acid sequence of the 14-3-3eta albumen such as SEQUENCE NO.1 institute Show.Preferably, it is 14-3-3eta albumen that second epitope and first epitope, which are separately selected from amino acid fragment, The relative specificity segment of sequence: 1-6aa, 27-38aa, 71-83aa, 112-119aa and 141-154aa.

In the present invention, the first antibody and secondary antibody are separately selected from monoclonal antibody and/or polyclonal Antibody, preferably monoclonal antibody.

In some embodiments of the invention, the reagent further includes the 14-3-3eta albumen sterling as calibration object, institute State calibration object be calibrated product dilution proportionally gradient dilution at various concentration working calibration product solution.

In some preferred embodiments of the invention, the secondary antibody or its binding fragment and specific binding pair A member in member combines, and another Yuan combination in the donor and specific binding pair member.Preferably, described second Antibody or its binding fragment are in conjunction with biotin, and the donor is in conjunction with Streptavidin.

In some embodiments of the invention, receptor and first antibody in combination or its combination in the component a The concentration of segment is 10-200 μ g/mL, preferably 20-150 μ g/mL, more preferable 30-100 μ g/mL, most preferably 40-80 μ g/mL； And/or secondary antibody or the concentration of its binding fragment are 0.1-8 μ g/mL, preferably 0.2-6 μ g/mL in the component b, more preferably 0.4-4 μ g/mL, most preferably 0.6-2 μ g/mL；And/or the concentration of donor described in component c is 5-20 μ g/mL, preferably 8-15 μ G/mL, more preferable 10-12 μ g/mL.

In the present invention, it is non-particulate forms, and in aqueous Jie that the receptor, which includes olefin(e) compound and metallo-chelate, It is solvable in matter；And/or the receptor is the high molecular particle filled with luminophor and lanthanide series.

In the present invention, it is non-particulate forms, and aqueous that the donor, which is photoactivation or chemical activation sensitizer, It is solvable in medium；And/or the donor is the high molecular particle filled with Photoactive compounds, can generate list under light excitation Line state oxygen.

In some further specific preferred embodiments of the invention, Anti-CCP antibody and 14- are detected using containing The homogeneous immunological detection reagent suit of 3-3eta albumen detects the concentration of 14-3-3eta albumen by homogeneous immunodetection, Comprising:

Step R1 by sample to be tested and component a2 and combines b2 and mixes, obtains third mixture；

Third mixture is mixed with component c2, obtains the 4th mixture by step R2；

Step R3 contacts energy or reactive compound with the 4th mixture, and the donor is excited to generate single line State oxygen, the receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence and/or intensity of chemiluminescence signal described in detecting step R3, to judge to survey sample to be tested In with the presence or absence of 14-3-3eta albumen and/or determine 14-3-3eta albumen content.

In some embodiments of the invention, the method also includes the production 14-3-3eta albumen before step R1 The step of standard working curve.

In some further embodiments of the invention, in step R4, the letter of chemiluminescence described in detecting step R3 Number intensity, and determine based on 14-3-3eta protein standard working curve the content of 14-3-3eta albumen in sample to be tested.

In the present invention, the step of not separating and/or wash between step R1 and R2 and between step R2 and R3.

In some embodiments, in step R3, the 4th mixture is irradiated using the exciting light of 600-700nm wavelength, is swashed It sends out donor and generates singlet oxygen, receptor reacts the transmitting light for generating 520-620nm with the singlet oxygen touched.

The 17th aspect of the present invention provides a kind of each in rheumatoid arthritis (RA) biomarker group for detecting The chemiluminescence immune detection system of biomarker comprising using reagent set as described in respect of the second aspect of the invention or make Method with kit as described in the third aspect of the present invention or as described in the method as described in terms of the present invention the 13 to the 16th To detect in sample to be tested with the presence or absence of each biomarker and/or determination in rheumatoid arthritis (RA) biomarker group The content of each biomarker in rheumatoid arthritis (RA) biomarker group.

According to certain embodiments of the present invention, the system comprises:

Reaction unit is used for reagent set or third aspect present invention described in sample to be tested and second aspect of the present invention Reagent in the kit chemically reacts；

Excitation and reading plotter generate active oxygen, receptor using the excitation donor microballoon of 600-700nm wavelength Microballoon generates the transmitting light of 520-620nm with the reactive oxygen species received, records the optical signal of above-mentioned transmitting light；

Processor surveys sample to be tested according to the presence of the optical signal of the transmitting light recorded and/or intensity judgement In with the presence or absence of object to be measured molecule and/or determine object to be measured molecule content.

In some embodiments of the invention, the processor is fitted using cubic spline interpolation fitting, directly to Out in sample to be tested object to be measured molecule concentration value.

The 18th aspect of the present invention provides a kind of for detecting the homogeneous immunologic detection method of Anti-CCP, uses this It invents detection system described in the 17th aspect and passes through as described in the present invention for detecting the reagent set of Anti-CCP Homogeneous immune detection detects the concentration of Anti-CCP comprising:

Sample to be tested is mixed with component a1, obtains the first mixture by step R1；

First mixture is mixed with component b1, obtains the second mixture by step R2；

Step R3 mixes the second mixture with component c1, and the third for obtaining producing detectable chemiluminescence signal is mixed Close object；

Step R4, the intensity of chemiluminescence signal described in detecting step R3, so that it is determined that the content of Anti-CCP antibody.

In some embodiments of the invention, the method also includes the production Anti-CCP antibody marks before step R1 The step of quasi- working curve.

In some further embodiments of the invention, in step R4, the letter of chemiluminescence described in detecting step R3 Number intensity, and determine based on Anti-CCP antibody standard working curve the content of Anti-CCP antibody in sample to be tested.

The 19th aspect of the present invention provides a kind of for detecting the homogeneous immunologic detection method of Anti-CCP, uses it Using detection system described in the 17th aspect of the present invention and as described in the present invention for detecting the reagent of 14-3-3eta albumen It is set with to detect the concentration of 14-3-3eta albumen by homogeneous immunodetection comprising:

Step R1 by sample to be tested and component a2 and combines b2 and mixes, obtains third mixture；

Third mixture is mixed with component c2, obtains the 4th mixture by step R2；

Step R3 contacts energy or reactive compound with the 4th mixture, and the donor is excited to generate single line State oxygen, the receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence and/or intensity of chemiluminescence signal described in detecting step R3, to judge to survey sample to be tested In with the presence or absence of 14-3-3eta albumen and/or determine 14-3-3eta albumen content.

In some embodiments of the invention, the method also includes the production 14-3-3eta albumen before step R1 The step of standard working curve.

In some further embodiments of the invention, in step R4, the letter of chemiluminescence described in detecting step R3 Number intensity, and determine based on 14-3-3eta protein standard working curve the content of 14-3-3eta albumen in sample to be tested.

It according to certain embodiments of the present invention, can also include other reagents in heretofore described reagent set.

According to some preferred embodiments, heretofore described reagent set includes for detecting the equal of anti-CCP antibody Phase immunologic function test reagent comprising:

First chamber, the first chamber include component a3, and the component a3 with singlet oxygen by that can react life It is constituted at the receptor and the first antigen in combination of detectable signal, wherein first antigen can be with anti-cyclic citrulline The first recognition site of epitope of peptide antibody is specifically bound；

Second chamber, the second chamber include component b3, and the component b3 with anti-cyclic citrullinated peptide by that can resist One in the second antigen and specific pairs object in combination of the second recognition site of epitope specific binding of body A member is constituted；

Third composition, the third composition include component c3, and the component c3 is single by that can generate in excited state Another member in the donor of line state oxygen and specific pairs object in combination is constituted.

In the present invention, first antigen and second antigen are identical or different, and the two contains independently selected from synthesis The ring-like peptide of citrulling, the line style peptide containing citrulling synthesize the shape on a peptide chain by the single peptide fragment containing citrulling of at least two At polypeptide, citrulling peptide fragment mixture and citrullinated albumen containing the single peptide fragment containing citrulling of at least two.

In some embodiments of the invention, first antigen and second antigen are separately selected from synthesis Cyclic peptide containing cyclic citrulline, the line style peptide containing citrulling are synthesized by the single peptide fragment containing citrulling of at least two in a peptide chain The polypeptide of upper formation and citrulling peptide fragment mixture containing the single peptide fragment containing citrulling of at least two.

Preferably, first antigen and second antigen are separately to be closed by the 2-4 peptide fragments containing citrulling The polypeptide formed on Cheng Yi peptide chain or the citrulling peptide fragment mixture containing the 2-4 single peptide fragments containing citrulling；Into one The preferably described peptide fragment containing citrulling of step is selected from SEQ ID No.2-5.

In some preferred embodiments of the invention, first antigen is combined by intermediate with receptor, described Intermediate is hydrophilic macromolecule substance.

In some embodiments, the intermediate is protein, and it is pure to be preferably selected from hemocyanin, ovalbumin, ox blood Albumen or bovine thyroglobulin.

Preferably, the intermediate is selected from dendritic macromole, polycarboxylate, poly- sulfydryl and polyethylene glycol.

In some preferred examples, second antigen is in conjunction with biotin, and the donor and Streptavidin knot It closes.

In some embodiments of the invention, total concentration of the component a3 in first chamber is 10-200 μ g/mL, It is preferred that 20-150 μ g/mL, more preferable 25-100 μ g/mL；And/or concentration of the component b3 in second chamber is 0.1- 10 μ g/mL, preferably 0.5-5 μ g/mL, more preferable 1-3 μ g/mL.

In the present invention, it is non-particulate forms, and in aqueous Jie that the receptor, which includes olefin(e) compound and metallo-chelate, It is solvable in matter；And/or the receptor is the high molecular particle filled with luminophor and lanthanide series.

In the present invention, it is non-particulate forms, and aqueous that the donor, which is photoactivation or chemical activation sensitizer, It is solvable in medium；And/or the donor is the high molecular particle filled with Photoactive compounds, can generate list under light excitation Line state oxygen.

It is adopted in some further preferred embodiments of the invention corresponding to the mentioned reagent in kit of the present invention It is set with the homogeneous immunological detection reagent containing detection Anti-CCP antibody and Anti-CCP is detected by homogeneous immunodetection Concentration comprising:

Step R1 reacts to obtain the first mixture after mixing sample to be tested with first chamber and second chamber；

Step R2 reacts to obtain the second mixture after mixing the first mixture with third composition；

Step R3 contacts energy or reactive compound with second mixture, and the donor is excited to generate single line State oxygen, the receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence and/or intensity of chemiluminescence signal described in detecting step R3, to judge to survey sample to be tested In with the presence or absence of cyclic citrullinated peptid and/or determine cyclic citrullinated peptid content.

In some embodiments of the invention, the method also includes the anti-cyclic citrullinated peptide of production before step R1 is anti- The step of body standard working curve.

In some further embodiments of the invention, in step R4, the letter of chemiluminescence described in detecting step R3 Number intensity, and determine that based on the cyclic citrullinated peptid standard working curve, anti-cyclic citrullinated peptide is anti-in sample to be tested The content of body.

In some embodiments of the invention, the second mixture is irradiated using the exciting light of 600-700nm wavelength, excitation supplies Body generates singlet oxygen, and receptor reacts the transmitting light for generating 520-620nm, the letter of detection transmitting light with the singlet oxygen touched Number intensity, to judge in sample to be tested with the presence or absence of cyclic citrullinated peptid and/or determine cyclic citrullinated peptid Content.

According to some preferred embodiments, heretofore described reagent set includes for detecting Anti-carp antibody Homogeneous immunological detection reagent comprising:

Component a4, it includes the first antigens that can be specifically bound with the epitope binding site of anti-Carp antibody；

Component b4, it includes anti-immunity complex antibody, the anti-immunity complex antibody being capable of specific recognition and knot Close the anti-Carp antibody formed in the first immune complex with the first antigen, nonrecognition it is free, unbonded antigen it is anti- Carp antibody.

In the present invention, first antigen or the anti-immunity complex antibody are combined with receptor, and the receptor can It is reacted with singlet oxygen and generates detectable chemiluminescence signal.

In the present invention, it is non-particulate forms, and in aqueous Jie that the receptor, which includes olefin(e) compound and metallo-chelate, It is solvable in matter；And/or the receptor is the high molecular particle filled with luminophor and lanthanide series.

In some embodiments of the invention, the reagent set also includes component c4, and it includes can be in excited state Generate the donor of singlet oxygen；It is preferred that the donor is in conjunction with a member in specific binding pair member, and specifically bind Another member in member is matched in conjunction with first antigen or the anti-immunity complex antibody；It is further preferred that described Donor is in conjunction with Streptavidin, and correspondingly the first antigen or the anti-immunity complex antibody are in conjunction with biotin.

In the present invention, it is non-particulate forms, and aqueous that the donor, which is photoactivation or chemical activation sensitizer, It is solvable in medium；And/or the donor is the high molecular particle filled with Photoactive compounds, can generate list under light excitation Line state oxygen.

It will be understood by a person skilled in the art that the anti-immunity complex antibody is by identifying that it is compound that epitope is immunized with first Anti- Carp antibody in object combines, and the identification epitope is comformational epitope and/or linear epitope.The anti-immunity compound is anti- Body identifies the constant region of anti-Carp antibody in the first immune complex.The anti-immunity complex antibody nonrecognition first The chain moiety of anti-Carp antibody in immune complex.The anti-immunity complex antibody specific recognition first is immunized compound The Fc section of anti-Carp antibody in object.

In the present invention, the anti-immunity complex antibody is polyclonal antibody and/or monoclonal antibody；Preferably, described Anti-immunity complex antibody is monoclonal antibody.

In some embodiments of the invention, the preparation method of the polyclonal antibody includes: employment immune complex pair Animal is immunized, and the animal blood serum for containing the polyclonal antibody is obtained；The animal blood serum is obtained through affinitive layer purification The polyclonal antibody of specific recognition people's immune complex.

In some embodiments of the invention, the preparation method of the monoclonal antibody include: will be through people's immune complex The spleen cell of mouse after immune is cultivated after merging with murine myeloma cell, is examined to cell culture supernatant It surveys, retains positive cell strain.

In the present invention, first antigen is selected from the carbamylation peptide fragment of synthesis, by the single carbamylation peptide fragment of at least two Synthesize formed on a peptide chain polypeptide, the carbamylation peptide fragment mixture containing the single carbamylation peptide fragment of at least two and Carbamylation albumen.

Preferably, first antigen is selected from the carbamylation peptide fragment of synthesis, is closed by the single carbamylation peptide fragment of at least two The polypeptide formed on Cheng Yi peptide chain and the carbamylation peptide fragment mixture containing the single carbamylation peptide fragment of at least two.

It is formed on a peptide chain it is further preferred that first antigen is synthesized by 2-4 carbamylation peptide fragment Polypeptide or carbamylation peptide fragment mixture containing the single carbamylation peptide fragment of 2-4；It is preferred that the carbamylation peptide fragment is selected from Sequence No.2-5。

In some preferred embodiments of the invention, first antigen is combined by intermediate with receptor, described Intermediate is hydrophilic macromolecule substance.

In the present invention, the intermediate be protein, be preferably selected from hemocyanin, ovalbumin, bovine serum albumin(BSA) or Bovine thyroglobulin.Preferably, the intermediate is selected from dendritic macromole, polycarboxylate, poly- sulfydryl and polyethylene glycol.

In some embodiments of the invention, the concentration of the receptor and anti-immunity complex antibody in combination is 10-200 μ g/mL, preferably 20-150 μ g/mL, more preferable 25-100 μ g/mL；And/or first antigen and in combination The concentration of a member in specific binding pair member is 0.1-10 μ g/mL, preferably 0.5-5 μ g/mL, more preferable 1-3 μ g/mL.

It is adopted in some further preferred embodiments of the invention corresponding to the mentioned reagent in kit of the present invention It is set with the homogeneous immunological detection reagent containing detection Anti-CCP antibody and Anti-CCP is detected by homogeneous immunodetection Concentration comprising following steps:

S1 is formed by the first antigen in conjunction with the anti-Carp antibody in sample to be tested by the first antigen-anti-Carp antibody The first immune complex constituted；

S2 is formed by anti-immunity complex antibody in conjunction with first immune complex by the first antigen-anti-Carp The second immune complex that antibody-anti-immunity complex antibody is constituted；

S3, the second immune complex of detection whether there is；If the second immune complex exists, show in sample to be tested There are anti-Carp antibody.

In some embodiments, detecting the second immune complex by chemiluminescent method in step S3 whether there is.

In the present invention, for first immune complex through the first antigen in conjunction with donor, corresponding described second is immune For compound through anti-immunity complex antibody in conjunction with receptor, the receptor can be reacted with singlet oxygen generates detectableization Luminous signal is learned, the donor can generate singlet oxygen in excited state.

How in some embodiments, first immune complex by the first antigen with receptor in conjunction with, described accordingly the Through anti-immunity complex antibody in conjunction with donor, the receptor can react generation with singlet oxygen to be examined two immune complexs The chemiluminescence signal of survey, the donor can generate singlet oxygen in excited state.

In some specific embodiments, described method includes following steps:

T1, by the first antigen in conjunction with biotin with the anti-Carp antibody in sample to be examined in conjunction with, formed by biology The third immune complex that plain-the first antigen-anti-Carp antibody is constituted；

T2, the anti-immunity of the anti-Carp antibody in the specific recognition third immune complex in conjunction with receptor is compound Object antibody is combined with third immune complex, and formation is resisted by the-the first antigen of biotin-anti-Carp antibody-anti-immunity compound The 4th immune complex that body-receptor is constituted；

T3 is formed affine by donor-strepto- by the donor in conjunction with Streptavidin in conjunction with the 4th immune complex The 5th immune complex that the-the first antigen of element-biotin-anti-Carp antibody-anti-immunity complex antibody-receptor is constituted；

T4, the 5th immune complex of detection whether there is；If the 5th immune complex exists, exist in sample to be tested Anti- Carp antibody；

Alternatively,

R1, by the first antigen in conjunction with receptor with the anti-Carp antibody in sample to be examined in conjunction with, formed by receptor-the The 6th immune complex that one antigen-anti-Carp antibody is constituted；

R2 answers the anti-immunity of the anti-Carp antibody in the 6th immune complex of specific recognition in conjunction with biotin It closes object antibody to combine with the 6th immune complex, formation is resisted by the-the first antigen of receptor-anti-Carp antibody-anti-immunity compound The 7th immune complex that body-biotin is constituted；

R3 is formed in conjunction with the biotin in same 7th immune complex of donor in conjunction with Streptavidin by receptor- First antigen-anti-Carp antibody-anti-immunity complex antibody-biotin-Streptavidin-donor is constituted the 8th immune Compound；

R4, the 8th immune complex of detection whether there is；If the 8th immune complex exists, exist in sample to be tested Anti- Carp antibody；

Wherein, it in the presence of the 5th immune complex or the 8th immune complex, is excited with energy or reactive compound Donor generates singlet oxygen, and the receptor is reacted with singlet oxygen generates detectable chemiluminescence signal.

In some embodiments, the method also includes anti-Carp antibody standard is made before step T1 or step R1 The step of working curve.

In some further embodiments, in step T4 or step R4, the intensity of the chemiluminescence signal is detected, And the content of anti-Carp antibody in sample to be tested is determined based on anti-Carp antibody standard working curve.

In some embodiments of the invention, using 600-700nm wavelength exciting light irradiate the 5th immune complex or 8th immune complex, excited donor generate singlet oxygen, and receptor is reacted with the singlet oxygen touched generates 520-620nm's Emit light, the signal value of detection transmitting light, to judge to survey in sample to be tested with the presence or absence of anti-Carp antibody and/or anti-Carp The concentration of antibody.

According to some preferred embodiments, heretofore described reagent set includes for detecting Anti-carp antibody Homogeneous immunological detection reagent comprising:

Component a5, it includes the receptor and in combination first for generating detectable signal can be reacted with singlet oxygen The epitope of antigen, first antigen can be specifically bound with the first binding site of epitope of anti-Carp antibody；

Component b5, it includes can specifically bind with the second binding site of epitope of anti-Carp antibody second Antigen, the first binding site of epitope and epitope the second binding site non-overlapping of anti-Carp antibody；

Component c5 comprising can be in the donor of excited state generation singlet oxygen.

In the present invention, first antigen and the second antigen are carbamylation antigen；It is preferred that the carbamylation antigen is Carbamylation human serum albumins；The further preferred human serum albumins is as shown in SEQ ID No.1.

In some embodiments of the invention, the reagent further includes the anti-Carp antibody sterling as calibration object, described Calibration object be calibrated product dilution proportionally gradient dilution at various concentration working calibration product solution.

A member knot in some preferred embodiments of the invention, in second antigen and specific binding partners It closes, and another Yuan combination in the donor and specific binding partners.Preferably, second antigen and biotin knot It closes, and the donor is in conjunction with Streptavidin.

In some embodiments of the invention: the total concentration of the receptor and the first antigen in combination is 10-200 μ G/mL, preferably 20-150 μ g/mL, more preferable 25-100 μ g/mL；And/or second antigen and specificity in combination are tied The total concentration for closing a member in counter pair is 0.1-10 μ g/mL, preferably 0.5-5 μ g/mL, more preferable 1-3 μ g/mL.

In the present invention, it is non-particulate forms, and in aqueous Jie that the receptor, which includes olefin(e) compound and metallo-chelate, It is solvable in matter；And/or the receptor is the high molecular particle filled with luminophor and lanthanide series.

In the present invention, it is non-particulate forms, and aqueous that the donor, which is photoactivation or chemical activation sensitizer, It is solvable in medium；And/or the donor is the high molecular particle filled with Photoactive compounds, can generate list under light excitation Line state oxygen.

It is adopted in some further preferred embodiments of the invention corresponding to the mentioned reagent in kit of the present invention It is set with the homogeneous immunological detection reagent containing detection Anti-CCP antibody and Anti-CCP is detected by homogeneous immunodetection Concentration comprising following steps:

Step R1 by sample to be tested and component a5 and combines b5 and mixes, obtains third mixture；

Third mixture is mixed with component c5, obtains the 4th mixture by step R2；

Step R3 contacts energy or reactive compound with the 4th mixture, and the donor is excited to generate single line State oxygen, the receptor can be reacted with the singlet oxygen received generates detectable chemiluminescence signal；

Step R4, the presence and/or intensity of chemiluminescence signal described in detecting step R3, to judge to survey sample to be tested In with the presence or absence of anti-Carp antibody and/or determine the content of anti-Carp antibody.

In some embodiments of the invention, the method also includes the anti-Carp antibody marks of production before step R1 The step of quasi- working curve.

In some further embodiments of the invention, in step R4, the letter of chemiluminescence described in detecting step R3 Number intensity, and determine based on anti-Carp antibody standard working curve the content of anti-Carp antibody in sample to be tested.

In the present invention, the step of not separating and/or wash between step R1 and R2 and between step R2 and R3.

In some embodiments of the invention, mixed using the exciting light irradiation the 4th of 600-700nm wavelength in step R3 Object is closed, excited donor generates singlet oxygen, and receptor reacts the transmitting light for generating 520-620nm with the singlet oxygen touched.

According to some preferred embodiments, heretofore described reagent set includes for detecting Anti-carp antibody Homogeneous immunological detection reagent comprising:

Component a6, it includes can be with the antigen in conjunction with the anti-Carp antibody of target and the second anti-Carp antibody specificity；

Component b6, it includes the second anti-Carp antibody.

In the present invention, the antigen or the second anti-Carp antibody are combined with receptor；Preferably, the antigen with Receptor combines；The receptor can be reacted with singlet oxygen generates detectable chemiluminescence signal.

In the present invention, it is non-particulate forms, and in aqueous Jie that the receptor, which includes olefin(e) compound and metallo-chelate, It is solvable in matter；And/or the receptor is the high molecular particle filled with luminophor and lanthanide series.

In some embodiments of the invention, the reagent set also includes component c6, and it includes can be in excited state Generate the donor of singlet oxygen；It is preferred that the donor is in conjunction with a member in specific binding partners, and specifically binds and match Another member in object is combined with the antigen or the second anti-Carp antibody；It is further preferred that the donor and chain Mould Avidin combines, and correspondingly the second anti-Carp antibody is in conjunction with biotin.

In the present invention, it is non-particulate forms, and aqueous that the donor, which is photoactivation or chemical activation sensitizer, It is solvable in medium；And/or the donor is the high molecular particle filled with Photoactive compounds, can generate list under light excitation Line state oxygen.

In the present invention, the second anti-Carp antibody is polyclonal antibody and/or monoclonal antibody；Preferably, described Two anti-Carp antibody are monoclonal antibody.

In some embodiments of the invention, the antigen is selected from the carbamylation peptide fragment of synthesis, by the single ammonia of at least two Formylated peptide fragment synthesizes polypeptide, the carbamylation peptide containing the single carbamylation peptide fragment of at least two formed on a peptide chain Section mixture and carbamylation albumen.

Preferably, the antigen is selected from the carbamylation peptide fragment of synthesis, is synthesized by the single carbamylation peptide fragment of at least two The polypeptide formed on one peptide chain and the carbamylation peptide fragment mixture containing the single carbamylation peptide fragment of at least two.

It is further preferred that the antigen is to synthesize the polypeptide formed on a peptide chain by 2-4 carbamylation peptide fragment Or the carbamylation peptide fragment mixture containing the single carbamylation peptide fragment of 2-4；It is preferred that the carbamylation peptide fragment is selected from SEQ ID No.2-5。

In some preferred embodiments of the invention, the antigen is combined by intermediate with receptor, the centre Body is hydrophilic macromolecule substance.Preferably, the intermediate is protein, is preferably selected from hemocyanin, ovalbumin, ox blood Pure albumen or bovine thyroglobulin.It is further preferable that the intermediate is selected from dendritic macromole, polycarboxylate, poly- mercapto Base and polyethylene glycol.

In some embodiments of the invention, the total concentration of the antigen and receptor in combination is 0.005-0.1 μ g/mL；And/or the total concentration of the second anti-Carp antibody and a member in specific binding partners in combination For 0.025-0.1 μ g/mL；And/or another 's in the donor and specific binding partners in combination is total dense Spend 5-20 μ g/mL.

It is adopted in some further preferred embodiments of the invention corresponding to the mentioned reagent in kit of the present invention It is set with the homogeneous immunological detection reagent containing detection Anti-CCP antibody and Anti-CCP is detected by homogeneous immunodetection Concentration method the following steps are included:

M1 makes the epitope knot of the target anti-Carp antibody competition and antigen in the second anti-Carp antibody and sample to be tested It closes, the first immune complex for being made of respectively the formation of epitope and antigen the anti-Carp antibody of antigen-the second and by resisting The second immune complex that the anti-Carp antibody of original-target is constituted；

M2, the second immune complex of detection whether there is；If the second immune complex exists, show in sample to be tested There are anti-Carp antibody.

In some embodiments of the invention, the step M1 includes by the second anti-Carp antibody, sample to be tested and antigen It is uniformly mixed, reaction makes the epitope of target anti-the Carp antibody competition and antigen in the second anti-Carp antibody and sample to be tested In conjunction with, respectively by epitope and antigen formed the first immune complex being made of the anti-Carp antibody of antigen-the second and by The second immune complex that the anti-Carp antibody of antigen-is constituted.

In some further embodiments of the invention, the second anti-Carp antibody, sample to be tested and antigen are mixed equal Even step includes by the second anti-Carp antibody, sample to be tested and antigen while being uniformly mixed；Alternatively, first by the second anti-Carp Antibody and sample to be tested are mixed to form the 1st mixture, then antigen is mixed with the 1st mixture.

In some embodiments of the invention, the step M1 the following steps are included:

P1 mixes antigen with sample to be tested, and reaction makes the epitope knot of the anti-Carp of the target in sample to be tested and antigen The second immune complex for being formed and being made of the anti-Carp antibody of antigen-target is closed, the 11st mixture is obtained；

P2 mixes the second anti-Carp antibody with the 11st mixture, and reaction mixes the second anti-Carp antibody with the 11st In conjunction with the not epitope of the antigen in conjunction with the anti-Carp in sample to be tested in object, formed by the anti-Carp antibody of antigen-the second The first immune complex constituted.

In some embodiments, detecting the second immune complex by chemiluminescent method in step M2 whether there is.

In some embodiments, step M2 include by the chemiluminescence signal value of the 1st mixture with by medium with step M1 The chemiluminescence signal value for the control immune complex that the antigen of amount and the second anti-Carp antibody are formed is compared, and is thus sentenced Disconnected second immune complex whether there is.

In some embodiments of the invention, the antigen is in conjunction with receptor, the second anti-Carp antibody and biotin In conjunction with the receptor can be reacted with singlet oxygen generates detectable chemiluminescence signal.

It in some embodiments of the invention, further include step M0 before step M1, using sample diluting liquid according to 1: The volume ratio of (4-20) dilutes sample to be tested；Preferably, using sample diluting liquid according to 1:(6-16) volume ratio dilution it is to be measured Sample；It is highly preferred that using sample diluting liquid according to 1:(8-12) volume ratio dilute sample to be tested.

In some embodiments of the invention, described method includes following steps:

The second anti-Carp antibody in conjunction with biotin is mixed to form the 2nd mixture with sample to be tested by R1；

R2 mixes the antigen for being combined with receptor with the 2nd mixture, makes the in conjunction with biotin second anti-Carp antibody With the anti-Carp antibody competition of target in sample to be tested in conjunction with the epitope of antigen for being combined with receptor, pass through antigen table respectively Position is immunized with the antigen formation for being combined with receptor by the third that the anti-Carp antibody-biotin of receptor-antigen-the second is constituted compound Object and the 4th immune complex being made of the anti-Carp antibody of receptor-antigen-, to form the 3rd mixture；

R3 mixes the donor in conjunction with Streptavidin with the 3rd mixture, make donor in conjunction with Streptavidin with Biotin in third immune complex combines, and by receptor-antigen-the second anti-Carp antibody-biotin-, strepto- is affine for formation The 5th immune complex that element-donor is constituted, to form the 4th mixture；

R4 generates singlet oxygen with energy or reactive compound excited donor, and the receptor reacts life with singlet oxygen At detectable chemiluminescence signal；Detecting the 4th immune complex whether there is；If the 4th immune complex exists, to There are anti-Carp antibody in test sample sheet.

In some embodiments of the invention, described method includes following steps:

T1 mixes the antigen for being combined with receptor with sample to be tested, reaction, make the anti-Carp of the target in sample to be tested with Be combined with the antigen of receptor epitope combine formed the 4th be made of the anti-Carp antibody of receptor-antigen-target be immunized it is compound Object obtains the 12nd mixture；

T2 mixes the second anti-Carp antibody in conjunction with biotin with the 12nd mixture, and reaction makes and biotin knot The anti-Carp's in the unbonded sample to be tested in the second anti-Carp antibody competition and the 12nd mixture closed is combined with receptor Antigen epitope combine, formation be immunized by the third that the anti-Carp antibody-biotin of antigen-the second for being combined with receptor is constituted Compound, to form the 13rd mixture；

Donor in conjunction with Streptavidin is mixed with the 13rd mixture, makes the donor in conjunction with Streptavidin by T3 In conjunction with the biotin in third immune complex, formed by the anti-Carp antibody-biotin of receptor-antigen-the second-strepto- parent The 5th immune complex constituted with element-donor, to form the 14th mixture；

T4 generates singlet oxygen with energy or reactive compound excited donor, and the receptor reacts life with singlet oxygen At detectable chemiluminescence signal；Detecting the 4th immune complex whether there is；If the 4th immune complex exists, to There are anti-Carp antibody in test sample sheet.

In some embodiments of the invention, anti-the method also includes making target before step R1 or step T1 The step of Carp antibody standard working curve.

In some embodiments of the invention, in step R4 or step T4, the intensity of the chemiluminescence signal is detected, And the content of the anti-Carp antibody of target in sample to be tested is determined based on anti-Carp antibody standard working curve.

In some embodiments of the invention, mixed using exciting light the 4th mixture of irradiation of 600-700nm wavelength or the 14th Object is closed, excites the donor in the 5th immune complex to generate singlet oxygen, receptor reacts generation with the singlet oxygen touched The transmitting light of 520-620nm, detects the signal value of the transmitting light of the 4th mixture or the 14th mixture, and by it with step R2 or The chemiluminescence signal value for the control immune complex that the antigen of step T1 and T2 moderate and the second anti-Carp antibody are formed into Row compares, and thus the concentration in sample to be tested with the presence or absence of the anti-Carp antibody of target and/or the anti-Carp antibody of target is surveyed in judgement.

III, embodiment

To keep the present invention easier to understand, below in conjunction with embodiment, present invention be described in more detail, these realities Apply example only serve it is illustrative, it is not limited to application range of the invention.If raw material used in the present invention or component nothing Specified otherwise can be made by commercial sources or conventional method.

In the method for the invention, all reagents after combination or mixing, can carry out according to actual needs mix and/ Or it incubates.Specifically, the temperature of the incubation can be the arbitrary temp in 25-45 DEG C of temperature range, and incubative time can be Overnight or 10-20min.