阅读说明：本技术 一种封闭抗体的抗原包被酶标板的制备方法及其试剂盒 (Preparation method of antigen-coated enzyme label plate for blocking antibody and kit thereof ) 是由 张道兵 于 2021-09-03 设计创作，主要内容包括：本发明公开了一种人封闭抗体的抗原包被酶标板的制备方法及其试剂盒,用基因工程方法制备特异性重组抗原--HLAⅠ重组抗原和HLAⅡ重组抗原,几种抗原提取纯化后,制成混合抗原包被酶标板,与封闭抗体阳性对照品、阴性对照品、酶标记的抗体、稀释液、清洗液、显色液A、显色液B和终止液组成检测试剂盒,试剂盒建立了快捷简便测试人封闭抗体的检测方法,试剂盒主要用于供科学研究和临床实验的研究使用。(The invention discloses a preparation method of an antigen-coated enzyme label plate of a human blocking antibody and a kit thereof, wherein specific recombinant antigens, namely HLA I recombinant antigen and HLA II recombinant antigen, are prepared by a genetic engineering method, a mixed antigen-coated enzyme label plate is prepared after extracting and purifying a plurality of antigens, and the mixed antigen-coated enzyme label plate, a blocking antibody positive reference substance, a negative reference substance, an enzyme-labeled antibody, a diluent, a cleaning solution, a developing solution A, a developing solution B and a stop solution form a detection kit.)

1. A preparation method of an antigen-coated ELISA plate for blocking an antibody is characterized by comprising the following steps:

(1) transforming BL21 competent escherichia coli by using human HLA-A (NM-001242758) cDNA clone (vector is pDONR223), extracting plasmid DNA, sequencing and identifying an expression product of the BL21 competent escherichia coli, and expressing a large number of HLA-A protein fragments which are HLA I recombinant protein fragments;

(2) transforming BL21 competent escherichia coli by using human HLA-B (NM _005514) cDNA clone (a vector is pDONR223), extracting plasmid DNA, sequencing and identifying an expression product of the BL21 competent escherichia coli, and expressing a large number of HLA-B protein fragments which are HLA I recombinant protein fragments;

(3) BL21 competent Escherichia coli was transformed with human HLA-DRB1 (NM-001243965) cDNA clone (vector pDONR223), plasmid DNA was extracted, the expression product of BL21 competent Escherichia coli was sequenced and identified, and a large amount of HLA II protein was expressed.

(4) Diluting HLA I recombinant protein antigen and HLA II recombinant protein antigen with buffer solution of pH7-10, coating the antigen in microporous plate at 20-300 μ l/hole for 2-40 hr, washing the plate with PBST for 2-6 times for 1-10min, and drying;

(5) filling the wells with PBS containing 2-20% calf serum, incubating at 37 deg.C, sealing for 0.5-5 hr, washing the plate for 2-6 times, and sealing; the plate is an enzyme-labeled reaction plate coated by HLA I antigen and HLA II antigen, and can be used for the specificity detection of closed antibody IgG.

2. The method for preparing the antigen-coated elisa plate for blocking the antibody according to claim 1, wherein the HLA-a recombinant protein fragment is prepared by the steps of:

prokaryotic expression of HLA-A: transforming BL21 competent escherichia coli by using human HLA-A (NM-001242758) cDNA clone (vector is pDONR223), screening positive clone according to kanamycin resistance, extracting plasmid DNA of the positive clone for identification to obtain a fragment with the same molecular weight as that of a target fragment, primarily judging the positive result, extracting the plasmid DNA by using PCR positive clone, and sequencing to obtain a result which shows that the HLA-AcDNA fragment is correctly cloned to the pDONR223 and is homologous with a humanized HLA I gene in GenBank;

identification of HLA-a protein fragments: after being induced, the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection and ultrasonic cracking, and thallus protein is identified by Westernblotting and can generate strong positive reaction with HumanHLAclass I antibody, so that the recombinant protein has stronger immunogenicity;

large-scale expression and purification of HLA-A recombinant protein: after a large amount of induction, expression and affinity chromatography purification, a large amount of HLA-A recombinant protein fragments are obtained.

3. The method for preparing the antigen-coated elisa plate for blocking the antibody according to claim 1, wherein the HLA-B recombinant protein fragment is prepared by the steps of:

prokaryotic expression of HLA-B: transforming BL21 competent escherichia coli by using a human HLA-B (NM _005514) cDNA clone (a vector is pDONR223), screening positive clones according to kanamycin resistance, extracting plasmid DNA of the positive clones for identification to obtain fragments with the molecular weight consistent with that of a target fragment, and primarily judging the fragments to be positive; extracting plasmid DNA by PCR positive cloning, and sequencing to show that the HLA-BcDNA fragment is correctly cloned to pDONR223 and is homologous with human HLA I gene in GenBank;

identification of HLA-B protein fragments: after being induced, the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection and ultrasonic cracking, and thallus protein is identified by Westernblotting and can generate strong positive reaction with HumanHLAclass I antibody, so that the recombinant protein has stronger immunogenicity;

and (3) large-scale expression and purification of HLA-B recombinant protein: after a large amount of induction, expression and affinity chromatography purification, a large amount of HLA-B recombinant protein fragments are obtained.

4. The method for preparing the antigen-coated elisa plate for blocking the antibody according to claim 1, wherein the HLA ii recombinant protein is prepared by the steps of:

prokaryotic expression of HLA ii: transforming BL21 competent escherichia coli by human HLA-DRB1 (NM-001243965) cDNA clone (vector is pDONR223), screening positive clone according to kanamycin resistance, extracting plasmid DNA of the positive clone for identification to obtain a fragment with the same molecular weight as that of a target fragment, primarily judging the positive, extracting the plasmid DNA by PCR positive clone, and sequencing to obtain a result which shows that the HLA II cDNA fragment is correctly cloned to the pDONR223 and is homologous with a humanized HLA II gene in GenBank;

identification of HLA ii protein fragments: the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection after induction and ultrasonic cracking, and thallus protein is identified by Westernblotting and can generate strong positive reaction with HumanHLA-DR antibody, so that the recombinant protein has strong immunogenicity;

large-scale expression and purification of HLA II recombinant protein: after a large amount of induction, expression and affinity chromatography purification, a large amount of HLA II recombinant protein is obtained.

5. An enzyme-linked immunoassay kit for blocking an antibody is characterized by comprising an antigen-coated enzyme label plate of a human blocking antibody, a positive control, a negative control, an enzyme-labeled secondary antibody, a diluent, a cleaning solution, a developing solution A, a developing solution B and a stop solution;

the ELISA plate in the antigen-coated ELISA plate of the human blocking antibody is a 96-microporous polystyrene plate, and the immobilized specific recombinant protein is a mixed antigen consisting of HLA I recombinant antigen and HLA II recombinant antigen;

the positive control is a blocking antibody positive control product formed by combining human HLA I antibody containing 30% glycerol, 5% BSA and 2% sucrose and human HLA-DR antibody.

6. The ELISA kit of claim 2 wherein the negative control is phosphate buffer pH7.4 containing 30% glycerol, 5% BSA, 2% sucrose; the enzyme-labeled secondary antibody is a mouse anti-human IgG labeled by Horse Radish Peroxide (HRP); the diluent is phosphate buffer solution with pH6-10 containing 2-20% calf serum and 0.5-10% antihuman IgM; the cleaning solution is phosphate buffer solution containing 5mL of Tween 20 per liter; the color developing liquid is TMB-H₂O₂A system; the stop solution is a concentrated sulfuric acid solution with 0.5-5 mol/L.

7. The ELISA kit for detecting a blocking antibody of claim 2, wherein the detection step of the blocking antibody by the ELISA kit for detecting a blocking antibody of the human blocking antibody comprises:

(1) adding the sample to be tested and the positive control into the micropores at one time, incubating, and combining the blocking antibody with the capture antigen on the surface of the solid phase carrier, namely the antigen pre-coated in the micropores;

(2) washing, adding the enzyme-labeled secondary antibody into the hole, and performing secondary incubation, wherein the enzyme-labeled secondary antibody is combined with the detection antibody in the previous step;

(3) washing off the unbound enzyme-labeled secondary antibody, adding enzyme substrate 3,3 ', 5, 5' -Tetramethylbenzidine (TMB), converting the liquid in the pores into blue under the catalysis of peroxidase, adding stop solution, and converting into yellow. The shade of the color is positively correlated with the content of the enclosed antibody in the sample;

(4) and (3) measuring the absorbance (OD value) at the wavelength of 450nm by using a microplate reader, judging whether the blocked antibody in the sample is negative or positive, wherein the judgment value (OD) is equal to the average OD value multiplied by 2.1 of the negative control, and the OD value of the sample is more than the OD value judged to be positive and less than or equal to the OD value judged to be negative.

Technical Field

The invention belongs to the technical field of in-vitro detection reagents, and relates to a preparation method of an antigen-coated enzyme label plate for a human blocking antibody and a kit thereof.

Background

Pregnancy is a successful semi-allogenic organ transplant. However, the spontaneous abortion is more and more common in modern women because of the influence of various factors, which leads to the failure of normal pregnancy. Repeated Spontaneous Abortion (RSA) refers to spontaneous abortion occurring 3 or more times continuously, and the incidence rate is 0.4% -1.0%, such as 5% calculated by 2 times of continuous occurrence. Research shows that 40-80% of RSA patients are related to immune factors, one part of the immune factors is related to autoantibodies, the other part of the immune factors is considered to be inappropriate for immunization of both female and fetal, and the lack of Blocking Antibody (BA) is a main factor. BA is closely related to recurrent abortion, and has effects of preventing helper T lymphocyte from cytotoxic effect on embryo trophoblast, and preventing recognition of fetal antigen by helper T lymphocyte, thereby preventing attack of maternal immune system on embryo. The BA in the serum of normal pregnant women mainly includes anti-HLA-DR, anti-TLX, anti-FC receptor antibody and anti-idiotypic antibody (Ab 2). The ELISA method is used for detecting HLA-I and II antibodies of RSA patients in a broad spectrum, IgG antibodies are detected, including non-reactive antibodies and IgG antibodies possibly existing before active immunotherapy of husband lymphocytes, IgM antibodies are not included, and the higher Ab2 antibody level can be indirectly expected due to the strong positive HLA-I and II antibodies, so that the method is used for indirectly detecting the blocking effect.

HLA is a glycoprotein antigen also known as a histocompatibility antigen. The antigen consists of a series of closely linked genes encoded by a complex of histocompatibility antigens, also known as HLA genes. Located on the short arm of chromosome 6, and has 6 loci, at least 4 gene regions: namely HLA-A, HLA-B, HLA-C and HLA-D. HLA-D is further divided into HLA-DR, HLA-DQ, HLA-DP subregions. Antigens encoded by HLA-A, HLA-B, HLA-C genes are referred to as class I antigens. Antigens encoded by HLA-DR, HLA-DQ, HLA-DP genes are referred to as class II antigens. The exons for coding the antigen-binding peptide are HLA-A, exon 2-4 of gene-B, HLA-DQB1, and exon 2 of gene-DRB 1.

Disclosure of Invention

The invention relates to a preparation method of a specific recombinant antigen HLA I and HLA II coated reaction plate, which is prepared by the following steps:

(1) obtaining of HLA-A recombinant protein:

prokaryotic expression of HLA-A: transforming BL21 competent escherichia coli by using human HLA-A (NM-001242758) cDNA clone (vector is pDONR223), screening positive clone according to kanamycin resistance, extracting plasmid DNA of the positive clone for identification to obtain a fragment with the same molecular weight as that of a target fragment, primarily judging the positive result, extracting the plasmid DNA by using PCR positive clone, and sequencing to obtain a result which shows that the HLA-A cDNA fragment is correctly cloned to the pDONR223 and is homologous with a humanized HLA I gene in GenBank;

identification of HLA-a protein fragments: after being induced, the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection and ultrasonic cracking, and the mycoprotein is identified by Western blotting and can generate strong positive reaction with Human HLA class I antibodyy, so that the recombinant protein has strong immunogenicity;

large-scale expression and purification of HLA-A recombinant protein: obtaining a large amount of HLA-A recombinant protein after a large amount of induction, expression and affinity chromatography purification;

(2) obtaining of HLA-B recombinant protein:

prokaryotic expression of HLA-B: transforming BL21 competent escherichia coli by using a human HLA-B (NM _005514) cDNA clone (a vector is pDONR223), screening positive clones according to kanamycin resistance, extracting plasmid DNA of the positive clones for identification to obtain fragments with the molecular weight consistent with that of a target fragment, and primarily judging the fragments to be positive; extracting plasmid DNA by PCR positive cloning, and sequencing to show that the HLA-B cDNA segment is correctly cloned to pDONR223 and is homologous with human HLA I gene in GenBank;

identification of HLA-B protein fragments: after being induced, the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection and ultrasonic cracking, and the mycoprotein is identified by Western blotting and can generate strong positive reaction with Human HLA class I antibodyy, so that the recombinant protein has strong immunogenicity;

and (3) large-scale expression and purification of HLA-B recombinant protein: obtaining a large amount of HLA-B recombinant protein after a large amount of induction, expression and affinity chromatography purification;

(3) obtaining of HLA ii recombinant protein:

prokaryotic expression of HLA ii: transforming BL21 competent escherichia coli by human HLA-DRB1 (NM-001243965) cDNA clone (vector is pDONR223), screening positive clone according to kanamycin resistance, extracting plasmid DNA of the positive clone for identification to obtain a fragment with the same molecular weight as that of a target fragment, primarily judging the positive, extracting the plasmid DNA by PCR positive clone, and sequencing to obtain a result which shows that the HLA II cDNA fragment is correctly cloned to the pDONR223 and is homologous with a humanized HLA II gene in GenBank;

identification of HLA ii protein fragments: after being induced, the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection and ultrasonic cracking, and the thallus protein is identified by Western blotting and can generate strong positive reaction with Human HLA-DR antibody, so that the recombinant protein has strong immunogenicity;

large-scale expression and purification of HLA II recombinant protein: obtaining a large amount of HLA II recombinant protein after a large amount of induction, expression and affinity chromatography purification;

(4) diluting HLA I recombinant protein antigen and HLA II recombinant protein antigen with buffer solution of pH7-10, coating the antigen in microporous plate at 20-300 μ l/hole for 2-40 hr, washing the plate with PBST for 2-6 times for 1-10min, and drying;

(5) filling the wells with PBS containing 2-20% calf serum, incubating at 37 deg.C, sealing for 0.5-5 hr, washing the plate for 2-6 times, and sealing; the plate is an enzyme-labeled reaction plate coated by HLA I antigen and HLA II antigen, and can be used for the specificity detection of closed antibody IgG.

The invention also provides a blocking antibody ELISA detection kit, which comprises: the kit is composed of an ELISA plate coated by the mixed recombinant antigens with other specificities, a positive control substance of a blocking antibody and a second antibody marked by horseradish peroxidase, and is specifically divided into the following parts: (1) enzyme label plate: coating by mixed recombinant antigens; (2) sample diluent: buffer solution with pH6-10 containing 2-20% calf serum and 0.5-10% antihuman IgM; (3) a positive control; (4) a negative control; (5) horseradish peroxidase-labeled secondary antibody; (6) cleaning fluid; (7) color development liquid: TMB-H₂O₂A system; (8) stopping liquid: 0.5-5mol/L concentrated sulfuric acid solution.

Further, preparation of blocking antibody positive control: the Human HLA class I antibody and Human HLA-DR antibody purchased were dissolved in 0.01M PBS (pH7.4) containing 30% glycerol, 5% BSA, 2% sucrose and 0.1% sodium thimerosal.

Further, preparation of blocking antibody negative control: 0.01M phosphate buffer pH7.4 containing 30% glycerol, 5% BSA, 2% sucrose and 0.1% thimerosal;

further, the enzyme-labeled secondary antibody was a commercially available horseradish peroxide (HRP) -labeled mouse anti-human IgG.

Further, the diluent was phosphate buffer solution of pH7.4 containing 8% calf serum and 2% anti-human IgM.

Further, the ELISA detection step of the kit for the blocking antibody comprises the following steps:

(1) adding the sample to be tested and the positive control into the micropores at one time, incubating, and combining the blocking antibody with the capture antigen on the surface of the solid phase carrier, namely the antigen pre-coated in the micropores;

(2) washing, adding an enzyme-labeled secondary antibody into the hole, performing secondary incubation, and combining the enzyme-labeled secondary antibody with the detection antibody in the previous step;

(3) washing off the unbound enzyme-labeled secondary antibody, adding enzyme substrate 3,3 ', 5, 5' -Tetramethylbenzidine (TMB), converting the liquid in the pores into blue under the catalysis of peroxidase, adding stop solution, and converting into yellow. The shade of the color is positively correlated with the content of the enclosed antibody in the sample;

(4) and (3) measuring the absorbance (OD value) at the wavelength of 450nm by using a microplate reader, judging whether the blocked antibody in the sample is negative or positive, wherein the judgment value (OD) is equal to the average OD value multiplied by 2.1 of the negative control, and the OD value of the sample is more than the OD value judged to be positive and less than or equal to the OD value judged to be negative.

The present invention selects partial gene sequences of HLA I and HLA II protein coding related to blocking antibody detection, and adopts gene recombination technology, prokaryotic expression and affinity chromatography to obtain several new recombinant proteins. The modified recombinant protein not only retains the antigenicity thereof, but also improves the prokaryotic expression quantity of the recombinant protein. And meanwhile, the detection rate of the blocking antibody can be ensured by coating a plurality of recombinant antigens, and false positive interference caused by the existence of IgM in a sample can be reduced by adding the anti-human IgM diluent. The invention successfully uses an ELISA method to conveniently and rapidly detect the blocking antibody.

Compared with the prior art, the invention has the following advantages:

the use is convenient and fast, and complex instruments are not needed; the method has simple steps, and the obtained result is accurate and reliable, so that positive control is provided, and the accuracy of the result is ensured.

Detailed Description

In order to make those skilled in the art better understand the solution of the present invention, the following description clearly and completely describes the technical solution in the embodiment of the present invention with reference to examples, and it is obvious that the described examples are only a part of examples of the present invention, and not all examples. All other embodiments obtained by a person skilled in the art based on the examples of the present invention without any inventive step shall fall within the scope of protection of the present invention.

Examples

An enzyme-linked immunoassay kit for blocking antibodies comprises an antigen-coated enzyme label plate of a human blocking antibody, a positive control, a negative control, an enzyme-labeled secondary antibody, a diluent, a cleaning solution, a developing solution A, a developing solution B and a stop solution.

The preparation method of the antigen-coated ELISA plate of the human blocking antibody comprises the following steps: (1) obtaining of HLA-A recombinant protein:

prokaryotic expression of HLA-A: transforming BL21 competent escherichia coli by using human HLA-A (NM-001242758) cDNA clone (vector is pDONR223), screening positive clone according to kanamycin resistance, extracting plasmid DNA of the positive clone for identification to obtain a fragment with the same molecular weight as that of a target fragment, primarily judging the positive result, extracting the plasmid DNA by using PCR positive clone, and sequencing to obtain a result which shows that the HLA-A cDNA fragment is correctly cloned to the pDONR223 and is homologous with a humanized HLA I gene in GenBank;

identification of HLA-a protein fragments: after being induced, the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection and ultrasonic cracking, and the mycoprotein is identified by Western blotting and can generate strong positive reaction with Human HLA class I antibodyy, so that the recombinant protein has strong immunogenicity;

large-scale expression and purification of HLA-A recombinant protein: obtaining a large amount of HLA-A recombinant protein after a large amount of induction, expression and affinity chromatography purification;

(2) obtaining of HLA-B recombinant protein:

prokaryotic expression of HLA-B: transforming BL21 competent escherichia coli by using a human HLA-B (NM _005514) cDNA clone (a vector is pDONR223), screening positive clones according to kanamycin resistance, extracting plasmid DNA of the positive clones for identification to obtain fragments with the molecular weight consistent with that of a target fragment, and primarily judging the fragments to be positive; extracting plasmid DNA by PCR positive cloning, and sequencing to show that the HLA-B cDNA segment is correctly cloned to pDONR223 and is homologous with human HLA I gene in GenBank;

identification of HLA-B protein fragments: after being induced, the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection and ultrasonic cracking, and the mycoprotein is identified by Western blotting and can generate strong positive reaction with Human HLA class I antibodyy, so that the recombinant protein has strong immunogenicity;

and (3) large-scale expression and purification of HLA-B recombinant protein: obtaining a large amount of HLA-B recombinant protein after a large amount of induction, expression and affinity chromatography purification;

(3) obtaining of HLA ii recombinant protein:

prokaryotic expression of HLA ii: transforming BL21 competent escherichia coli by human HLA-DRB1 (NM-001243965) cDNA clone (vector is pDONR223), screening positive clone according to kanamycin resistance, extracting plasmid DNA of the positive clone for identification to obtain a fragment with the same molecular weight as that of a target fragment, primarily judging the positive, extracting the plasmid DNA by PCR positive clone, and sequencing to obtain a result which shows that the HLA II cDNA fragment is correctly cloned to the pDONR223 and is homologous with a humanized HLA II gene in GenBank;

identification of HLA ii protein fragments: after being induced, the escherichia coli BL21 transformed with the recombinant plasmid is subjected to thallus collection and ultrasonic cracking, and the thallus protein is identified by Western blotting and can generate strong positive reaction with Human HLA-DR antibody, so that the recombinant protein has strong immunogenicity;

large-scale expression and purification of HLA II recombinant protein: obtaining a large amount of HLA II recombinant protein after a large amount of induction, expression and affinity chromatography purification;

(4) diluting HLA I protein antigen and HLA II protein antigen with carbonate buffer solution with pH9.6, coating 96-well plate with 100 μ l/well, placing in wet box at 4 deg.C for 10-24 hr, washing plate with PBST for 4 times, each time for 3min, and patting to dry to obtain ELISA plate coated with HLA recombinant mixed antigen;

(5) filling the wells with PBS containing 10% calf serum, incubating at 37 deg.C, sealing for 1.5h, washing the plate for 4 times, and sealing; and obtaining the enzyme-labeled reaction plate coated by the HLA recombinant mixed antigen.

It should be noted that the reagents in step (4) and step (5) can be selected within the given ranges, which are the optimum ratios, such as: the pH value of the carbonate buffer solution in the step (4) is 7-10, the 96-pore plate is loaded with 20-300 mul/pore, the plate washing times are 2-6 times, and each time lasts for 1-10 min; the PBS in the step (5) contains 2-20% of calf serum, the blocking time is 0.5-5h, and the plate washing times are 2-6, so the steps can be as follows:

(4) diluting HLA I protein antigen and HLA II protein antigen with carbonate buffer solution with pH7, coating 96-well plate, 20 μ l/well, placing in a wet box at 4 deg.C for 2 hr, washing plate with PBST for 2 times, each time for 1min, and patting to dry to obtain enzyme-labeled plate coated with HLA recombinant mixed antigen;

(5) filling the wells with PBS containing 2% calf serum, incubating at 37 deg.C, sealing for 0.5h, washing the plate for 2 times, and sealing; and obtaining the enzyme-labeled reaction plate coated by the HLA recombinant mixed antigen.

Or the following steps:

(4) diluting HLA I protein antigen and HLA II protein antigen with carbonate buffer solution with pH10, coating 96-well plate with 300 μ l/well, placing in a wet box at 4 deg.C for 40 hr, washing the plate with PBST for 6 times, each time for 10min, and patting to dry to obtain enzyme-labeled plate coated with HLA recombinant mixed antigen;

(5) filling the holes with PBS containing 20% calf serum, incubating at 37 ℃, sealing for 5h, washing the plate for 6 times, and sealing; and obtaining the enzyme-labeled reaction plate coated by the HLA recombinant mixed antigen.

The other reagents in the kit are prepared according to the following steps:

(1) coating solution (0.05mol/L, pH9.6 sodium carbonate-sodium bicarbonate buffer): na (Na)₂CO₃1.5g，NaHCO₃2.9g of thimerosal sodium and 1.0g of thimerosal sodium, purified water is added to 1000mL, and the pH is adjusted to 9.6.

(2) Sample dilutions (0.01 MPBS ph7.4 with 8% calf serum and 2% anti-human IgM): NaCl8.0g, KH₂PO₄0.2g，Na₂HPO₄·12H₂Adding purified water to 1000mL of O2.9 g, KCl 0.2g and thiomersalate sodium 1g, adjusting the pH to 7.4 to prepare 0.01M PBS solution; sample dilutions were made with 80mL calf serum, 20mL anti-human IgM, and 900mL PBS.

(3) Sealing liquid: (10% Calf serum/PBS solution) Calf serum 100mL, 1 XPBS (pH7.4)900 mL.

(4) Washing solution (PBST, ph 7.4): NaCl8.0g, KH₂PO₄0.2g，Na₂HPO₄·12H₂O2.9 g, KCl 0.2g, Tween 205mL, thimerosal 1g, purified water 1000mL, pH adjusted to 7.4.

(5) Enzyme-labeled secondary antibody: horseradish peroxide (HRP) -labeled mouse anti-human IgG was purchased and used by diluting 5000-fold with PBS.

(6) Positive control: the purchased Human HLA class I antibody and Human HLA-DR antibody were dissolved in a protective solution containing 30% glycerol, 5% BSA, 2% sucrose and 0.1% thimerosal in PBS (0.01M), pH7.4, and diluted 5000-fold with the protective solution when used.

(7) Negative control: 0.01M PBS pH7.4 containing 30% glycerol, 5% BSA, 2% sucrose, and 0.1% thimerosal.

(8) Substrate solution (TMB-H)₂O₂Urea solution):

substrate a (3,3 ', 5, 5' -tetramethylbenzidine, TMB): TMB200mg, absolute ethyl alcohol 100mL, purified water to 1000 mL;

② substrate B buffer solution (0.1mol/L citric acid-0.2 mol/L disodium hydrogen phosphate buffer solution, pH5.0-5.4): na (Na)₂HPO₄14.6g, 9.33g of citric acid and 6.4mL of 0.75% urea hydrogen peroxide, adding purified water to 1000mL, and adjusting the pH value to 5.0-5.4.

(9) Stopping liquid: (2 mol/LH)₂SO₄): 600mL of purified water, 100mL of concentrated sulfuric acid (slowly added dropwise with stirring), and 900mL of purified water.

In this example, the preparation process of the microplate is as follows:

1. main instrument

(1) Enzyme-linked immunosorbent assay (ELISA) instrument

(2) Water bath pot

2. Method of producing a composite material

(1) Selection of optimal titer of enzyme-labeled secondary antibody

Coating and washing with 100ng/mL human IgG;

diluting enzyme-labeled anti-human IgG monoclonal antibody with diluent, adding decibel into coated hole, maintaining and washing;

thirdly, adding a substrate for color development, adding acid to stop reaction, reading the absorbance (A), and taking the dilution of the enzyme-labeled antibody with the value of A being 1.0 as the optimal titer of the enzyme-labeled secondary antibody, wherein the optimal titer is 1: 2000.

(2) Chessboard titration method for selecting optimal titer of coating antigen

Firstly, a plurality of recombinant antigens are diluted in a coating solution in series, coated and washed.

And diluting the strong positive reference serum, the weak positive reference serum and the negative reference serum by using a diluent according to a 10-fold increasing series, adding the sample, preserving the heat and washing.

And thirdly, adding an enzyme-labeled secondary antibody diluted according to the optimal titer, preserving heat and washing.

Adding substrate for color development, adding acid to stop reaction and reading A value.

Selecting the dilution of the coating antigen with the A value of strong positive reference serum between 0.8 and 1.0 and the A value of negative reference serum less than 0.1 as the optimal titer, wherein the HLA-A antigen is 15 to 55 mu g/hole, the HLA-B antigen is 10 to 50 mu g/hole and the HLA II antigen is 10 to 40 mu g/hole.

(3) Preparation method of specific recombinant antigen coated reaction plate

Diluting HLA I protein antigen and HLA II protein antigen with a carbonate buffer solution with the pH value of 9.6, coating a 96-well plate with 100 mu l/well, placing the plate in a wet box at the temperature of 4 ℃ for 10-24 hours, washing the plate for 4 times by PBST (PBST), and then patting the plate dry to obtain the ELISA plate coated by the HLA recombinant mixed antigen.

Filling the holes with PBS containing 10% calf serum, incubating at 37 ℃, sealing for 1.5h, washing the plate for 4 times, and sealing; and obtaining the enzyme-labeled reaction plate coated by the HLA recombinant mixed antigen.

After the enzyme label plate is prepared, the steps of detecting the blocking antibody in the serum by using the kit and adopting an ELISA method are as follows:

sample adding: blank holes are respectively set, and no solution is added; adding 100 mu L of negative control into 2 wells of negative control wells; adding 100 mu L of positive control into 2 wells of the positive control well; add 100 uL sample diluent in advance, then add 5 uL sample to be tested. Gently shaking and mixing, covering with enzyme label plate, and standing at 37 deg.C for 30 min.

Discarding liquid in the hole, spin-drying, washing the plate for 5 times, soaking for 1-2min each time, and spin-drying at a rate of 200 μ L per hole.

Adding 150 μ L of enzyme-labeled conjugate (except blank wells) into each well, mixing, covering with enzyme-labeled plate, and standing at 37 deg.C for 30 min.

Discarding liquid in the hole, spin-drying, washing the plate for 5 times, soaking for 1-2min each time, and spin-drying at a rate of 200 μ L per hole.

Add 50. mu.L of each of the substrate solutions A and B to each well, and develop in the dark at 37 ℃ for 15 min.

The reaction was stopped by adding 50. mu.L of stop solution to each well.

The blank well was zeroed and absorbance at 450nm was read with a microplate reader.

As a result, the determination value (OD) was equal to the negative control average OD value × 2.1, and the sample OD value was greater than the determination value of positive and less than negative.

It is noted that some of the steps may be selected differently than the specific examples given above. These are all made by those skilled in the art based on their basic skills in understanding the idea of the present invention, and are not to be exemplified herein.

Finally, it is to be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not intended to be limiting. It will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention, and these changes and modifications are to be considered as within the scope of the invention.