CN1773282A - Autoantibody detection method for the diagnosis of immune-mediated type 1 diabetes - Google Patents

Abstract

The present invention relates to a self-body antibody detection method for immune mediated diabetes type A diagnosis, belonging to the field of biological medicine technology. Said invention includes the following steps: (1), using reverse transcriptive enzyme to make mRNA undergo the process of reverse transcription. Then using high fidelity DNA polymerase to respectively synthesize target DNA, using ligase and protein expression plasmid to make connection, making it be transformed into colibacillus to respectively express target protein; (2), using maltose-binding protein affinity chromatographic column and GST affinity chromotographic column to purify and separate out expression protein, dividing every purified expression protein into two portions; (3), coating antigen: making four antigens be mixed in the coating buffer solution, adding coating buffer solution in every enzyme-labeled well, standing still; (4), closing; and (5), using double-antigen sandwich method to determine antibody. Said invention can once detect any one or several kinds of antibodies of GAD65, IA2, Phogrin and ICA 69.

Description

Autoantibody detection method for the diagnosis of immune-mediated type 1 diabetes

technical field

The invention relates to a detection method used in the technical field of biomedicine, in particular to an autoantibody detection method used in the diagnosis of immune-mediated type 1 diabetes.

Background technique

Diabetes type diagnosis is carried out according to the etiology of diabetes. Diabetes is divided into 4 types, namely type 1 diabetes, type 2 diabetes, gestational diabetes and other special types of diabetes. Their harm to patients and treatment methods are completely different. Diagnosis and typing before treatment are very important for patients. Type 1 diabetes is also called juvenile-onset diabetes, because it often occurs before the age of 35, accounting for less than 10% of diabetes. The cells of the pancreas that produce insulin in the patient's body have been damaged, and there is an absolute lack of insulin. Patients need to use insulin from the onset treatment, and lifelong use. Because type 1 and type 2 diabetes are not only different in etiology, genetic predisposition, clinical manifestations, but also in treatment, it is very important for diabetic patients to carry out accurate type diagnosis of diabetes, choose treatment method and estimate prognosis before treatment. Especially for patients with type 1 diabetes, they need to be diagnosed and treated as early as possible, and mistakes in typing may bring irreversible consequences to patients. As early as the 1980s, it was observed that specific insulin cell autoantibodies could be detected in the blood of patients diagnosed with type 2 diabetes. Such patients were prone to failure of secondary oral hypoglycemic drugs and had to rely on insulin. treatment phenomenon. A large number of subsequent studies have shown that these patients actually belong to type 1 diabetes mellitus (LADA), and the cause of the disease is autoimmune disorders. However, because the clinical manifestations are difficult to distinguish from type 2 diabetes patients, and there is no relevant antibody detection method, they are usually misdiagnosed as 2 diabetes mellitus. Type 2 diabetes, while treated according to type 2 diabetes, will seriously damage the patient's health and life. In 1997, the American Diabetes Association (ADA) pointed out in the diagnosis and classification of diabetes that type 1 diabetes is caused by autoimmune mediation. Over the past ten years, a variety of autoantibodies against protein molecules in pancreatic beta cells have been discovered and confirmed. Including antibodies to various autoantigens such as GAD ₆₅ , IA2, Phogrin, ICA69, Insulin, and GM2-1. The total positive rate of the first three antibodies can reach 80% before or just after the onset of type 1 diabetes, and the positive rate of ICA69 alone is 20-43%.

Found through literature search to prior art, take Roche GAD or IA2 antibody assay kit as example, in its product manual (Diaplets Anti-GAD Roche Instruction Manual 2 April 2000, anti-GAD antibody enzyme-linked immunoassay operation manual, Roche product , April 2, 2002; DiapletsAnti-IA-2 Roche Instruction Manual 2 April 2000 Anti-IA2 Antibody Enzyme-Linked Immunoassay Operation Manual, Roche Products, April 2, 2002) The assay method of the antibody mentioned in: 1. Remove the streptavidin-coated ELISA plate and add biotinylated GAD to react for 1 hour. 2. Wash the plate 3 times. 3. Add the sample to be tested and react for 1 hour. 4. Wash the plate 3 times. 5. Add peroxidase-labeled anti-human IgG reagent to react for 1 hour. 6. Wash the plate 3 times. 7. Add ABTS for color reaction for half an hour. 8. Take a reading after adding the stop solution. Its disadvantage is that it can only be used for the single determination of multiple autoantibodies such as GAD ₆₅ and IA ₂ in patients with type 1 diabetes. Although the disease can be predicted and diagnosed to a certain extent, each antibody determination costs 100-150 RMB. The diagnostic rate is relatively low, and the joint detection of two or more antibodies is expensive, and the operation steps are many and the time is long, which is not a small burden for the tester and the tester.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide an autoantibody detection method for the diagnosis of immune-mediated type 1 diabetes, which can detect any one of anti-GAD ₆₅ , IA ₂ , Phogrin, and ICA69 antibodies at one time At the same time, due to the double-antigen sandwich method (antigen-antibody-detection antigen one-step reaction method), the whole operation process is simple and accurate, and the diagnosis rate of type 1 diabetes is significantly improved, the detection time is shortened, and medical expenses are reduced.

The present invention is achieved through the following technical solutions, and the present invention comprises the following steps:

(1) Use reverse transcriptase to reverse-transcribe mRNA, then use high-fidelity DNA polymerase to synthesize target DNA, connect with ligase and protein expression plasmid, and transform into Escherichia coli to express target protein respectively;

(2) Purify and isolate the expressed protein with a maltose-binding protein affinity chromatography column and a GST affinity chromatography column, and divide each purified expressed protein into two parts;

(3) Antigen coating: mix four kinds of antigens in coating buffer, add coating buffer to each enzyme-labeled well, and place;

(4) Blocking: Pour off the protein solution in the enzyme-labeled well, add blocking buffer, place it, wash the plate and save it for later use;

(5) Determination of antibodies by double-antigen sandwich method.

The mRNA refers to the total mRNA containing IA2, Phogrin, ICA69 and the mRNA of the human brain hippocampus that is commercialized by Gene Company for GAD ₆₅ protein expression; the target DNA refers to GAD ₆₅ , IA2, Phogrin The four target DNAs of ICA69 and ICA69; the target proteins refer to the four target proteins of GAD ₆₅ , IA2, Phogrin and ICA69.

In the step (2), each purified expressed protein is divided into two parts, one part is cryopreserved as a coating antigen for later use, and the other part is biotinylated with a biotinylation kit, and cryopreserved for later use. Used as a test antigen.

The coating of the antigen refers to: mixing the four antigens in the coating buffer of pH 8.5, adding 60 μl of the coating buffer to each enzyme-labeled well, and standing at 10°C for 1 hour.

In the step (4), add 300 μl of blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum, place at 10° C. for 1 hour, wash the plate and store it at 4° C. for later use.

The method of the double-antigen sandwich method is as follows: take out the spare ELISA plate, add the detection antigen diluted to the working concentration for antibody detection in the measurement hole, and the added detection antigen can be added as required. Any two, any three or all four are used to determine the corresponding antibodies, add the sample to be tested, and react on a shaker; pour off the reaction solution, add streptavidin peroxidase complex, and react under the same conditions; wash Plate, color development, stop color development, readout.

The double-antigen sandwich method specifically refers to adding 25 μl of the detection antigen diluted to the working concentration for antibody detection into the assay well.

The double-antigen sandwich method specifically refers to adding 25 μl of the sample to be tested and reacting for 1 hour at 22-25° C. on a shaker at 60 rpm.

The double-antigen sandwich method specifically refers to adding 100 μl of streptavidin-peroxidase complex and reacting under the same conditions for 20 minutes.

The plate washing specifically refers to: wash the plate 5 times by machine, conditions: 350 μl/well, shake for half a minute, and stand still for 2 minutes; the color development specifically refers to: add 100 μl TMB reaction solution, and place it in the dark at room temperature 15 minutes; said termination of color development specifically refers to: adding 100 μl of 0.5M sulfuric acid, and gently shakes it several times to mix; said reading specifically refers to: reading the measured value at 450nm.

Generally, there are many methods for detecting antibodies by using antigens, such as radioimmunoassay, fluorescence assay, enzyme-linked immunoassay, and the like. However, the traditional method of ELISA is an indirect assay of antigen-antibody-second antibody, which has many steps and long process, is prone to false positives, and has large errors; radioimmunoassay has a short shelf life and is easy to detect due to the short shelf life of the assay kit. It is inconvenient to promote and use on a large scale due to environmental pollution and other reasons; the fluorescence measurement method has high requirements for instruments and high operating costs and is not conducive to routine measurement. Therefore, the double-antigen sandwich method is selected in this method, that is, each antibody has two binding arms, and the variable regions on the two binding arms are the same. During the antigen-antibody reaction, one binding arm of the antibody specifically binds to the coated antigen, and the other binding arm specifically binds to the detection antigen (biotinylated antigen) to form a bridging form of antigen-antibody-antigen. After adding the streptavidin-peroxidase complex, the streptavidin specifically binds to the biotin on the detection antigen. Peroxidase acts on the substrate that can produce a color reaction, and finally the concentration of the antibody can be judged by the level of the color reaction, so as to achieve the purpose of antibody detection. The working principle of multi-antibody joint detection: four kinds of diabetes self-antigens are coated in the same enzyme-labeled well, if four mixed detection antigens are added during antibody determination, then when the serum samples of diabetic patients are added, those In the antibody-positive sample, as long as any antibody corresponding to the coated antigen is contained, specific antigen-antibody binding will occur. Whether there is a positive antibody can be concluded through the color reaction; if the added detection antigen is three out of the four, then it can be detected whether the three antibodies are positive during the determination process; if the detection antigen is added Any two kinds can be changed into the determination of two kinds of antibodies; if only a single detection antigen is added, it is the same as the current commercialized kit for single antibody detection.

In order to simplify the determination process, reduce operating time, and reduce the occurrence of false positives, a one-step reaction method that is conducive to rapid determination and a relatively successful double-antigen sandwich method for heteroantigens have been successfully developed on the basis of this double-antigen sandwich method. The difference between the determination principle of the double antigen sandwich method of heteroantigen and the ordinary double antigen sandwich method is that the coating antigen and the detection antigen used in the ordinary double antigen sandwich method are exactly the same antigen, while the different antigen refers to the antigen used for the target antibody. The antigen portion detected is the same, but the fusion protein portion is different. Because during protein expression, in order to make the expressed protein active, a fusion protein with good water solubility must be introduced into the expression system, which is connected with the expressed target protein in the primary structure. If the ordinary double-antigen sandwich method is used, because the fusion protein is also the same, once the antibody against the fusion protein part is contained in the sample to be measured, a bridged form of fusion protein-antibody-fusion protein will be generated in the measurement, so that will manifest itself as a false positive. Two methods can be adopted to prevent the emergence of this form: one is to use the double-antigen sandwich method of heteroantigen; the other is to add an excessive amount of free protein that is partially identical to the fusion protein in the assay system, and use competitive inhibition to prevent the fusion protein. - Production of bridged forms of antibody-fusion proteins.

The beneficial effects of the present invention are: 1. The detection rate of type 1 diabetes is improved: due to the increase of two autoantibody measurements, it is not because of the autoimmunity caused by these two relatively common autoantibodies of GAD ₆₅ and IA2 Diabetes can also be detected. 2. Simplicity: Since four autoantibodies can be measured at one time, it saves the time spent on the separate determination of multiple antibodies. 3. Fast and accurate: Due to the use of a double-antigen sandwich one-step assay, the 6 hours required by the Roche kit is shortened to 2.5 hours. In addition, due to the reduction of the reaction steps, the operation error is correspondingly reduced. 4. The variation coefficients of all inter-batch and intra-batch antibody assays are less than 10%, and the stability fully meets the antibody assay requirements.

Detailed ways

Example 1: Determination of anti-GAD ₆₅ , IA ₂ , Phogrin, and ICA69 antibodies

1. Preparation of four antigens:

(1) mRNA was extracted from 1 gram of fresh pancreas tissue for the protein expression of IA ₂ , Phogrin, and ICA69; the mRNA used for GAD ₆₅ protein expression was commercial human brain hippocampus mRNA (Clontech Company Cat No. 6578-1).

(2) RNA reverse transcription: GIBCO's THERMOSCRIPT reverse transcription kit was used.

(3) PCR amplification of each target gene: primers used for amplification of each target gene

_GAD65 :P1:ACAGGATTGGCATCTCCG,

P2: GGTCGACTTGGTGAGCAAGGTTA

ICA69:P1:GAAGGATTCTTGTCAGGACACA,

P2: GGTGTCGACTCATGCATTGAGCA

IA2:P1:GGATCCCATGGTGACACTAC,

P2: AAGCTTACACAGAGATGCTCACACAG

Phogrin: P1: TCATCGGATCCCTCCGCCATAGCTC,

P2: GATCAAGCTTCCTGACAACATC

A high-fidelity DNA amplification system (PLATINUM Taq DNA Polymerase High Fidelity GIBCO Company) was used to amplify the DNA of each reverse transcription product and synthesize the corresponding cDNA.

(4) Cloning of each amplified target gene into a replication plasmid: each cDNA product was immediately ligated with pGEM-Teazy (Cat No A1360 of Promega Company), and transformed into JM109 for blue and white spot screening. Take the white spot and use a plasmid extraction kit to extract each plasmid carrying the target gene.

(5) Each replicated target gene is digested with a DNA endonuclease and then cloned into the expression plasmid again:

Each extracted plasmid was digested with a DNA endonuclease, and the digested product was subjected to 1.5% agarose electrophoresis, and the target DNA was recovered with a QIAGEN Gel DNA Recovery Kit (QIAGEN Cat No 28704). The recovered DNA was ligated with a ligation kit (Cat No A1360 of Promega Company) and protein expression plasmid pMAL ^TM -c2x and transformed into TB1 competent cells (CatNo #E4122S of Protein Expression System Biolabs Company). In addition, in order to achieve better results in antibody assays in the future, each target gene was also connected to the protein expression plasmid pET-42a and transformed into JM109 _DE3 competent cells (Protein Expression System Novegen Company Cat No 70561-3).

(6) Protein expression: each competent state after transformation was plated on an LB medium plate, cultivated overnight at 37° C., inoculated each single colony into the LB liquid culture medium, and cultured and expressed at 15° C. and 60 rpm for 6 hours. All proteins expressed in pMAL ^TM -c2x expression system obtained soluble expression proteins with MBP fusion protein. Four kinds of six proteins were successfully expressed in this protein expression: IA2 with MBP, GAD ₆₅ with MBP, ICA69 with MBP, Phogrin with MBP, IA2 with GST, and Phogrin with GST.

(7) Protein purification: The expressed protein with MBP uses the protein purification part in the protein expression system of Biolabs company, that is, the maltose binding protein affinity chromatography column, to carry out protein purification on each protein with MBP; the expressed protein with GST The protein purification part of Novegen's protein expression system, that is, the GST affinity chromatography column, was used for protein purification. The purified protein was concentrated to 1 mg/ml with a centrifuge concentrator tube, and the protein concentration was measured with a BCA assay kit from Pierce Company.

(8) Biotinylation of the four antigens: Take 1ml of each of GAD ₆₅ , ICA69 (the fusion protein is MBP), IA ₂ and Phogrin (the fusion protein is GST), use the Pierce biotinylation kit and follow the steps required by the operation manual Each antigen was biotinylated, and the biotinylated protein solutions were added with 50% glycerol and 2% bovine serum albumin and stored at -20°C.

(9) Take 1ml of the purified four kinds of antigens (both have MBP) and add the same amount of complete Freund's reagent to fully mix them with two 5ml syringes, and use the subcutaneous injection method to treat 3 to 4 kg of white rabbits. immunity. One month later, the same protein and incomplete Freund's reagent were mixed in the same way for booster immunization, and then booster immunization was performed one week later. Two white rabbits were immunized with each antigen. After the antibodies were produced, blood was bled from the heart. After centrifugation, the serum was taken as reference serum for antibody determination, and stored at -20°C for future use.

2. Determination of antibodies:

(1) Coating of antigens: The antigens used for coating that were stored at low temperature were taken out from the refrigerator. These four antigens were all expressed proteins with MBP fusion protein, and the concentration was 1 mg/ml. The four antigens were diluted with pH 8.5 carbonate coating buffer and mixed. The final dilution ratio of each antigen was: GAD ₆₅ and IA2 were 1:100, and ICA69 and Phogrin were both 1:200. The sample volume in the enzyme-labeled wells was 60 μl/well, and placed at 10°C for 90 minutes.

(2) Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 μl of carbonate blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum at a pH of 8.5, place at 10°C for 1 hour, and wash the plate Store at 4°C for later use in the determination of antibodies.

(3) Determination of antibodies by double antigen sandwich method:

Take out the spare ELISA plate, add 25μl of the detection antigen diluted to the working concentration for antibody detection in the assay well, the added detection antigen can contain any one, any two, any three or all four as required To determine the corresponding antigen, add 25 μl of the sample to be tested, and react at 22° C. on a shaker at 60 rpm for 1 hour. The working final concentration of each detected antigen was IA2 20 μg/ml; GAD ₆₅ 15 μg/ml; ICA69 10 μg/ml; Phogrin 7.5 μg/ml. Antibody diluent was phosphate buffer saline solution with pH 7.5 containing 5 mg/ml MBP protein and 2% BSA.

(4) The reaction solution was discarded, and 100 μl of streptavidin-peroxidase complex (Rockland Company, Cat No. S000-03) with a concentration of 0.5 μg/ml was added to react for 20 minutes under the same conditions.

(5) Plate washing: wash the plate 5 times by machine, condition: 350ul/well, shake for half a minute, rest for 2 minutes;

(6) Color development: Add 100 μl TMB (Sigma Company, Cat No.T-8768) reaction solution, and place it at room temperature in the dark for 15 minutes;

(7) Termination of color development: add 100 μl of 0.5M sulfuric acid, shake gently a few times to mix;

(8) Reading: Read the measured value at 450nm.

The invention improves the detection rate of type 1 diabetes: due to the addition of the detection of two autoantibodies, the autoimmune diabetes not caused by two common autoantibodies, GAD ₆₅ and IA2, can also be detected. Since the four autoantibodies can be measured at one time, the time spent on the separate determination of multiple antibodies is eliminated. The 6 hours required with the Roche kit was shortened to 2.5 hours due to the use of a double-antigen sandwich one-step assay. In addition, due to the reduction of the reaction steps, the operation error is correspondingly reduced.

Example 2: Determination of Anti-GAD ₆₅ , IA2, Phogrin, and ICA69 Antibodies

1. Preparation of four antigens:

(1) Extract mRNA from 1 gram of fresh pancreatic tissue, which is commercial human brain mRNA (Clontech Company Cat No 6578-1).

(2) RNA reverse transcription: GIBCO's THERMOSCRIPT reverse transcription kit was used.

(3) PCR amplification of each target gene: primers used for each target gene

_GAD65 :P1:ACAGGATTGGCATCTCCG,

P2: GGTCGACTTGGTGAGCAAGGTTA

ICA69:P1:GAAGGATTCTTGTCAGGACACA,

P2: GGTGTCGACTCATGCATTGAGCA

IA2:P1:GGATCCCATGGTGACACTAC,

P2: AAGCTTACACAGAGATGCTCACACAG

Phogrin: P1: TCATCGGATCCCTCCGCCATAGCTC,

P2: GATCAAGCTTCCTGACAACATC

A high-fidelity DNA amplification system (PLATINUM Taq DNA Polymerase High Fidelity GIBCO Company) was used to amplify the DNA of each reverse transcription product and synthesize the corresponding cDNA.

(4) Cloning of each amplified target gene into a replication plasmid: each cDNA product was immediately ligated with pGEM-Teazy (Cat No A1360 of Promega Company), and transformed into JM109 for blue and white spot screening. Take the white spot and use a plasmid extraction kit to extract each plasmid carrying the target gene.

(5) Each replicated target gene is digested with a DNA endonuclease and then cloned into the expression plasmid again:

Each extracted plasmid was digested with a DNA endonuclease, and the digested product was subjected to 1.5% agarose electrophoresis, and the target DNA was recovered with a QIAGEN Gel DNA Recovery Kit (QIAGEN Cat No 28704). The recovered DNA was ligated with the protein expression plasmid pMAL ^TM -c2x using a ligation kit (Promega cat No A1360) and transformed into TB1 competent cells (protein expression system Biolabs company cat No #E4122S). In addition, in order to achieve better results in antibody assays in the future, each target gene was also connected to the protein expression plasmid pET-42a and transformed into JM109 _DE3 competent cells (Protein Expression System CatNo 70561-3 from Novegen Company).

(6) Protein expression: each competent state after transformation was plated on an LB medium plate, cultivated overnight at 37° C., inoculated each single colony into the LB liquid culture medium, and cultured and expressed at 15° C. and 60 rpm for 6 hours. All proteins expressed in pMAL ^TM -c2x expression system obtained soluble expression proteins with MBP fusion protein. Four kinds of six proteins were successfully expressed in this protein expression: IA2 with MBP, GAD ₆₅ with MBP, ICA69 with MBP, Phogrin with MBP, IA2 with GST, and Phogrin with GST.

(7) Protein purification: The expressed protein with MBP uses the protein purification part in the protein expression system of Biolabs company, that is, the maltose binding protein affinity chromatography column, to carry out protein purification on each protein with MBP; the expressed protein with GST The protein purification part of Novegen's protein expression system, that is, the GST affinity chromatography column, was used for protein purification. The purified protein was concentrated to 1 mg/ml with a centrifuge concentrator tube, and the protein concentration was measured with a BCA assay kit from Pierce Company.

(8) Biotinylation of the four antigens: Take 1ml of each of GAD ₆₅ , ICA69 (the fusion protein is MBP), IA ₂ and Phogrin (the fusion protein is GST), use the Pierce biotinylation kit and follow the steps required by the operation manual Each antigen was biotinylated, and the biotinylated protein solutions were added with 50% glycerol and 2% bovine serum albumin and stored at -20°C.

(9) Take 1ml of the purified four kinds of antigens (both have MBP) and add the same amount of complete Freund's reagent to fully mix them with two 5ml syringes, and use the subcutaneous injection method to treat 3 to 4 kg of white rabbits. immunity. One month later, the same protein and incomplete Freund's reagent were mixed in the same way for booster immunization, and then booster immunization was performed one week later. Two white rabbits were immunized with each antigen. After the antibodies were produced, blood was bled from the heart. After centrifugation, the serum was taken as reference serum for antibody determination, and stored at -20°C for future use.

2. Determination of antibodies:

(1) Coating of antigens: The antigens used for coating that were stored at low temperature were taken out from the refrigerator. These four antigens were all expressed proteins with MBP fusion protein, and the concentration was 1 mg/ml. The four antigens were diluted with pH 8.5 carbonate coating buffer and mixed. The final dilution ratio of each antigen was: GAD ₆₅ and IA2 were 1:100, and ICA69 and Phogrin were both 1:200. The sample volume in the enzyme-labeled wells was 60 μl/well, and placed at 10°C for 90 minutes.

(2) Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 ul of carbonate blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum at a pH of 8.5, place it at 10°C for 1 hour, and wash the plate Store at 4°C for later use in the determination of antibodies.

(3) Determination of antibodies by double antigen sandwich method:

Take out the spare ELISA plate, add 25μl of the detection antigen diluted to the working concentration for antibody detection in the assay well, the added detection antigen can contain any one, any two, any three or all four as required To determine the corresponding antigen, add 25 μl of the sample to be tested, and react at 25° C. on a shaker at 60 rpm for 1 hour. The working final concentration of each detected antigen was IA2 20 μg/ml; GAD ₆₅ 15 μg/ml; ICA69 10 μg/ml; Phogrin 7.5 μg/ml. Antibody diluent was phosphate buffer saline solution with pH 7.5 containing 5 mg/ml MBP protein and 2% BSA.

(4) Pour off the reaction solution, add 100 ul streptavidin peroxidase complex (Rockland Company, Cat No. S000-03) with a concentration of 0.5 μg/ml, and react under the same conditions for 20 minutes.

(5) Plate washing: wash the plate 5 times by machine, conditions: 350 μl/well, shake for half a minute, rest for 2 minutes;

(6) Color development: add 100ul TMB (Sigma Company, Cat No.T-8768) reaction solution, and place it in the dark at room temperature for 15 minutes;

(7) Termination of color development: add 100ul 0.5M sulfuric acid, shake gently a few times to mix;

(8) Reading: Read the measured value at 450nm.

The invention improves the detection rate of type 1 diabetes: due to the addition of the detection of two autoantibodies, the autoimmune diabetes not caused by two common autoantibodies, GAD ₆₅ and IA2, can also be detected. Since the four autoantibodies can be measured at one time, the time spent on the separate determination of multiple antibodies is eliminated. The 6 hours required with the Roche kit was shortened to 2.5 hours due to the use of a double-antigen sandwich one-step assay. In addition, due to the reduction of the reaction steps, the operation error is correspondingly reduced.

Example 3: Determination of Anti-GAD ₆₅ , IA2, Phogrin, and ICA69 Antibodies

1. Preparation of four antigens:

(1) Take 1 gram of fresh pancreatic tissue and use a strong denaturing agent to extract mRNA, which is commercial human brain mRNA (Clontech Company Cat No 6578-1).

(2) RNA reverse transcription: GIBCO's THERMOSCRIPT reverse transcription kit was used.

(3) PCR amplification of each target gene: primers used for each target gene

_GAD65 :P1:ACAGGATTGGCATCTCCG,

P2: GGTCGACTTGGTGAGCAAGGTTA

ICA69:P1:GAAGGATTCTTGTCAGGACACA,

P2: GGTGTCGACTCATGCATTGAGCA

IA2:P1:GGATCCCATGGTGACACTAC,

P2: AAGCTTACACAGAGATGCTCACACAG

Phogrin: P1: TCATCGGATCCCTCCGCCATAGCTC,

P2: GATCAAGCTTCCTGACAACATC

A high-fidelity DNA amplification system (PLATINUM Taq DNA Polymerase High Fidelity GIBCO Company) was used to amplify the DNA of each reverse transcription product and synthesize the corresponding cDNA.

(4) Cloning of each amplified target gene into a replication plasmid: each cDNA product was immediately ligated with pGEM-Teazy (Cat No A1360 of Promega Company), and transformed into JM109 for blue and white spot screening. Take the white spot and use a plasmid extraction kit to extract each plasmid carrying the target gene.

(5) Each replicated target gene is digested with a DNA endonuclease and then cloned into the expression plasmid again:

Each extracted plasmid was digested with a DNA endonuclease, and the digested product was subjected to 1.5% agarose electrophoresis, and the target DNA was recovered with a QIAGEN Gel DNA Recovery Kit (QIAGEN Cat No 28704). The recovered DNA was ligated with the protein expression plasmid pMAL ^TM -c2x using a ligation kit (Promega cat No A1360) and transformed into TB1 competent cells (protein expression system Biolabs company cat No #E4122S). In addition, in order to achieve better results in antibody assays in the future, each target gene was also connected to the protein expression plasmid pET-42a and transformed into JM109 _DE3 competent cells (Protein Expression System CatNo 70561-3 from Novegen Company).

(6) Protein expression: after transformation, each competent state was plated on a sub-LB medium plate, cultivated overnight at 37° C., inoculated each single colony into LB liquid culture medium, and cultured and expressed at 15° C., 60 rpm for 6 hours. All proteins expressed in pMAL ^TM -c2x expression system obtained soluble expression proteins with MBP fusion protein. Four kinds of six proteins were successfully expressed in this protein expression: IA2 with MBP, GAD ₆₅ with MBP, ICA69 with MBP, Phogrin with MBP, IA2 with GST, and Phogrin with GST.

(7) Protein purification: The expressed protein with MBP uses the protein purification part in the protein expression system of Biolabs company, that is, the maltose binding protein affinity chromatography column, to carry out protein purification on each protein with MBP; the expressed protein with GST The protein purification part of Novegen's protein expression system, that is, the GST affinity chromatography column, was used for protein purification. The purified protein was concentrated to 1 mg/ml with a centrifuge concentrator tube, and the protein concentration was measured with a BCA assay kit from Pierce Company.

(8) Biotinylation of the four antigens: Take 1ml of each of GAD ₆₅ , ICA69 (the fusion protein is MBP), IA ₂ and Phogrin (the fusion protein is GST), use the Pierce biotinylation kit and follow the steps required by the operation manual Each antigen was biotinylated, and the biotinylated protein solutions were added with 50% glycerol and 2% bovine serum albumin and stored at -20°C.

(9) Take 1ml of the purified four kinds of antigens (both have MBP) and add the same amount of complete Freund's reagent to fully mix them with two 5ml syringes, and use the subcutaneous injection method to treat 3 to 4 kg of white rabbits. immunity. One month later, the same protein and incomplete Freund's reagent were mixed in the same way for booster immunization, and then booster immunization was performed one week later. Two white rabbits were immunized with each antigen. After the antibodies were produced, blood was bled from the heart. After centrifugation, the serum was taken as reference serum for antibody determination, and stored at -20°C for future use.

2. Determination of antibodies:

(1) Coating of antigens: The antigens used for coating that were stored at low temperature were taken out from the refrigerator. These four antigens were all expressed proteins with MBP fusion protein, and the concentration was 1 mg/ml. The four antigens were diluted with pH 8.5 carbonate coating buffer and mixed. The final dilution ratio of each antigen was: GAD ₆₅ and IA2 were 1:100, and ICA69 and Phogrin were both 1:200. The sample volume in the enzyme-labeled wells was 60 μl/well, and placed at 10°C for 90 minutes.

(2) Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 ul of carbonate blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum at a pH of 8.5, place it at 10°C for 1 hour, and wash the plate Store at 4°C for later use in the determination of antibodies.

(3) Determination of antibodies by double antigen sandwich method:

Take out the spare ELISA plate, add 25μl of the detection antigen diluted to the working concentration for antibody detection in the assay well, the added detection antigen can contain any one, any two, any three or all four as required To determine the corresponding antigen, add 25 μl of the sample to be tested, and react at 23° C. on a shaker at 60 rpm for 1 hour. The working final concentration of each detected antigen was IA2 20 μg/ml; GAD ₆₅ 15 μg/ml; ICA69 10 μg/ml; Phogrin 7.5 μg/ml. Antibody diluent was phosphate buffer saline solution with pH 7.5 containing 5 mg/ml MBP protein and 2% BSA.

(4) The reaction solution was discarded, and 100 μl of streptavidin-peroxidase complex (Rockland Company, Cat No. S000-03) with a concentration of 0.5 μg/ml was added to react for 20 minutes under the same conditions.

(5) Plate washing: wash the plate 5 times by machine, conditions: 350 μl/well, shake for half a minute, rest for 2 minutes;

(6) Color development: Add 100 μl TMB (Sigma Company, Cat No.T-8768) reaction solution, and place it at room temperature in the dark for 15 minutes;

(7) Termination of color development: add 100 μl of 0.5M sulfuric acid, shake gently a few times to mix;

(8) Reading: Read the measured value at 450nm.

The invention improves the detection rate of type 1 diabetes: due to the addition of the detection of two autoantibodies, the autoimmune diabetes not caused by two common autoantibodies, GAD ₆₅ and IA2, can also be detected. Since the four autoantibodies can be measured at one time, the time spent on the separate determination of multiple antibodies is eliminated. The 6 hours required with the Roche kit was shortened to 2.5 hours due to the use of a double-antigen sandwich one-step assay. In addition, due to the reduction of the reaction steps, the operation error is correspondingly reduced.

Example 4: Joint determination of anti-GAD ₆₅ , IA2, and ICA69 antibodies

(1) Antigen preparation: mRNA was extracted from human pancreas, mRNA was reverse-transcribed with reverse transcriptase, and four target DNAs of GAD ₆₅ , IA2, Phogrin, and ICA69 were synthesized with high-fidelity DNA polymerase , connected with ligase and protein expression plasmid, transformed into Escherichia coli to express GAD ₆₅ , IA2, Phogrin, and ICA69 four target proteins, and finally purified and separated the expressed protein with maltose binding protein affinity chromatography column, and each A purified expression protein is divided into two parts, one part is stored at low temperature for coating antigen, and the other part is biotinylated with a biotinylation kit, and stored at low temperature for detection of antigen;

(2) Determination of antibodies:

① Coating of antigens: Dilute the four antigens with pH 8.5 carbonate coating buffer and mix them. The final dilution ratio of each antigen is: GAD ₆₅ and IA2 are both 1:100, ICA69 and Phogrin are both 1:100 : 200. The sample volume in the enzyme-labeled well is 60 μl/well, and placed at 10°C for 90 minutes;

② Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 μl of blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum, place at 10°C for 1 hour, wash the plate and store it at 4°C for use in antibodies determination;

③ Determination of antibodies by double-antigen sandwich method:

a. Take out the spare ELISA plate, add 25 μl of GAD ₆₅ , IA2, ICA69 mixed detection antigen to the assay well, the detection antigen mixing method is GAD ₆₅ : IA2 : ICA69 : dilution buffer according to 1: 1: 1: 1 The volume ratio was mixed so that the working final concentration of each detection antigen was 20 μg/ml for IA2; 15 μg/ml for GAD ₆₅ ; 10 μg/ml for ICA69. Add 25 μl of the sample to be tested, and react for 1 hour at 22°C on a shaker at 60 rpm;

b. Pour off the reaction solution, add 100 μl streptavidin peroxidase complex, and react under the same conditions for 20 minutes;

c. Plate washing: Machine washes the plate 5 times, conditions: 350 μl/well, shaking for half a minute, resting for 2 minutes;

d. Color development: add 100 μl TMB reaction solution, and place it at room temperature in the dark for 15 minutes;

e. Terminate color development: add 100 μl 0.5M sulfuric acid, shake gently a few times to mix;

f. Reading: Read the measured value at 450nm.

Implementation effect: In the 40 samples tested in the experiment, any anti-GAD ₆₅ , IA ₂ , and ICA69 antibodies in each sample were detected by the combined detection of the three antibodies, and the sensitivity and specificity were 100%. .

Example 5: Joint determination of anti-GAD ₆₅ , IA2, and Phogrin antibodies

(1) Antigen preparation: mRNA was extracted from human pancreas, mRNA was reverse-transcribed with reverse transcriptase, and four target DNAs of GAD ₆₅ , IA2, Phogrin, and ICA69 were synthesized with high-fidelity DNA polymerase , connected with ligase and protein expression plasmid, transformed into Escherichia coli to express GAD ₆₅ , IA2, Phogrin, and ICA69 four target proteins, and finally purified and separated the expressed protein with maltose binding protein affinity chromatography column, and each A purified expression protein is divided into two parts, one part is stored at low temperature for coating antigen, and the other part is biotinylated with a biotinylation kit, and stored at low temperature for detection of antigen;

(2) Determination of antibodies:

① Coating of antigens: Dilute the four antigens with pH 8.5 carbonate coating buffer and mix them. The final dilution ratio of each antigen is: GAD ₆₅ and IA2 are both 1:100, ICA69 and Phogrin are both 1:100 : 200. The sample volume in the enzyme-labeled well is 60 μl/well, and placed at 10°C for 90 minutes;

② Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 μl of blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum, place at 10°C for 1 hour, wash the plate and store it at 4°C for use in antibodies determination;

③ Determination of antibodies by double-antigen sandwich method:

a. Take out the spare ELISA plate, add 25μl GAD ₆₅ , IA2, ICA69 mixed detection antigen to the measurement well, the detection antigen mixing method is GAD ₆₅ : IA2 : Phogrin : dilution buffer according to 1: 1: 1: 1 The volume ratio was mixed so that the working final concentration of each detection antigen was IA2 20 μg/ml; GAD ₆₅ 15 μg/ml; Phogrin 7.5 μg/ml. Add 25 μl of the sample to be tested, and react at 22.5°C on a shaker at 60 rpm for 1 hour;

b. Pour off the reaction solution, add 100 μl streptavidin peroxidase complex, and react under the same conditions for 20 minutes;

c. Plate washing: Machine washes the plate 5 times, conditions: 350 μl/well, shaking for half a minute, resting for 2 minutes;

d. Color development: add 100 μl TMB reaction solution, and place it at room temperature in the dark for 15 minutes;

e. Terminate color development: add 100 μl 0.5M sulfuric acid, shake gently a few times to mix;

f. Reading: Read the measured value at 450nm.

Implementation effect: In the 40 samples tested in the experiment, any anti-GAD ₆₅ , IA ₂ , and Phogrin antibodies in each sample were detected by the combined detection of the three antibodies, and the sensitivity and specificity were 100%. .

Example 6: Joint determination of anti-GAD ₆₅ , ICA69, and Phogrin antibodies

(1) Antigen preparation: mRNA was extracted from human pancreas, mRNA was reverse-transcribed with reverse transcriptase, and four target DNAs of GAD ₆₅ , IA2, Phogrin, and ICA69 were synthesized with high-fidelity DNA polymerase , connected with ligase and protein expression plasmid, transformed into Escherichia coli to express GAD ₆₅ , IA2, Phogrin, and ICA69 four target proteins, and finally purified and separated the expressed protein with maltose binding protein affinity chromatography column, and each A purified expression protein is divided into two parts, one part is stored at low temperature for coating antigen, and the other part is biotinylated with a biotinylation kit, and stored at low temperature for detection of antigen;

(2) Determination of antibodies:

① Coating of antigens: Dilute the four antigens with pH 8.5 carbonate coating buffer and mix them. The final dilution ratio of each antigen is: GAD ₆₅ and IA2 are both 1:100, ICA69 and Phogrin are both 1:100 : 200. The sample volume in the enzyme-labeled well is 60 μl/well, and placed at 10°C for 90 minutes;

② Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 μl of blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum, place at 10°C for 1 hour, wash the plate and store it at 4°C for use in antibodies determination;

③ Determination of antibodies by double-antigen sandwich method:

a. Take out the spare ELISA plate and add 25 μl of GAD ₆₅ , IA2, ICA69 mixed detection antigen to the assay well. The detection antigen mixing method is GAD ₆₅ : ICA69 : Phogrin : dilution buffer at 1:1:1:1 The volume ratio was mixed so that the working final concentration of each detection antigen was GAD ₆₅ 15 μg/ml; ICA69 10 μg/ml; Phogrin 7.5 μg/ml. Add 25 μl of the sample to be tested, and react for 1 hour at 23°C on a shaker at 60 rpm;

b. Pour off the reaction solution, add 100 μl streptavidin peroxidase complex, and react under the same conditions for 20 minutes;

c. Plate washing: Machine washes the plate 5 times, conditions: 350 μl/well, shaking for half a minute, resting for 2 minutes;

d. Color development: add 100 μl TMB reaction solution, and place it at room temperature in the dark for 15 minutes;

e. Terminate color development: add 100 μl 0.5M sulfuric acid, shake gently a few times to mix;

f. Reading: Read the measured value at 450nm.

Implementation effect: In the 40 samples tested in the experiment, any anti-GAD ₆₅ , ICA69, and Phogrin antibodies in each sample were detected by the combined detection of the three antibodies, and the sensitivity and specificity were 100%.

Example 7: Joint determination of anti-GAD ₆₅ and IA2 antibodies

(1) Antigen preparation: mRNA was extracted from human pancreas, mRNA was reverse-transcribed with reverse transcriptase, and four target DNAs of GAD ₆₅ , IA2, Phogrin, and ICA69 were synthesized with high-fidelity DNA polymerase , connected with ligase and protein expression plasmid, transformed into Escherichia coli to express GAD ₆₅ , IA2, Phogrin, and ICA69 four target proteins, and finally purified and separated the expressed protein with maltose binding protein affinity chromatography column, and each A purified expression protein is divided into two parts, one part is stored at low temperature for coating antigen, and the other part is biotinylated with a biotinylation kit, and stored at low temperature for detection of antigen;

(2) Determination of antibodies:

① Coating of antigens: Dilute the four antigens with pH 8.5 carbonate coating buffer and mix them. The final dilution ratio of each antigen is: GAD ₆₅ and IA2 are both 1:100, ICA69 and Phogrin are both 1:100 : 200. The sample volume in the enzyme-labeled well is 60 μl/well, and placed at 10°C for 90 minutes;

② Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 μl of blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum, place at 10°C for 1 hour, wash the plate and store it at 4°C for use in antibodies determination;

③ Determination of antibodies by double-antigen sandwich method:

a. Take out the spare ELISA plate, add 25 μl of GAD ₆₅ , IA2 mixed detection antigen in the assay well, the mixing method of detection antigen is GAD ₆₅ : IA2 : dilution buffer is mixed in a volume ratio of 1:1:2, so that each The working final concentration of detection antigen is GAD ₆₅ 15μg/ml; IA2 20μg/ml. Add 25 μl of the sample to be tested, and react for 1 hour at 25°C on a shaker at 60 rpm;

b. Pour off the reaction solution, add 100 μl streptavidin peroxidase complex, and react under the same conditions for 20 minutes;

c. Plate washing: Machine washes the plate 5 times, conditions: 350 μl/well, shaking for half a minute, resting for 2 minutes;

d. Color development: add 100 μl TMB reaction solution, and place it at room temperature in the dark for 15 minutes;

e. Terminate color development: add 100 μl 0.5M sulfuric acid, shake gently a few times to mix;

f. Reading: Read the measured value at 450nm.

Implementation effect: In the 40 samples tested in the experiment, any anti-GAD ₆₅ and IA ₂ antibodies in each sample can be detected by double-antibody combined detection, and the sensitivity and specificity are 100%.

Embodiment 8: Determination of anti-GAD ₆₅ antibody

(1) Antigen preparation: mRNA was extracted from human pancreas, mRNA was reverse-transcribed with reverse transcriptase, and four target DNAs of GAD ₆₅ , IA2, Phogrin, and ICA69 were synthesized with high-fidelity DNA polymerase , connected with ligase and protein expression plasmid, transformed into Escherichia coli to express GAD ₆₅ , IA2, Phogrin, and ICA69 four target proteins, and finally purified and separated the expressed protein with maltose binding protein affinity chromatography column, and each A purified expression protein is divided into two parts, one part is stored at low temperature for coating antigen, and the other part is biotinylated with a biotinylation kit, and stored at low temperature for detection of antigen;

(2) Determination of antibodies:

① Coating of antigens: Dilute the four antigens with pH 8.5 carbonate coating buffer and mix them. The final dilution ratio of each antigen is: GAD ₆₅ and IA2 are both 1:100, ICA69 and Phogrin are both 1:100 : 200. The sample volume in the enzyme-labeled well is 60 μl/well, and placed at 10°C for 90 minutes;

② Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 μl of blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum, place at 10°C for 1 hour, wash the plate and store it at 4°C for use in antibodies determination;

③ Determination of antibodies by double-antigen sandwich method:

a. Take out the spare microplate, add 25 μ l GAD ₆₅ detection antigen in the assay hole, the dilution method of detection antigen is GAD ₆₅ : the dilution buffer is mixed by 1: 3 volume ratio, make the working final concentration of GAD ₆₅ detection antigen be 15 μg/ml. Add 25 μl of the sample to be tested, and react for 1 hour at 24°C on a shaker at 60 rpm;

b. Pour off the reaction solution, add 100 μl streptavidin peroxidase complex, and react under the same conditions for 20 minutes;

c. Plate washing: machine washes the plate 5 times, conditions: 350 μl/well, shaking for half a minute, resting for 2 minutes;

d. Color development: add 100 μl TMB reaction solution, and place it at room temperature in the dark for 15 minutes;

e. Terminate color development: add 100 μl 0.5M sulfuric acid, shake gently a few times to mix;

f. Reading: Read the measured value at 450nm.

Implementation effect: in the 20 samples tested in the experiment, any anti-GAD ₆₅ antibody in each sample can be detected by the antibody detection, and the sensitivity and specificity are both 100%.

Embodiment 9: Determination of anti-IA2 antibody

(1) Antigen preparation: mRNA was extracted from human pancreas, mRNA was reverse-transcribed with reverse transcriptase, and four target DNAs of GAD ₆₅ , IA2, Phogrin, and ICA69 were synthesized with high-fidelity DNA polymerase , connected with ligase and protein expression plasmid, transformed into Escherichia coli to express GAD ₆₅ , IA2, Phogrin, and ICA69 four target proteins, and finally purified and separated the expressed protein with maltose binding protein affinity chromatography column, and each A purified expression protein is divided into two parts, one part is stored at low temperature for coating antigen, and the other part is biotinylated with a biotinylation kit, and stored at low temperature for detection of antigen;

(2) Determination of antibodies:

① Coating of antigens: Dilute the four antigens with pH 8.5 carbonate coating buffer and mix them. The final dilution ratio of each antigen is: GAD ₆₅ and IA2 are both 1:100, ICA69 and Phogrin are both 1:100 : 200. The sample volume in the enzyme-labeled well is 60 μl/well, and placed at 10°C for 90 minutes;

② Blocking: Pour off the protein solution in the enzyme-labeled wells, add 300 μl of blocking buffer containing 5% non-fat milk powder and 15% fetal bovine serum, place at 10°C for 1 hour, wash the plate and store it at 4°C for use in antibodies determination;

③ Determination of antibodies by double-antigen sandwich method:

a. Take out the spare ELISA plate, add 25μl IA2 detection antigen to the measurement well, the dilution method of the detection antigen is IA2: the dilution buffer is mixed at a volume ratio of 1:3, so that the working final concentration of the IA2 detection antigen is 20 μg/ml . Add 25 μl of the sample to be tested, and react for 1 hour at 23°C on a shaker at 60 rpm;

b. Pour off the reaction solution, add 100 μl streptavidin peroxidase complex, and react under the same conditions for 20 minutes;

c. Plate washing: Machine washes the plate 5 times, conditions: 350 μl/well, shaking for half a minute, resting for 2 minutes;

d. Color development: add 100 μl TMB reaction solution, and place it at room temperature in the dark for 15 minutes;

e. Terminate color development: add 100 μl 0.5M sulfuric acid, shake gently a few times to mix;

f. Reading: Read the measured value at 450nm.

Implementation effect: in the 20 samples tested in the experiment, any anti-IA2 antibody in each sample can be detected by the antibody detection, and the sensitivity and specificity are both 100%.

Claims (10)

1. an autoantibody detection method for immunization-mediated type 1 diabetes diagnosis, characterized in that, comprising the following steps: (1) Use reverse transcriptase to reverse-transcribe mRNA, then use high-fidelity DNA polymerase to synthesize target DNA, connect with ligase and protein expression plasmid, and transform into Escherichia coli to express target protein respectively; (2) Purify and isolate the expressed protein with a maltose-binding protein affinity chromatography column and a GST affinity chromatography column, and divide each purified expressed protein into two parts; (3) Antigen coating: mix four kinds of antigens in coating buffer, add coating buffer to each enzyme-labeled well, and place; (4) Blocking: Pour off the protein solution in the enzyme-labeled well, add blocking buffer, place it, wash the plate and save it for later use; (5) Determination of antibodies by double-antigen sandwich method. 2. the autoantibody detection method that is used for immunization-mediated type 1 diabetes diagnosis according to claim 1, is characterized in that, described mRNA refers to the total mRNA that contains IA2, Phogrin, ICA69 and is used for GAD ₆₅ protein expression is the mRNA of human brain hippocampus commercialized by a gene company; the target DNA refers to the four target DNAs of GAD ₆₅ , IA2, Phogrin, and ICA69; the target protein refers to GAD ₆₅ , IA2, Phogrin, ICA69 four target proteins. 3. The autoantibody detection method for immunization-mediated type 1 diabetes diagnosis according to claim 1, characterized in that, in the step (2), each purified expressed protein is divided into two parts, A part was stored at low temperature for coating antigen, and the other part was biotinylated with a biotinylation kit, and stored at low temperature for antigen detection. 4. The autoantibody detection method for immunization-mediated type 1 diabetes diagnosis according to claim 1, characterized in that, the coating of the antigen refers to: mixing four kinds of antigens in a pH8.5 coating within the buffer. 5. the autoantibody detection method that is used for immunization-mediated type 1 diabetes diagnosis according to claim 1, is characterized in that, described blocking buffer refers to containing 5% fat-free milk powder, 15% fetal calf serum blocking. buffer. 6. the autoantibody detection method that is used for immunization-mediated type 1 diabetes diagnosis according to claim 1, is characterized in that, described double-antigen sandwich method, its method is: take out spare microtiter plate, in measuring hole Add the detection antigen diluted to the working concentration for antibody detection, and the added detection antigen can contain any one, any two, any three or all four as required to determine the corresponding antibody, and add the test product , react on a shaker; pour off the reaction solution, add streptavidin peroxidase complex, and react under the same conditions; wash the plate, develop color, stop color development, and read. 7. The autoantibody detection method for the diagnosis of type 1 diabetes mellitus mediated by immunity according to claim 6, wherein the double-antigen sandwich method specifically refers to the reaction at 22-25°C on a shaker. 8 . The autoantibody detection method for diagnosis of immune-mediated type 1 diabetes according to claim 6 , wherein the plate washing specifically refers to: plate washing by machine, 350 μl/well, oscillating, static. 9. The autoantibody detection method for the diagnosis of type 1 diabetes mellitus mediated by immunity according to claim 6, characterized in that said color development specifically refers to adding TMB reaction solution and placing it at room temperature in the dark. 10. The autoantibody detection method for immunization-mediated type 1 diabetes diagnosis according to claim 6, characterized in that, the termination of color development specifically refers to adding 0.5M sulfuric acid, shaking gently for a few times to make the mixture uniform.