Anti-beta2-glycoprotein i antibody detection reagent and chemiluminescence detection kit

Abstract

The application provides an anti-beta2-glycoprotein I antibody detection reagent and a chemiluminescence detection kit, wherein the anti-beta2-glycoprotein I antibody detection reagent comprises a biotin-labeled beta2-glycoprotein I antigen prepared by the reaction of biotin with the beta2-glycoprotein I antigen under the catalysis of a Sortase A enzyme and with an LPETG label connected. The biotin with the specific label is used for the site-specific labeling of the N terminal of the beta2-glycoprotein I antigen, does not affect the whole amino acid sequence region (domains I-V) of the beta2-glycoprotein I antigen, and meanwhile, the purity of the connection product is high, the batch difference is small, the positive detection rate of the anti-beta2-glycoprotein I antibody magnetic microparticle chemiluminescence detection kit can be improved, the detection result accuracy is improved, and the batch difference is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of in vitro diagnostic reagents, and particularly relates to an anti-beta2-glycoprotein I antibody detection reagent and a chemiluminescence detection kit. BACKGROUND

[0002] Anti-phospholipid antibody is a general term for a group of autoantibodies against various negatively charged phospholipids, among which, anticardiolipin antibody (ACA) is the most representative, and its target antigen is negatively charged cardiolipin in cell membrane and mitochondrial membrane, which is a glycerophospholipid structure. In pathological state, such phospholipids are distributed outside the cell membrane, and when they are combined with beta2-glycoprotein 1 in serum, the antigen sites are exposed, inducing the production of corresponding autoantibodies, i.e. anti-beta2-glycoprotein 1 antibody (anti-beta2-GPI antibody).

[0003] Anti-beta2-glycoprotein 1 antibody can be used as a marker of autoimmune thrombosis, and also provides serological evidence for distinguishing autoimmune diseases and infectious diseases. The normal range of anti-beta2-glycoprotein 1 antibody in serum is 0-20 RU / ml, and if it is higher than this value, it is mainly seen in anti-phospholipid antibody syndrome (its sensitivity is 30%-60%, and its specificity is 98%) and systemic lupus erythematosus (SLE) patients. Simultaneous determination of anti-beta2-glycoprotein 1 antibody and ACA can make the diagnosis rate of anti-phospholipid antibody syndrome reach 95%. In addition, anti-beta2-glycoprotein 1 antibody higher than the normal value can also be seen in habitual abortion, rheumatoid arthritis, Kawasaki disease, etc.

[0004] At present, the commonly used methods for detecting anti-beta2-glycoprotein 1 antibody include enzyme-linked immunosorbent assay (ELISA), immunoblotting and solid-phase radioimmunoassay. Among them, enzyme-linked immunosorbent assay is the most commonly used, but enzyme-linked immunosorbent assay is complex, and needs to be coated, washed and the like, which is time-consuming and laborious.

[0005] Magnetic microparticle chemiluminescence method is a new analysis method combining magnetic separation technology, chemiluminescence technology and immune analysis technology. Its principle is to use the free energy released by chemical reaction to excite the intermediate, so that it releases photons when returning from the excited state to the ground state, and the photons are determined for quantitative analysis. This method fully utilizes the rapidity and automation of magnetic separation technology, the high sensitivity of chemiluminescence technology, and the specificity of immune analysis. Magnetic microparticle chemiluminescence method needs to use biotin-labeled beta2-glycoprotein I antigen to specifically bind with anti-beta2-glycoprotein 1 antibody in the sample, and then react with streptavidin magnetic beads and alkaline phosphatase-labeled human IgG antibody to produce a fluorescent signal, which is related to the content of anti-beta2-glycoprotein 1 antibody. The performance of biotin-labeled beta2-glycoprotein I antigen directly affects the accuracy of the detection result and the batch difference, especially the positive detection rate.

[0006] However, the existing conventional biotin labeling technology has certain randomness, the connection efficiency cannot be guaranteed, and the protein activity is affected, there are disadvantages such as reducing the purity of the connection product, and the batch difference of the chemiluminescence detection kit composed of the same is large. The traditional coupling technology may bind to the lysine site on the whole amino acid sequence region (domain I~V) of the beta2-glycoprotein I antigen, and after biotin labeling, spatial steric hindrance may be formed, thereby causing the anti-beta2-glycoprotein I antibody chemiluminescence detection kit composed of the same to have missed detection, the detection result is not high in accuracy, and the magnetic microparticle chemiluminescence kit for detecting the anti-beta2-glycoprotein I antibody on the market is very limited. SUMMARY

[0007] The technical problem solved by the present application is to overcome the shortcomings of the prior art, and to provide an anti-beta2-glycoprotein I antibody detection reagent with high positive detection rate, high detection result accuracy and small batch difference, and a magnetic microparticle chemiluminescence detection kit containing the detection reagent.

[0008] To solve the above technical problems, the present application adopts the following technical solutions:

[0009] The present application provides an anti-beta2-glycoprotein I antibody detection reagent, which comprises a biotin-labeled beta2-glycoprotein I antigen and a buffer solution with a pH value of 7-8, wherein the biotin-labeled beta2-glycoprotein I antigen is prepared by reacting biotin with a LPETG tag and beta2-glycoprotein I antigen under the catalysis of Sortase A enzyme, and the biotin is connected to the N-terminal end of the beta2-glycoprotein I antigen through the LPETG tag.

[0010] In the embodiments of the present application, the buffer solution is 0.01-0.02M phosphate buffer solution containing 0.5wt%-1.5wt% bovine serum albumin.

[0011] In the embodiments of the present application, the concentration of the biotin-labeled beta2-glycoprotein I antigen in the anti-beta2-glycoprotein I antibody detection reagent is 0.5-1.0 μg / mL, for example, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, or 1.0 μg / mL.

[0012] In the embodiments of the present application, the anti-beta2-glycoprotein I antibody detection reagent is composed of the biotin-labeled beta2-glycoprotein I antigen and the buffer solution with a pH value of 7-8.

[0013] Preferably, the LPETG tag is connected to the biotin through PEGn, n is an integer between 10 and 20, for example, n is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0014] Further preferably, n is an integer between 10 and 15, and further preferably, n is an integer between 11 and 13.

[0015] In an embodiment of the present application, the amino acid sequence of the Sortase A enzyme is shown as SEQ ID NO: 1.

[0016] In an embodiment of the present application, the amino acid sequence of the β2-glycoprotein I antigen is shown as SEQ ID NO: 2.

[0017] In an embodiment of the present application, the method for preparing the biotin-labeled β2-glycoprotein I antigen is as follows: biotin connected with LPETG tag, Sortase A enzyme and β2-glycoprotein I antigen are reacted in TBS buffer solution containing 8-15 mM CaCl2, pH 7-8, at 24-26℃ and 500-1000 rpm for 2-4 h, and the obtained reaction solution is subjected to gel filtration chromatography column to remove Sortase A enzyme and free biotin connected with LPETG tag.

[0018] Further, the initial concentration of biotin connected with LPETG tag in the TBS buffer solution is 0.5-2 mM, the initial concentration of Sortase A enzyme in the TBS buffer solution is 40-80 μM, and the initial concentration of β2-glycoprotein I antigen in the TBS buffer solution is 15-50 μM.

[0019] According to some specific embodiments of the present application, the gel filtration chromatography column is Superdex 75 Increase 10 / 300 GL.

[0020] The second aspect of the present application also provides an anti-β2-glycoprotein I antibody detection kit, which is a magnetic microparticle chemiluminescence detection kit, comprising R1 reagent, R2 reagent, M reagent and chemiluminescence substrate, the R1 reagent is the anti-β2-glycoprotein I antibody detection kit described above, the M reagent is SA magnetic bead solution, the R2 reagent is enzyme-labeled secondary antibody solution, and the chemiluminescence substrate is the chemiluminescence substrate corresponding to the enzyme-labeled secondary antibody.

[0021] In the embodiment of the present application, the concentration of the M reagent is 0.4-1 mg / mL, for example, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL.

[0022] In the embodiment of the present application, the particle size of the SA magnetic beads in the M reagent is 0.5-1.5 μm, for example, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm.

[0023] In the embodiment of the present application, the concentration of the R2 reagent is 0.05-0.2 μg / mL, and the enzyme-labeled secondary antibody is an alkaline phosphatase-labeled mouse anti-human IgG solution.

[0024] In the embodiment of the present application, the chemiluminescent substrate is AMPPD (1,2-dioxetane derivative).

[0025] In the embodiment of the present application, the kit comprises sample diluent, calibrators and quality control, wherein the sample diluent is 5-10 mM phosphate buffer with pH of 7-7.5. The anti-β2-glycoprotein I antibody is diluted into the first calibrator with a concentration of 20 RU / mL and the second calibrator with a concentration of 200 RU / mL by using 5-10 mM phosphate buffer with pH of 7-7.5. The anti-β2-glycoprotein I antibody is diluted into the first quality control with a concentration of 10 RU / mL and the second quality control with a concentration of 80 RU / mL by using 5-10 mM phosphate buffer with pH of 7-7.5.

[0026] Compared with the prior art, the present application has the following advantages:

[0027] The present application labels the N terminal of the β2-glycoprotein I antigen by using biotin with specific label, without affecting the whole amino acid sequence region (domain I-V) of the β2-glycoprotein I antigen, and the purity of the connection product is high, the batch difference is small, which can improve the positive detection rate and the detection result accuracy of the anti-β2-glycoprotein I antibody magnetic microparticle chemiluminescence detection kit, and reduce the batch difference. DETAILED DESCRIPTION

[0028] The inventors of the present application found in previous studies that the existing chemical coupling biotin technology has a certain randomness, which cannot guarantee the connection efficiency, and also affects the activity of the protein, and causes the problem of impure connection product. The traditional coupling technology can combine with the lysine sites on the whole amino acid sequence region (domain I~V) of the β2-glycoprotein I antigen, and after biotin labeling, spatial steric hindrance can be formed, thereby causing the anti-β2-glycoprotein I antibody detection kit composed of the same to have missed detection, and the detection result is not accurate. At the same time, the existing biotin labeling technology is randomly combined, the connection product is not pure, and the problem of large batch difference of the detection kit composed of the same is easily caused. Therefore, the inventors of the present application have carried out a large number of research and experimental verification, and the biotin is labeled at the N-terminal of the β2-glycoprotein I antigen by using a specific label, thereby overcoming the above problems, and providing an anti-β2-glycoprotein I antibody magnetic microparticle chemiluminescence detection kit with high positive detection rate and detection result accuracy and small batch difference. Specifically, the technical scheme adopted by the inventors of the present application is that the anti-β2-glycoprotein I antibody detection kit comprises a biotin-labeled β2-glycoprotein I antigen and a buffer solution with a pH value of 7~8, the biotin-labeled β2-glycoprotein I antigen is prepared by reacting biotin with a LPETG label and β2-glycoprotein I antigen under the catalysis of Sortase A enzyme, and the biotin is connected to the N-terminal of the β2-glycoprotein I antigen through the LPETG label.

[0029] Further, the present application selects a specific chain length hydrophilic biotin for enzyme-catalyzed site coupling, which further ensures that the antigen-antibody reaction site is not affected, and the coupling product is uniform, stable, and has small batch difference. Experimental verification shows that when the PEGn connecting the biotin and the LPETG label is PEG10~15, it has obviously better effect, and the PEG12 has the best effect, which can effectively avoid the shielding of the antigen reaction site. Chain length that is too long can cause false positive problems, and chain length that is too short can also shield the reaction site, thereby affecting the performance of the kit.

[0030] The present application will be further described below in conjunction with the examples. However, the present application is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are the conventional conditions in the industry. The technical features involved in each embodiment of the present application can be combined with each other as long as they do not conflict with each other.

[0031] In the following examples and comparative examples, the implementation conditions not mentioned are the conventional conditions in the industry, and the reagents not mentioned are commercial products.

[0032] Example 1

[0033] The embodiment provides an anti-beta2-glycoprotein I antibody detection reagent and a chemiluminescence detection kit containing the detection reagent.

[0034] The detection reagent in the embodiment is a biotin-labeled beta2-glycoprotein I antigen, and a preparation method thereof comprises the following steps:

[0035] (1) Biotin-PEG12-LPETG (biotin-polyethylene glycol-LPETG tag) with an LPETG tag is synthesized by Suzhou Xiruima Chemical Co., Ltd.

[0036] (2) 1 mM of Biotin-PEG12-LPETG, 50 μM of Sortase A enzyme (the sequence is shown as SEQ ID NO: 1) and 25 μM of beta2-glycoprotein I antigen (the sequence is shown as SEQ ID NO: 2) are set in a 10 mM CaCl2TBS buffer with a pH of 7.5, the temperature is set to 25 DEG C, the rotation speed is set to 800 rpm, the reaction is carried out for 3 hours, 20 mM Tris buffer (pH 7.4) containing 150 mM NaCl is used, a Superdex 75 Increase 10 / 300 GL column is first equilibrated at a flow rate of 0.6 mL / min for at least 3 times the column volume (72 mL), then the sample is loaded, and then elution is carried out at a flow rate of 0.8 mL / min, the eluate containing the molecular weight of 54-56 kDa target protein conjugate is collected, and Biotin-PEG12-LPET-beta2-glycoprotein I antigen with a concentration greater than 0.1 mg / mL is obtained by concentration, and biotin is specifically labeled at the N-terminal end of beta2-glycoprotein I.

[0037] (3) The Biotin-PEG12-LPET-beta2-glycoprotein I antigen is diluted into a Biotin-PEG12-LPET-beta2-glycoprotein I antigen solution with a concentration of 0.8 μg / mL by using 0.02 M phosphate buffer with a pH of 7.5 containing 1 wt% bovine serum albumin, and the Biotin-PEG12-LPET-beta2-glycoprotein I antigen solution is R1 reagent.

[0038] Three batches were prepared. The R1 reagent of each batch was mixed with M reagent (0.6 mg / mL SA magnetic beads: 1 μm streptavidin magnetic beads), R2 reagent (enzyme-labeled secondary antibody: 0.1 μg / mL alkaline phosphatase-labeled mouse anti-human IgG), chemiluminescent substrate (AMPPD), sample diluent (8 mM phosphate buffer, pH 7.2), calibrators (anti-β2-glycoprotein I antibody was diluted with 8 mM phosphate buffer, pH 7.2, to a concentration of 20 RU / mL for the first calibrator and 200 RU / mL for the second calibrator), and quality controls (anti-β2-glycoprotein I antibody was diluted with 8 mM phosphate buffer, pH 7.2, to a concentration of 10 RU / mL for the first quality control and 80 RU / mL for the second quality control) to form an anti-β2-glycoprotein I antibody chemiluminescent detection kit, and were recorded as lot #1, lot #2, and lot #3, respectively.

[0039] Comparative Example 1

[0040] This comparative example provides another anti-β2-glycoprotein I antibody detection reagent and an anti-β2-glycoprotein I antibody chemiluminescent detection kit containing the detection reagent.

[0041] The anti-β2-glycoprotein I antibody detection reagent of this comparative example was prepared by dissolving sulfo-NHS-PEG12-Biotin reagent in DMSO to 10 mg / mL, continuously adding drop by drop to a 25 μM β2-glycoprotein I antigen solution dissolved in 20 mM PB buffer (pH 7.4) containing 150 mM NaCl, and reacting at room temperature for 30 min. After the reaction was completed, free sulfo-NHS-PEG12-Biotin was removed by dialysis using 0.1 M PBS buffer (pH 7.4) to obtain Biotin-PEG12-β2-glycoprotein I antigen. The Biotin-PEG12-β2-glycoprotein I antigen was diluted with 0.02 M phosphate buffer (pH 7.5) containing 1 wt% bovine serum albumin to a concentration of 0.8 μg / mL to obtain a Biotin-PEG12-LPET-β2-glycoprotein I antigen solution, which was the R1 reagent.

[0042] Three batches were prepared. The R1 reagent of each batch was mixed with M reagent (0.6 mg / mL SA magnetic beads: 1 μm streptavidin magnetic beads), R2 reagent (enzyme-labeled secondary antibody: 0.1 μg / mL alkaline phosphatase-labeled mouse anti-human IgG), chemiluminescent substrate (AMPPD), sample diluent (8 mM phosphate buffer, pH 7.2), calibrators (anti-β2-glycoprotein I antibody was diluted with 8 mM phosphate buffer, pH 7.2, to a concentration of 20 RU / mL for the first calibrator and 200 RU / mL for the second calibrator), and quality controls (anti-β2-glycoprotein I antibody was diluted with 8 mM phosphate buffer, pH 7.2, to a concentration of 10 RU / mL for the first quality control and 80 RU / mL for the second quality control) to form an anti-β2-glycoprotein I antibody chemiluminescent detection kit, and were recorded as lot #1, lot #2, and lot #3, respectively.

[0043] The method for using the anti-β2-glycoprotein I antibody chemiluminescence detection kit of Example 1 and Comparative Example 1 is as follows:

[0044] Step 1: Place the kit into the corresponding position of the reagent compartment of the SMART 6500 full-automatic chemiluminescence instrument (HORIBO), and input the kit information into the instrument system through the barcode scanner or set through the instrument software.

[0045] Step 2: Place the calibrators into the sample compartment of the instrument. Identify the calibrator information through the barcode scanner, and assign the calibrator position in the instrument system.

[0046] Step 3: Place the quality control samples and samples to be tested into the sample compartment of the instrument, and edit the corresponding detection information through the instrument software. (Clinical samples are diluted to the concentration range of the calibrators using the sample diluent before detection.)

[0047] Step 4: Start the running program, and all calibrator, quality control sample and sample to be tested processing steps will be automatically executed.

[0048] For example, when the device detects the sample to be tested, the steps of the device automatic execution are as follows:

[0049] Step 4.1: Add 50 μL of M reagent and 50 μL of R1 reagent into the detection tube in turn, then add 20 μL of the sample to be tested, mix, and incubate at 37°C for 20 min;

[0050] Step 4.2: Add a magnetic field to make the system after incubation in step 4.1 settle in the magnetic field, remove the supernatant, and add 500 μL of the instrument-matched washing solution for 3-5 times of washing;

[0051] Step 4.3: Remove the magnetic field, then add 135 μL of R2 reagent to the system after washing in step 4.2, mix, and incubate at 37°C for 15 min;

[0052] Step 4.4: Add a magnetic field to make the system after incubation in step 4.3 settle in the magnetic field, remove the supernatant, and add 500 μL of the washing solution for 3-5 times of washing, remove the magnetic field, and shake to fully suspend the magnetic particles;

[0053] Step 4.4: Add a magnetic field to make the suspended magnetic particles settle in the magnetic field, remove the supernatant, remove the magnetic field, then add 150 μL of chemiluminescence substrate, remove the magnetic field, fully suspend, and incubate at 37°C for 5 min to detect the relative luminescence intensity value. The instrument software automatically performs standard curve fitting with the concentration of the calibrators of different concentrations as the abscissa X and the luminescence value as the ordinate Y, and automatically outputs the test concentration value of the test sample. All steps can be fully automated.

[0054] The anti-β2-glycoprotein I antibody chemiluminescence detection kit of Example 1 and the SMART6500 full-automatic chemiluminescence instrument (Hualibo) of Comparative Example 1 were used to detect 51 clinical positive samples and 32 clinical negative samples, respectively, and the detection was carried out according to the method and instrument described above, and the software automatically generated the corresponding luminescence value (RLU) and the calculated concentration value of each sample. The batch precision CV values of 3 batches of reagents of Example 1 and Comparative Example 1 were calculated, and the cut off≤20RU / mL was judged as negative, and the cut off>20RU / mL was judged as positive. The detection results of Example 1 are shown in Table 1, and the coincidence rate of positive and negative is shown in Table 2.

[0055] Table 1

[0056]

[0057]

[0058]

[0059] Table 2

[0060]

[0061] The detection results of Comparative Example 1 are shown in Table 3, and the coincidence rate of positive and negative is shown in Table 4.

[0062] Table 3

[0063]

[0064]

[0065]

[0066] Table 4

[0067]

[0068] According to the above detection results, Example 1 uses Sortase A enzyme to perform Biotin-PEG12-LPETG site-specific biotin coupling labeling, which avoids the steric hindrance caused by biotin labeling and improves the detection rate compared with Comparative Example. At the same time, the sample deviation of less than 5RU / ml is less than or equal to ±25%, the sample deviation of more than 5RU / ml is less than or equal to ±15%, and the precision of 3 batches is less than 15%, and the batch difference is controllable. While Comparative Example 1 uses sulfo-NHS-PEG12-biotin chemical random coupling labeling, which may or may not label the site. When labeling the site, it is easy to cause some samples to be missed and form false negatives. At the same time, due to the randomness of labeling, the batch difference of labeling is large and difficult to control, with a deviation of more than ±15%, and the precision of 3 batches is more than 15%.

[0069] The above detailed description of the application is intended to be illustrative and not limiting. Other alternatives will be apparent to those of skill in the art without departing from the spirit of the application.

Claims

1. An anti-β2-glycoprotein I antibody detection reagent, characterized in that: The anti-β2-glycoprotein I antibody detection reagent includes a biotin-labeled β2-glycoprotein I antigen and a buffer solution with a pH value of 7-8. The biotin-labeled β2-glycoprotein I antigen is prepared by reacting biotin connected to an LPETG tag with the β2-glycoprotein I antigen under the catalysis of Sortase A. The biotin connected to the LPETG tag is Biotin-PEG12-LPETG, and the LPETG tag is connected to the biotin via PEG12. The biotin-labeled β2-glycoprotein I antigen is Biotin-PEG12-LPET-β2-glycoprotein I antigen, and the biotin is connected to the N-terminus of the β2-glycoprotein I antigen via the LPETG tag.

2. The anti-β2-glycoprotein I antibody detection reagent according to claim 1, characterized in that The buffer is a 0.01-0.02 M phosphate buffer containing 0.5 wt%-1.5 wt% of bovine serum albumin; and / or, the concentration of the biotin-labeled β2-glycoprotein I antigen in the anti-β2-glycoprotein I antibody detection reagent is 0.5-1.0 μg / mL; And / or, the anti-β2-glycoprotein I antibody detection reagent consists of the biotin-labeled β2-glycoprotein I antigen and the buffer solution with a pH value of 7-8.

3. The anti-β2-glycoprotein I antibody detection reagent according to claim 1, characterized in that The amino acid sequence of the Sortase A enzyme is shown in SEQ ID NO: 1; And / or, the amino acid sequence of the β2-glycoprotein I antigen is shown in SEQ ID NO:

2.

4. The anti-β2-glycoprotein I antibody detection reagent according to claim 1, characterized in that The preparation method of the biotin-labeled β2-glycoprotein I antigen comprises: reacting biotin connected to an LPETG tag, a Sortase A enzyme, and the β2-glycoprotein I antigen in a TBS buffer solution containing 8-15 mM CaCl2 and a pH of 7-8 at 24-26° C. and a rotation speed of 500-1000 rpm for 2-4 hours; and removing the Sortase A enzyme and free biotin connected to the LPETG tag from the obtained reaction solution through a gel filtration chromatography column.

5. The anti-β2-glycoprotein I antibody detection reagent according to claim 4, characterized in that The initial concentration of the biotin connected to the LPETG tag in the TBS buffer is 0.5-2 mM, the initial concentration of the Sortase A enzyme in the TBS buffer is 40-80 μM, and the initial concentration of the β2-glycoprotein I antigen in the TBS buffer is 15-50 μM.

6. The anti-β2-glycoprotein I antibody detection reagent according to claim 4, characterized in that The gel filtration chromatography column is Superdex 75 Increase 10 / 300 GL.

7. An anti-β2-glycoprotein I antibody detection kit, characterized in that: The anti-β2-glycoprotein I antibody detection kit is a magnetic particle chemiluminescence detection kit, which includes R1 reagent, R2 reagent, M reagent and a chemiluminescent substrate, wherein the R1 reagent is the anti-β2-glycoprotein I antibody detection reagent described in any one of claims 1 to 6, the M reagent is an SA magnetic bead solution, the R2 reagent is an enzyme-labeled secondary antibody solution, and the chemiluminescent substrate is a chemiluminescent substrate corresponding to the enzyme-labeled secondary antibody.

8. The anti-β2-glycoprotein I antibody detection kit according to claim 7, characterized in that: The concentration of the M reagent is 0.4~1.0 mg / mL, and / or the particle size of the SA magnetic beads in the M reagent is 0.5~1.5 μm, and / or the concentration of the R2 reagent is 0.05~0.2 μg / mL, the enzyme-labeled secondary antibody is an alkaline phosphatase-labeled mouse anti-human IgG solution, and / or the chemiluminescent substrate is AMPPD; and / or the kit contains a sample diluent, a calibrator and a quality control product, and the sample diluent is a 5~10 mM phosphate buffer with a pH of 7~7.5.

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