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

Abstract

The invention provides an anti-beta2-glycoprotein I antibody detection reagent and a chemiluminescence detection kit, and the anti-beta2-glycoprotein I antibody detection reagent comprises a biotin-labeled beta2-glycoprotein I antigen prepared by reacting biotin connected with an LPETG label with the beta2-glycoprotein I antigen under the catalytic action of Sortase A enzyme. The N terminal of the beta2-glycoprotein I antigen is labeled with the biotin with a specific label in a fixed point manner, all amino acid sequence regions (structural domains I-V) of the beta2-glycoprotein I antigen are not influenced, and meanwhile, the purity of a connection product is high, the difference between batches is small, and the method is suitable for large-scale production. The positive detection rate and the detection result accuracy of the anti-beta2-glycoprotein I antibody magnetic particle chemiluminescence detection kit can be improved, and the difference between batches can be reduced.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro diagnostic reagents, and in particular relates to an anti-β2-glycoprotein I antibody detection reagent and a chemiluminescence detection kit. Background Art

[0002] Antiphospholipid antibodies are a group of autoantibodies against various negatively charged phospholipids, among which cardiolipin antibodies (ACA) are the most representative. Its target antigen is negatively charged cardiolipin present in cell membranes and mitochondrial membranes, which is a glycerophospholipid structure. Under pathological conditions, this type of phospholipid is distributed outside the cell membrane. When it binds to β2-glycoprotein type 1 in the serum, the antigenic site is exposed, inducing the production of corresponding autoantibodies, namely anti-β2-glycoprotein 1 antibodies (anti-β2-GPI antibodies).

[0003] Anti-β2-glycoprotein 1 antibodies can be used as a marker for autoimmune thrombosis and provide serological evidence for distinguishing autoimmune diseases from infectious diseases. The normal range of anti-β2-glycoprotein 1 antibodies in serum is 0-20RU / ml. If it is higher than this value, it is mainly seen in patients with antiphospholipid antibody syndrome (its sensitivity is 30%-60%, specificity is 98%) and systemic lupus erythematosus (SLE). Simultaneous determination of anti-β2-glycoprotein 1 antibodies and ACA can make the diagnosis rate of antiphospholipid antibody syndrome reach 95%. In addition, anti-β2-glycoprotein 1 antibodies higher than normal values ​​can also be seen in habitual abortion, rheumatoid arthritis, Kawasaki disease, etc.

[0004] At present, the commonly used methods for detecting anti-β2-glycoprotein 1 antibodies are enzyme-linked immunosorbent assay (ELISA), immunoblotting and solid phase radioimmunoassay. Among them, ELISA is the most widely used, but ELISA is complicated to operate and requires coating, washing, etc., which is time-consuming and labor-intensive.

[0005] Magnetic particle chemiluminescence is an emerging analytical method that combines magnetic separation technology, chemiluminescence technology and immunoassay technology. Its principle is to use the free energy released by chemical reactions to excite intermediates, so that they release photons when they return from the excited state to the ground state, and perform quantitative analysis by measuring the photons. This method makes full use of the rapid and easy automation of magnetic separation technology, the high sensitivity of chemiluminescence technology, and the specificity of immunoassay. The magnetic particle chemiluminescence method requires the use of biotin-labeled β2-glycoprotein I antigen to specifically bind to the anti-β2-glycoprotein 1 antibody in the sample, and then react with streptavidin magnetic beads and alkaline phosphatase-labeled human IgG antibodies to produce a fluorescent signal to react with the anti-β2-glycoprotein 1 antibody content. The performance of the biotin-labeled β2-glycoprotein I antigen directly affects the accuracy of the test results and the batch-to-batch differences, especially the positive detection rate.

[0006] However, the existing conventional biotin labeling technology has a certain degree of randomness, the connection efficiency cannot be guaranteed, and it will also affect the activity of the protein. There are disadvantages such as reducing the purity of the connection product, which easily leads to large batch differences in the chemiluminescent detection kit composed of it. Traditional coupling technology may bind to the lysine site on the entire amino acid sequence region (domains I to V) of the β2-glycoprotein I antigen, and may form steric hindrance after biotinylation labeling, thereby causing the anti-β2-glycoprotein I antibody chemiluminescent detection kit composed of it to miss detection, and the detection result is not accurate, resulting in very limited magnetic particle chemiluminescent kits for detecting anti-β2-glycoprotein I antibodies on the market. Summary of the invention

[0007] The technical problem solved by the present invention is to overcome the deficiencies of the prior art and provide an anti-β2-glycoprotein I antibody detection reagent with high positive detection rate, high detection result accuracy and small batch difference, and a magnetic particle chemiluminescence detection kit containing the detection reagent.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides an anti-β2-glycoprotein I antibody detection reagent, which comprises a biotin-labeled β2-glycoprotein I antigen and a buffer solution with a pH value of 7 to 8. The biotin-labeled β2-glycoprotein I antigen is prepared by reacting biotin connected with an LPETG tag with the β2-glycoprotein I antigen under the catalysis of Sortase A enzyme, and the biotin is connected to the N-terminal end of the β2-glycoprotein I antigen through the LPETG tag.

[0010] In the embodiment of the present invention, the buffer is a 0.01-0.02 M phosphate buffer containing 0.5 wt % to 1.5 wt % of bovine serum albumin.

[0011] In an embodiment of the present invention, 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, for example, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, and 1.0 μg / mL.

[0012] In the embodiment of the present invention, the anti-β2-glycoprotein I antibody detection reagent is composed of the biotin-labeled β2-glycoprotein I antigen and the buffer solution with a pH value of 7-8.

[0013] Preferably, the LPETG tag is linked to the biotin via PEGn, where 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] More preferably, n is an integer between 10 and 15, and even more preferably, n is an integer between 11 and 13.

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

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

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

[0018] Furthermore, the initial concentration of the biotin connected with 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.

[0019] According to some specific embodiments of the present invention, the gel filtration chromatography column is Superdex 75Increase10 / 300GL.

[0020] The second aspect of the present invention also provides an anti-β2-glycoprotein I antibody detection kit, which is a magnetic particle chemiluminescence detection kit, comprising R1 reagent, R2 reagent, M reagent, and a chemiluminescent substrate, wherein the R1 reagent is the above-mentioned anti-β2-glycoprotein I antibody detection reagent, 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.

[0021] In an embodiment of the present invention, 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, and 1.0 mg / mL.

[0022] In an embodiment of the present invention, the particle size of the SA magnetic beads in the M reagent is 0.5 to 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, and 1.5 μm.

[0023] In the embodiment of the present invention, 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 invention, the chemiluminescent substrate is AMPPD (1,2-dioxetane derivative).

[0025] In an embodiment of the present invention, the kit includes a sample diluent, a calibrator and a quality control product, wherein the sample diluent is a 5-10 mM phosphate buffer at a pH of 7-7.5. The anti-β2-glycoprotein I antibody is diluted with a 5-10 mM phosphate buffer at a pH of 7-7.5 to form a first calibrator with a concentration of 20 RU / mL and a second calibrator with a concentration of 200 RU / mL. The anti-β2-glycoprotein I antibody is diluted with a 5-10 mM phosphate buffer at a pH of 7-7.5 to form a first quality control product with a concentration of 10 RU / mL and a second quality control product with a concentration of 80 RU / mL.

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

[0027] The present invention uses biotin with a specific tag to site-specifically label the N-terminus of the β2-glycoprotein I antigen without affecting the entire amino acid sequence region (domains I to V) of the β2-glycoprotein I antigen. At the same time, the connection product has high purity and small batch-to-batch differences. The present invention can improve the positive detection rate and detection result accuracy of the anti-β2-glycoprotein I antibody magnetic particle chemiluminescence detection kit and reduce batch-to-batch differences. DETAILED DESCRIPTION

[0028] The inventors of this application found in their previous studies that, due to the certain randomness of the existing chemical coupling biotin technology, not only the connection efficiency cannot be guaranteed, but also the protein activity will be affected, and the connection product will be impure. The traditional coupling technology may bind to the lysine sites on the entire amino acid sequence region (domains I to V) of the β2-glycoprotein I antigen, and may form steric hindrance after biotinylation labeling, thereby causing the anti-β2-glycoprotein I antibody detection kit composed of it to miss detection, and the detection result accuracy is not high. At the same time, the existing biotinylation labeling technology is randomly combined, and the connection product is impure, which easily causes the problem of large batch differences in the detection kit composed of it. For this reason, the inventors of this application have overcome the above problems through a lot of research and experimental verification, by fixed-point labeling of biotin with a specific label on the N-terminus of the β2-glycoprotein I antigen, and provide an anti-β2-glycoprotein I antibody magnetic particle chemiluminescent detection kit with high positive detection rate and detection result accuracy and small batch differences. Specifically, the technical solution adopted by the inventors of the present application is: 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 to 8, the biotin-labeled β2-glycoprotein I antigen is prepared by reacting biotin connected to an LPETG label with the β2-glycoprotein I antigen under the catalysis of Sortase A enzyme, and the biotin is connected to the N-terminal end of the β2-glycoprotein I antigen through the LPETG label.

[0029] Furthermore, the present invention selects a specific chain length of hydrophilic biotin for enzymatic site-specific coupling, further ensuring that the antigen-antibody reaction site is not affected, and the coupling product is uniform, stable, and has small batch-to-batch differences. It was verified by experiments that when the PEGn connecting the biotin and LPETG tags is PEG10-15, it has a significantly better effect, among which PEG12 has the best effect and can effectively avoid the antigen reaction site from being shielded. Too long a chain length may cause a false positive problem, and too short a chain length may also shield the reaction site, thereby affecting the performance of the kit.

[0030] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention 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 specified are conventional conditions in the industry, and the reagents not specified are all commercially available products.

[0032] Example 1

[0033] This embodiment provides an anti-β2-glycoprotein I antibody detection reagent and a chemiluminescence detection kit containing the detection reagent.

[0034] The detection reagent in this embodiment is a biotin-labeled β2-glycoprotein I antigen, and its preparation method includes the following steps:

[0035] (1) Suzhou Sirimas Chemicals Co., Ltd. was commissioned to synthesize biotin with an LPETG label: Biotin-PEG12-LPETG (biotin-polyethylene glycol-LPETG label).

[0036] (2) 1 mM Biotin-PEG12-LPETG, 50 μM Sortase A (sequence as shown in SEQ ID NO: 1) and 25 μM β2-glycoprotein I antigen (sequence as shown in SEQ ID NO: 2) were placed in 10 mM CaCl2 TBS buffer (pH 7.5) at 25°C and 800 rpm for 3 h. The Superdex 75 Increase was first equilibrated at a flow rate of 0.6 mL / min using 20 mM Tris buffer (pH 7.4) containing 150 mM NaCl. At least 3 column volumes (72 mL) of a 10 / 300GL column were used, and then the sample was loaded, and then eluted at a flow rate of 0.8 mL / min, and the eluate containing the target protein conjugate with a molecular weight of 54 to 56 kDa was collected and concentrated to obtain the Biotin-PEG12-LPET-β2-glycoprotein I antigen with a concentration greater than 0.1 mg / mL, and biotin was specifically labeled at the N-terminal end of β2-glycoprotein I.

[0037] (3) The Biotin-PEG12-LPET-β2-glycoprotein I antigen was diluted to a Biotin-PEG12-LPET-β2-glycoprotein I antigen solution with a concentration of 0.8 μg / mL using a 0.02 M phosphate buffer with a pH of 7.5 containing 1 wt % bovine serum albumin, i.e., reagent R1.

[0038] This embodiment repeats the labeling of 3 batches. The 3 batches of R1 reagents were respectively 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 at pH 7.2, calibrator: dilute the anti-β2-glycoprotein I antibody with 8 mM phosphate buffer at pH 7.2 to a first calibrator with a concentration of 20 RU / mL and a second calibrator with a concentration of 200 RU / mL, respectively, and quality control: dilute the anti-β2-glycoprotein I antibody with 8 mM phosphate buffer at pH 7.2 to a first quality control with a concentration of 10 RU / mL and a second quality control with a concentration of 80 RU / mL to form an anti-β2-glycoprotein I antibody chemiluminescent detection kit, which are correspondingly recorded as lot#1, lot#2 and lot#3.

[0039] Comparative Example 1

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

[0041] The preparation method of the anti-β2-glycoprotein I antibody detection reagent of this comparative example is as follows: sulfo-NHS-PEG12-Biotin reagent dissolved to 10 mg / mL in DMSO is continuously added dropwise to a 25 μM β2-glycoprotein I antigen solution dissolved in 20 mM PB buffer (pH 7.4) containing 150 mM NaCl, and the reaction is carried out at room temperature for 30 minutes. After the reaction is completed, the free sulfo-NHS-PEG12-Biotin is dialyzed with 0.1 M PBS buffer with a pH of 7.4 to obtain Biotin-PEG12-β2-glycoprotein I antigen, and the Biotin-PEG12-β2-glycoprotein I antigen is diluted with 0.02 M phosphate buffer with a pH of 7.5 containing 1 wt% of bovine serum albumin to a concentration of 0.8 μg / mL Biotin-PEG12-LPET-β2-glycoprotein I antigen solution, which is the R1 reagent.

[0042] In this comparative example, three batches were repeatedly marked, and the three batches of R1 reagent were respectively mixed with 0.6 mg / mL M reagent (same as M reagent in Example 1) and R2 reagent (same as R2 reagent in Example 1), chemiluminescent substrate (same as chemiluminescent substrate in Example 1), sample diluent (same as sample diluent in Example 1), calibrator (same as calibrator in Example 1) and quality control (same as quality control in Example 1) to form an anti-β2-glycoprotein I antibody chemiluminescent detection kit, which were correspondingly recorded as lot#1', lot#2' and lot#3'.

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

[0044] Step 1: Place the reagent kit into the corresponding position of the reagent compartment of the SMART 6500 fully automatic chemiluminescence analyzer (Haobo). The reagent kit information is input into the instrument system through a barcode scanner or set through the instrument supporting software.

[0045] Step 2: Place the calibrator in the instrument sample compartment. Use the barcode scanner to identify the calibrator information and assign the calibrator location in the instrument system.

[0046] Step 3: Place the quality control product / sample to be tested in the instrument sample compartment, and edit the corresponding test information through the instrument supporting software (clinical samples should be diluted to the calibration sample concentration range using sample diluent before testing).

[0047] Step 4: Start the running program and all calibration / quality control / test sample processing steps will be executed automatically.

[0048] Among them, taking the device detecting the sample to be tested as an example, the steps when the device automatically executes are:

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

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

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

[0052] Step 4.4: Add a magnetic field to allow the system incubated in step 4.3 to settle in the magnetic field, remove the supernatant, add 500 μL of cleaning solution and wash 3-5 times, remove the magnetic field, and shake to fully suspend the magnetic particles;

[0053] Step 4.4: Add a magnetic field to allow the suspended magnetic particles to settle in the magnetic field, remove the supernatant, remove the magnetic field, then add 150 μL of chemiluminescent 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 fits the standard curve with the concentration of the calibrator of different concentrations as the horizontal axis X and the luminescence value as the vertical axis 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 chemiluminescent detection kit and SMART6500 fully automatic chemiluminometer (Hao Ou Bo) of Example 1 and Comparative Example 1 were used to detect 51 clinical positive samples and 32 clinical negative samples, respectively. The detection was performed with reference to the method and instrument described above. The software automatically generated the luminescence value (RLU) corresponding to each sample and the back-calculated concentration value, and the inter-batch precision CV values ​​of the three batches of reagents tested in Example 1 and Comparative Example 1 were calculated, respectively. Cut off ≤ 20RU / mL was judged as negative, and cut off > 20RU / mL was judged as positive. The test results of Example 1 are shown in Table 1, and the positive and negative coincidence rates are statistically shown in Table 2.

[0055] Table 1

[0056]

[0057]

[0058]

[0059] Table 2

[0060]

[0061] The test results of comparative example 1 are shown in Table 3, and the positive and negative coincidence rates are shown in Table 4.

[0062] Table 3

[0063]

[0064]

[0065]

[0066] Table 4

[0067]

[0068] According to the above test results, Example 1 uses Sortase A enzyme to carry out Biotin-PEG12-LPETG fixed-point biotin coupling labeling, which avoids the steric hindrance caused by biotin labeling and improves the detection rate compared with the comparative example. At the same time, the sample deviation lower than 5RU / ml is ≤±25%, the sample deviation greater than 5RU / ml is ≤±15%, and the precision of 3 batches is less than 15%, and the batch difference is controllable. And comparative example 1 adopts sulfo-NHS-PEG12-biotin chemical random coupling labeling, which may be labeled on the site, and it may not be labeled on the site. When it is labeled on the site, it is easy to cause some samples to miss detection and form false negatives. At the same time, due to the randomness of the labeling, the batch difference of the labeling is large and difficult to control, the deviation is greater than ±15%, and the precision of 3 batches is greater than 15%.

[0069] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-β2-glycoprotein I antibody detection reagent, characterized in that: The anti-β2-glycoprotein I antibody detection reagent comprises a biotin-labeled β2-glycoprotein I antigen and a buffer solution with a pH value of 7 to 8. The biotin-labeled β2-glycoprotein I antigen is prepared by reacting biotin connected with an LPETG label with the β2-glycoprotein I antigen under the catalysis of Sortase A enzyme, and the biotin is connected to the N-terminal end of the β2-glycoprotein I antigen through the LPETG label.

2. The anti-β2-glycoprotein I antibody detection reagent according to claim 1, characterized in that: The buffer is a 0.01-0.02M phosphate buffer containing 0.5wt%-1.5wt% 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 LPETG tag is connected to the biotin via PEGn, where n is an integer between 10 and 20.

4. The anti-β2-glycoprotein I antibody detection reagent according to claim 2, characterized in that: The n is an integer between 10 and 15.

5. 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.

6. 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 the following steps: reacting biotin connected with an LPETG label, Sortase A enzyme and β2-glycoprotein I antigen in a TBS buffer solution containing 8-15 mM CaCl2 and a pH value 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 with an LPETG label from the obtained reaction solution through a gel filtration chromatography column.

7. The anti-β2-glycoprotein I antibody detection reagent according to claim 6, characterized in that: The initial concentration of the biotin connected with 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.

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

9. 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 8, 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.

10. The anti-β2-glycoprotein I antibody detection kit according to claim 9, 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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