阅读说明：本技术 一种凋亡相关斑点样蛋白（asc）作为缺血性脑卒中标志物的应用 (Application of apoptosis-related plaque-spotted protein (ASC) as cerebral arterial thrombosis marker ) 是由 段学军 张国军 史一君 樊荣 吕虹 李国歌 丁耀威 徐彬瑗 于 2020-11-04 设计创作，主要内容包括：本发明涉及一种新的缺血性卒中的标志物分子,用于辅助评估缺血性卒中的体外检测。本发明公开了具有胱天蛋白募集域(CARD)和热蛋白样结构域(PYD)的凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a caspase-recruitment domain,简称ASC)作为缺血性卒中标志物的用途；本发明进一步提供以ASC为缺血性卒中标志物的试剂盒,测量来源于人体的体液中ASC含量。本发明所述的以ASC为缺血性卒中标志物的试剂盒,可用于缺血性卒中患者的早期检测、病程进展和治疗预后的监控以及辅助诊断等。(The invention relates to a novel marker molecule for ischemic stroke, which is used for assisting in-vitro detection for evaluating ischemic stroke. The invention discloses an application of apoptosis-related speckle-like protein (ASC for short) with a caspase recruitment domain (CARD) and a pyrrotein-like domain (PYD) as an ischemic stroke marker; the invention further provides a kit for measuring the content of ASC in body fluid from a human body, wherein the ASC is used as an ischemic stroke marker. The kit with the ASC as the ischemic stroke marker can be used for early detection, disease course progression, treatment prognosis monitoring, auxiliary diagnosis and the like of ischemic stroke patients.)

1. The application of the kit for specifically determining the concentration of the protein ASC in preparing a reagent for distinguishing ischemic stroke.

2. Use according to claim 1, characterized in that said distinguishing comprises:

1) determining the concentration of protein ASC in the body fluid;

2) comparing the concentration of ASC of the protein determined in step 1) with a reference concentration of ASC of the protein, wherein a concentration of ASC of the protein above the reference concentration is indicative of ischemic stroke.

3. Use according to claim 2, wherein the body fluid is a serum, plasma or whole blood sample.

4. Use according to any of claims 1-3, characterized in that the concentration of ASC protein is determined by an immunoassay.

5. The use according to claim 4, wherein the immunoassay is a magnetic particle full-automatic chemiluminescence immunoassay.

6. A kit for detecting ischemic stroke, comprising: magnetic particles coated with FITC antibody, FITC labeled ASC antibody, acridinium ester labeled ASC detection antibody, ASC calibrator and quality control material.

7. The kit of claim 6, wherein the concentration of the ASC calibrator is 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, respectively.

8. The kit of claim 6, further comprising a diluent, a wash solution, a pre-excitation solution, and an excitation solution.

9. The kit according to claim 6, wherein the FITC antibody-coated magnetic microparticle mother solution has a concentration of 10 mg/mL.

10. The kit according to claim 6, wherein the concentration of the FITC-labeled ASC antibody stock solution is 1.3 mg/mL.

Technical Field

The invention relates to the field of medical inspection, in particular to apoptosis-related spot-like protein (ASC) with a caspase recruitment domain (CARD) and a pyrrotein-like domain (PYD) as an ischemic stroke detection marker and application thereof.

Background

Stroke is a disease in which the blood circulation of the brain is disturbed and the function and structure of the brain tissue are damaged due to the blockage or rupture of the cerebral vessels.

The incidence of stroke is rising at a rate of nearly 9% per year in our country. According to reports of nutrition and chronic disease states of Chinese residents (2015), the number of deaths of Chinese residents due to stroke in recent years is over 200 thousands, accounting for 22.8% of total deaths. People with hypertension, heart disease and diabetes are high risk groups for stroke. The World Health Organization (WHO) analyzes chronic diseases in China, and if the current death rate is kept unchanged, nearly 400 million people in China die of stroke every year by 2030 years; if the death rate is only increased by 1%, 600 million people will die of stroke in China every year by 2030. According to the Chinese stroke prevention and treatment report 2018, the number of people who suffer from stroke and have suffered from stroke at present in people over 40 years old in China is 1242 thousands, 70% of people who survive after healing have disabilities of different degrees, and huge market demands for treatment accompanying and rehabilitation after healing are brought. With the comprehensive attention of our government to stroke, the positive method of early prevention and early treatment of stroke is implemented, and the current situation and the development situation of stroke are gradually improved.

However, few biomarkers have long been available for the diagnosis of stroke. The diagnosis means of patients with mild brain injury such as concussion, traumatic brain injury, cerebral apoplexy and the like are mainly electron Computed Tomography (CT) and Magnetic Resonance Imaging (MRI). The CT scanning is conventionally used for assisting a doctor to evaluate brain injury, and can quickly, simply and accurately eliminate cerebral hemorrhage, subdural hematoma and tumor and stroke. The use of CT scanning has limitations, particularly with major trauma that is mild in symptoms. In some cases, for example: cerebral infarction is caused, and the brain does not form a focus during CT detection; the focus is less than 1cm, and the focus is not easy to be found by CT; the position of the focus is not easy to be found by CT; the density of damaged brain tissue is similar to normal brain tissue, and normal CT is easily diagnosed as negative, when enhanced scanning is required. Statistics reported in literatures indicate that in patients with mild brain injury or cerebral concussion who go to emergency treatment and seek medical treatment, a greater proportion of CT scanning results are negative, and the diagnosis positive rate of cerebral infarction is 60-84%.

In order to better monitor early warning of brain injury diseases, disease course progress management and treatment prognosis and aid diagnosis, the medical field pays more and more attention to the discovery and application of developing blood-based brain injury biomarkers. Recent studies have shown that 2 brain-specific protein biomarkers (glial fibrillary acidic protein, GFAP, and ubiquitin carboxyl-terminal hydrolase-L1, UCH-L1) appear in the blood soon after brain injury. In 2018, the U.S. Food and Drug Administration (FDA) ranks diagnosis of brain injury as urgent medical needs, examines the brain injury detection according to a breakthrough mechanical project, simplifies development, and accelerates examination of innovative breakthrough technology. FDA approved 510K marketing application for the Banyan BTITM (brain trauma index) kit based on GFAP and UCH-L1, allowing this diagnostic test to be used as a diagnostic instrument. The method is the first FDA approved cerebral concussion blood detection, is a great milestone for converting a brain injury treatment mode, and can provide objective and quantifiable information for doctors to avoid unnecessary CT scanning and guide patients to take medical care. In addition, the serum S-100B test is a good brain injury screening project and is known as the CRP in neurobiology. Because of the high sensitivity to head CT scanning abnormality, the S-100B detection is suitable for early evaluation of diseases such as Traumatic Brain Injury (TBI) and acute cerebral apoplexy. Nevertheless, blood-based biomarkers of brain injury are too few, especially biomarkers for stroke diagnosis.

To be clinically useful, the novel diagnostic marker as a single marker should generally be superior to or at least equivalent to other markers known in the art. The value of the new marker is expressed in the sensitivity and the specificity, and a serological marker with both sensitivity and specificity is searched for and applied to the clinical detection of the stroke, so as to achieve the aims of early diagnosis of the stroke, postoperative monitoring and prognosis evaluation, and solve the problem urgently needed in diagnosis and treatment of the stroke.

The invention discloses apoptosis-related spot-like protein (ASC) with a caspase recruitment domain (CARD) and a pyrin-like domain (PYD) as an ischemic stroke marker and application thereof, and shows a method for evaluating ischemic stroke diseases by measuring the content of the apoptosis-related spot-like protein in human blood and preparation and application of a related in vitro diagnostic kit.

Disclosure of Invention

In order to make up the defects of the detection method for the brain injury diseases in the prior art, in particular to the detection of ischemic stroke, the invention provides a novel marker for detecting the ischemic stroke.

Preferably, the invention aims to disclose an apoptosis-related spot-like protein (ASC) with a caspase recruitment domain (CARD) and a thermoprotein-like domain (PYD) as an ischemic stroke marker and application thereof, in particular to a method for evaluating ischemic stroke diseases by measuring the content of the apoptosis-related spot-like protein in human body fluid and preparation and application of a related in-vitro diagnostic kit.

In order to achieve the above objects, a first aspect of the present invention provides an apoptosis-related spot-like protein (ASC) having a caspase recruitment domain (CARD) and a thermoprotein-like domain (PYD) as an ischemic stroke marker for early detection and assisted diagnosis of ischemic stroke. Wherein an increased level of detection of ASC is indicative of ischemic stroke. The amino acid sequence of the apoptosis-related plaque sample application protein (ASC for short) with a caspase recruitment domain (CARD) and a pyrin-like domain (PYD) is as follows:

the invention provides a specific application of the protein, namely a method for establishing and measuring the concentration of apoptosis-related speckle-like protein (ASC) of a caspase recruitment domain (CARD) and a thermoprotein-like domain (PYD) in human body fluids such as serum, plasma and the like in vitro detection.

The invention also provides a technical scheme for measuring the concentration of the human body fluid apoptosis-related spot-like protein (ASC).

The invention discloses application of a reagent for specifically determining protein ASC concentration in preparation of a kit for distinguishing ischemic stroke.

Preferably, the distinguishing comprises:

1) determining the concentration of protein ASC in the body fluid;

2) comparing the concentration of protein ASC determined in step 1) with a reference concentration of protein ASC, wherein a concentration of protein ASC higher than the reference concentration is indicative of ischemic stroke.

Preferably, the body fluid is a serum, plasma or whole blood sample.

Preferably, the concentration of ASC protein is determined by immunoassay.

Preferably, the immunoassay is a magnetic particle full-automatic chemiluminescence immunoassay.

The invention discloses a kit for detecting ischemic stroke.

Preferably, the kit comprises: magnetic particles coated with FITC antibody, FITC labeled ASC antibody, acridinium ester labeled ASC detection antibody, ASC calibrator and quality control material.

Preferably, the concentration of the ASC calibrator is 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, or 10ng/mL, respectively.

Preferably, the kit further comprises a diluent, a cleaning solution, a pre-excitation solution and an excitation solution.

Preferably, the concentration of the mother solution of the FITC coated magnetic particles is 10 mg/mL.

Preferably, the concentration of the FITC-labeled ASC antibody stock solution is 1.3 mg/mL.

Preferably, the concentration of the magnetic particle mother solution of the FITC antibody-coated magnetic particles is 10mg/mL, and the concentration of the working solution after dilution by 20 times is 0.5 mg/mL.

Preferably, the concentration of the FITC-labeled ASC antibody in the mother solution is 1.3mg/mL, and the concentration of the working solution obtained by diluting the antibody 1000 times is 1.3. mu.g/mL.

Preferably, the concentration of the acridinium ester-labeled ASC detection antibody is 1.3mg/mL, and the concentration of the working solution obtained by diluting 4000 times is 0.33. mu.g/mL.

Preferably, the ASC concentration of the calibrator is 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, or 10ng/mL, respectively.

Preferably, the diluent is Tris buffer with the components of 50mM Tris and 150mM NaCl and the pH value of 7.4.

Preferably, the washing solution is Tris buffer added with Tween-20 with volume fraction of 0.05%.

Preferably, the pre-excitation liquid comprises 0.2M NaOH solution.

Preferably, the exciting liquid has a component of 0.06% H₂O₂。

Preferably, the technical scheme is represented by a magnetic particle full-automatic chemiluminescence immunoassay kit, which is characterized by comprising the following reagents related to the method of the first aspect of the disclosure: comprises the following components: FITC antibody coated on the magnetic particles, FITC marked ASC antibody, acridinium ester marked ASC detection antibody, calibrator and quality control product, wherein the main component of the calibrator and the quality control product is recombinant human ASC protein.

Preferably, the technical scheme of the invention is as follows: mixing 20 μ l sample with 50 μ l FITC labeled ASC antibody and 50 μ l acridinium ester labeled ASC detection antibody, and incubating at 37 deg.C for 30 min; then adding 50 mul of FITC antibody-coated magnetic particles, and continuing to incubate for 20 minutes at 37 ℃; removing supernatant through cleaning and magnetic separation; relative luminescence values (RLU) were read by adding 100. mu.l each of 0.06% hydrogen peroxide solution and 0.2M sodium hydroxide solution. The ASC concentration and the RLU form a certain proportional relation, and the measuring instrument automatically fits and calculates the ASC concentration.

Through the technical scheme, the invention provides a method for evaluating ischemic stroke diseases by measuring the content of apoptosis-related spot-like protein in human body fluid and preparation and application of a related magnetic particle full-automatic chemiluminescence immunoassay kit.

The technology of the invention has the following advantages: the apoptosis-related spot-like protein (ASC) with a caspase recruitment domain (CARD) and a thermoprotein-like structure domain (PYD) provided by the invention is used as an ischemic stroke marker, is a body fluid marker with sensitivity and specificity, is used for early diagnosis, postoperative monitoring and prognosis evaluation of ischemic stroke, and overcomes the defects of difficult diagnosis and follow-up of stroke caused by lack of body fluid markers.

The invention provides a magnetic particle full-automatic chemiluminescence immunoassay kit for selectively evaluating ischemic stroke diseases, which has good specificity, sensitivity, stability and convenience in use and is suitable for clinical examination.

Drawings

FIG. 1 calibration curve for detection of ASC protein.

FIG. 2 receiver operating characteristic Curve (ROC) of ASC.

FIG. 3 compares ASC protein concentrations in patients with ischemic stroke and in normal persons.

FIG. 4 shows the health of the magnetic particle full-automatic chemiluminescence immune ASC analysis kit.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Example 1 construction of a kit Using ASC protein as a marker for cerebral apoplexy

A magnetic particle full-automatic chemiluminescence immunoassay kit for measuring ASC content in human serum samples. Comprises the following components: FITC antibody coated on the magnetic particles, FITC marked ASC antibody, acridine ester marked ASC detection antibody, calibrator and quality control product, wherein the main component of the calibrator and the quality control product is recombinant human ASC protein.

Coating FITC antibody magnetic particles, wherein the concentration of a mother solution of the magnetic particles is 10mg/mL, and the concentration of a working solution diluted by 20 times is 0.5 mg/mL;

FITC-labeled ASC antibody, the concentration of the mother solution was 1.3mg/mL, and the concentration of the working solution obtained by diluting 1000-fold was 1.3. mu.g/mL.

The concentration of the acridinium ester-labeled ASC detection antibody was 1.3mg/mL, and the concentration of the working solution obtained by diluting the antibody 4000 times was 0.33. mu.g/mL.

The ASC concentrations of the calibrator were 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, and 10ng/mL, respectively.

The diluent is Tris buffer solution, the components of the Tris buffer solution are 50mM Tris and 150mM NaCl, and the pH value is 7.4;

the cleaning solution is Tris buffer solution added with Tween-20 with the volume fraction of 0.05 percent;

the pre-excitation liquid is 0.2M NaOH solution;

the exciting liquid contains 0.06% H₂O₂。

Example 2 method of Using magnetic particle full-automatic chemiluminescence immunoassay kit for measuring content of ASC in human serum sample

Mixing 20 μ l of calibrator or sample with 50 μ l of FITC-labeled ASC antibody and 50 μ l of acridinium ester-labeled ASC detection antibody, and incubating at 37 deg.C for 30 min; then adding 50 mul of FITC antibody-coated magnetic particles, and continuing to incubate for 20 minutes at 37 ℃; removing supernatant through cleaning and magnetic separation; relative luminescence values (RLU) were read by adding 100. mu.l each of 0.06% hydrogen peroxide solution and 0.2M sodium hydroxide solution. The concentration of ASC and RLU form a certain proportional relation, and the analyzer calculates the concentration of ASC in a fitting way.

Example 3 preparation of calibration curves

Calibrators were added at different concentrations according to the procedure of example 2, with the concentrations of calibrators being 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, and 10ng/mL, respectively. The rest of the procedure was the same as in example 2. Drawing a calibration curve according to the measured value by using statistical drawing software according to the relative luminous value measured by the calibrator, wherein the abscissa is the concentration of ASC protein in the sample to be measured, and the ordinate is the relative luminous value; the linear fit equation for the measured calibration curve is: y is 44449x +4947.3, where y is the relative luminescence of the sample well and x is the concentration of ASC protein in serum (ng/mL). Calibration curves as shown in fig. 1, the R-Square (i.e., the regression coefficient of the calibration curve) is 0.9962.

Example 4 detection of clinical samples Using magnetic particle full-automatic chemiluminescence immunoassay kit with serum marker ASC as ischemic Stroke marker

Blood samples were collected and processed according to conventional serum separation methods to obtain serum. The detection method described in example 1 was performed for the serum sample (n 101) of ischemic stroke patients and the serum sample (n 214) of normal individuals. The results are shown in Table 1 below.

Table 1: ASC value determined for 214 normal samples

Table 2: determination of ASC values in 101 ischemic Stroke patients

The results of the detection were subjected to the following statistical analysis using the software span 16.0.

Determination of optimal diagnostic threshold (Cut off value):

and calculating the Youden index according to the sensitivity and specificity of each possible tangent point in the statistical result, and selecting the maximum tangent point as a critical point, wherein the results are shown in the following table.

Table 3: statistical analysis of positive and negative sample measurements

Therefore, the serum ASC content ≧ 0.29ng/mL was determined as the optimal critical point on the receiver operating characteristic curve (ROC) for ASC, i.e., the Cut off value (shown in FIG. 2). The sensitivity of diagnosing ischemic stroke by ASC content is 80.20%, and the specificity is 71.03%.

Differences in ASC in serum of ischemic stroke patients and normal population: the comparison of the concentrations detected according to example 1 is shown in FIG. 2. The mean concentration (normal individuals) was 0.29ng/mL, the mean concentration (ischemic stroke) was 0.78ng/mL, P < 0.0001, ASC was higher in serum of patients with ischemic stroke than in normal individuals by t-test, and there were statistical differences (fig. 3).

Example 5 soundness of magnetic particle full-automatic chemiluminescence immunoassay kit for measuring ASC content in human serum sample

Robustness (validity) is one of the reliability indicators of immunoassays. Immunochemical assays in principle require that the immunochemical properties of the calibration reagent and the test substance are identical, even if there is some structural difference between the two, without their dose-response curves deviating from parallel to one another. High-value serum containing the substance to be measured is generally adopted, diluted according to a certain proportion and then the content of the serum is measured. This assay is of good robustness if the dilution factor is linear with the content. The integrity can be used to verify the presence of unknown impurities or cross-reactive species in the sample being tested.

In the test for the health of the present embodiment, a high ASC sample, i.e., a 95 # ischemic stroke patient serum sample (ASC 4.45ng/mL), was taken. Another sample with ASC approximate to 0 value, namely No. 167 normal individual serum sample (ASC is 0.05ng/mL), is taken, the No. 95 ASC high-value sample is diluted according to the following proportion to prepare a sample with serial dilution concentration, and the ASC content of the sample is determined by using the magnetic particle full-automatic chemiluminescence ASC immunoassay kit. The results are shown in table 4 below and fig. 4.

The results show that: the dilution factor is in linear relation with the content, and the correlation number is 0.998, which indicates that the analysis has good soundness.

Table 4: soundness of the kit of the invention

Example 6 comparison of serum markers ASC and S100B as ischemic stroke markers

Serum S100B was measured with a S100B kit and ASC was measured with the kit of the present invention for 50 patients with ischemic stroke and 49 normal persons. The results are given in the following table:

table 5: the kit and the S100B marker kit sample detection constant value

The 99 samples were counted and analyzed for ROC curves for S100B and ASC, as follows:

table 6: statistical analysis of determination results of the kit and the S100B marker kit

The AUC area of ASC is 0.7151, greater than the AUC area 0.6831 of S100B; this indicates that ASC has a greater advantage in distinguishing between ischemic stroke and normal population than S100B.

The various features described in the foregoing detailed description may be combined in any suitable manner without contradiction, and various combinations that are possible in the present disclosure will not be further described in order to avoid unnecessary repetition.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

The present invention is illustrated by the above examples, but the present invention is not limited to the above process steps, i.e., it is not meant to imply that the present invention must rely on the above process steps to be practiced. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.