WO2019169799A1

WO2019169799A1 - Biotinylated insulin antigen and biotinylation process thereof

Info

Publication number: WO2019169799A1
Application number: PCT/CN2018/093058
Authority: WO
Inventors: 张大准; 张永顶; 马伟民; 王洪涛; 马新民
Original assignee: 深圳市伯劳特生物制品有限公司
Priority date: 2018-03-07
Filing date: 2018-06-27
Publication date: 2019-09-12
Also published as: CN108440666B; CN108440666A

Abstract

Disclosed are a biotinylated insulin antigen having a structure as represented by formula 1 and a biotinylation process thereof. The biotinylated insulin antigen of the present invention improves the coating efficiency of an insulin antigen because it is amplified by pre-coated avidin, can significantly improve the sensitivity of an insulin antibody detection reagent or kit prepared thereby, improves the positive rate of insulin antibody detection, and reduces the probability of missed detection or false detection.

Description

Biotinylated insulin antigen and biotinylation process thereof

The present application claims priority to Chinese Patent Application No. 201810186407.6, entitled "Biotinylated insulin antigen and biotinylation process" thereof, filed on March 07, 2018, the entire contents of which is incorporated herein by reference. This is incorporated herein by reference.

Technical field

The invention relates to the field of enzyme-linked immunoassay, in particular to a biotinylated insulin antigen and a biotinylation process thereof.

Background technique

The detection of insulin autoantibodies can be used as a marker of autoimmune β-cell injury. It can be used to detect and prevent type 1 diabetes at an early stage. Now, the indirect enzyme-linked immunosorbent assay is widely used, and the insulin antigen is coated on the microplate. The serum to be tested is added, wherein the insulin autoantibody in the serum is reacted with the insulin antigen coated on the microplate, washed, the enzyme-labeled secondary antibody is added, washed, and the substrate is colored.

Biotinylation is the process of covalently linking biotin to proteins, nucleic acids, or other molecules. Biotin can bind to avidin or streptavidin, each avidin or streptavidin can bind four molecular biotin molecules, and has strong affinity, affinity constant k=1015L/mol, ratio The affinity between antigen and antibody (K=10.5-11L/mol) is at least 100 times higher, and the binding specificity of the two is high and the stability is good. Using the biotin-avidin system with strong specificity and good affinity, the protein can be biotinylated and then specifically bound to avidin/streptavidin to achieve signal Amplify and improve the sensitivity of detection. There are many mature biotinylation reagents on the market, such as the thermo company.

Sulfo-NHS-LC-Biotin is used for biotinylation of antibodies and proteins.

However, conventional insulin biotinylation methods, such as use

The insulin-biotin conjugate obtained by the Sulfo-NHS-LC-Biotin biotinylation kit has the disadvantages of insufficient sensitivity and low positive rate for the development of the insulin antibody detection kit, and there is a risk of missed detection or false detection. Can not meet the requirements.

Summary of the invention

In view of the above, the object of the present invention is to provide a biotinylated insulin antigen and a preparation process thereof, so that the insulin antigen can significantly improve the positive rate of detecting insulin autoantibodies (IAA);

Another object of the present invention is to provide the use of the above insulin antigen in the preparation of an enzyme-linked immunoassay kit, in particular, a kit for detecting diabetes;

In order to achieve the above object, the present invention provides the following technical solutions:

A biotinylated insulin antigen having the structure shown in Formula 1:

Wherein, the circle represents the insulin amino acid chain, 1≤x≤n, n represents the number of amino groups in the insulin amino acid chain, x and n are integers; the ellipse represents the macromolecular protein amino acid chain, y+z≤m, z≥10, y ≥ 1, m represents the number of amino groups on the amino acid chain of the macromolecular protein, and y, z and m are integers. If z/x is a non-integer, z/x takes an integer and adds 1 to the integer position.

In view of the fact that the biotinylation efficiency of insulin in the prior art is not high, and the effect is not good, the reagent or the kit for detecting insulin antibody detection is insufficient in sensitivity and the positive rate is low, and the present invention will be improved by an improved process. One insulin molecule is coupled to the amino acid amino group via SATA and SMCC, and the macromolecular protein is coupled with biotin to form a Biotin-macromolecule protein-insulin conjugate (structure 1), which binds multiple insulin molecules and The biotin molecule (shown in Figure 1), which is pre-coated with avidin amplification, greatly enhances the coating efficiency of the antigen insulin, thereby significantly increasing the sensitivity of the insulin antibody detection reagent or kit prepared thereby. Greatly improve the positive rate of insulin antibody detection, reduce the chance of missed detection or false detection.

Preferably, the macromolecular protein is HBA, BSA, OVA or casein; the x is preferably 1.

It has been experimentally verified that the biotinylated insulin obtained by the conventional method and the biotinylated insulin antigen of the present invention can obtain a higher IAA positive rate when used for detection, and prevent the sample from being leaked due to a lower concentration. Check or wrong judgment.

Therefore, the present invention proposes the use of the biotinylated insulin antigen in the preparation of an enzyme-linked immunoassay kit, particularly in the preparation of a diabetic enzyme-linked immunoassay kit.

According to the application, the present invention provides a diabetes antibody spectrum detecting kit comprising a protein chip coated with a diabetes autoantigen, which is passed through avidinized protein chip and biotinylated diabetes autoantigen The biotinylated diabetes autoantigen is coated with each other, the insulin antigen of the present invention. Among them, the avidin is preferably streptavidin.

Preferably, the kit further comprises other commonly used components of the enzyme-linked immunosorbent kit, such as an enzyme-labeled antibody, a sample diluent, a washing solution, and a color developing solution. The enzyme label in the enzyme-labeled antibody of the present invention may be selected from conventional enzymes and corresponding color developing solutions such as horseradish peroxidase and TMB color developer.

Preferably, the diabetes autoantigen is selected from one or more of IA-2, GAD, IC, CPH, ZnT8, and insulin; in a specific embodiment of the present invention, the diabetes autoantigen is IA-2, GAD, IC, CPH, ZnT8 and insulin.

In the coating, the diabetic autoantigen is coated with an antigen dilution buffer using CB buffer or Tris buffer; preferably, the buffer is selected from a CB buffer of pH 9.6 or a Tris buffer of pH 8.5. Liquid, more preferably, adding PEG or PVP, Proclin 300 to the buffer, and adding water-soluble cyclodextrin, can make the coating more stable, the antigen coating point is more regular, more round, and the CV is smaller.

The water-soluble cyclodextrin may be Captisol, 2-hydroxy-β-cyclodextrin or carboxymethyl-β-cyclodextrin at a concentration of 0.02%; the concentration of PEG or PVP is 5%, and the concentration of Proclin 300 is 0.05. %, the percentage is the mass percentage (w/v) except that Proclin 300 is a volume percentage.

In a specific embodiment of the invention, the dilution buffer of GAD and CPH is 0.02 M Tris buffer (5% PEG, 0.05% Proclin 300, 0.02% Captisol, and 15% glycerol) at pH 8.5, final concentration They are 8ug/ml and 30ug/ml respectively.

The dilution buffer of IA-2, IC, insulin, and ZnT8 was C9.6 buffer of PH9.6, and the final concentrations were 10 ug/ml, 10 ug/ml, 80 ug/ml, and 15 ug/ml, respectively.

In addition, the protein chip in the kit of the present invention further comprises one or more of a negative control point, a positive control point, a sample control point, an enzyme label control point, a reference curve point, and a position reference point; Specifically, there is at least one negative property control point (NC) and one positive nature control point (PC); at least one sample point control point (SC) and one enzyme standard control point (EC); at least three reference curve points (S1-S3) and a position reference point (Loc) coated by the chip itself.

In a specific embodiment, the protein chip of the present invention further comprises a negative property control point (NC) and a positive nature control point (PC); a sample point quality control point (SC) and an enzyme standard control point (EC) 3 reference curve points (S1-S3) and a position reference point (Loc) coated by the chip itself.

Among them, the positive control point can be human IgG, and the corresponding enzyme-labeled antibody is an anti-human IgG enzyme label. The positive control point can also be DNP coated with BSA, and the corresponding enzyme-labeled antibody is a mixture of an anti-human IgG enzyme label and an anti-DNP enzyme label.

The negative control point may be a small concentration of human IgG lower than the reaction signal value, or may be replaced by other unrelated proteins; the sample control point may be goat anti-human IgG or other anti-human IgG; The point may be human IgG, or other anti-rabbit antibody (the enzyme is labeled with rabbit anti-human IgG), such as a goat anti-rabbit IgG antibody. The reference curve points are low, medium and high concentrations of human IgG in the specific implementation process.

The positional reference point of the protein chip itself is a human IgG solution, which is mainly used for the positioning of the array on the protein chip.

Preferably, the above-mentioned negative property control points, positive property control points, sample control points, enzyme standard control points, reference curve points, and position reference points are also coated by a pre-biotinylated and avidinized bottom plate.

Meanwhile, the present invention also provides a preparation process of the biotinylated insulin antigen, which is specifically as follows:

Step 1. Insulin and S-acetylthioacetic acid succinimidyl ester (SATA, purchased from Thermo) are reacted at room temperature and purified by dialysis (by-products are small molecules that can pass through the dialysis bag, and the macromolecular insulin-SH-P cannot penetrate. Through the dialysis bag, the by-product can be removed by multiple dialysis methods to obtain insulin-SH-P, and the reaction formula is as follows:

In insulin-SH-P, insulin means insulin, SH means an active thiol group, and P means a protective group, ie

In the insulin structural formula, a circle represents an insulin amino acid chain, n represents the number of amino groups in the insulin amino acid chain, 1 ≤ x ≤ n, x and n are integers, and x is preferably 1.

insulin-SH-P is separated from PBS containing EDTA (metal ion in chelate solution to remove metal ions in solution) and hydroxylamine hydrochloride at room temperature, and the protective group is removed by purification on a Sephadex column to form active sulfhydryl groups. The group's insulin-SH, spare, the reaction is as follows:

Macromolecular protein, Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylic acid sodium salt (SMCC, available from Thermo) and succinic acid imide-twin Ethylene glycol-biotin was reacted at room temperature and purified by dialysis three times (by-product is that small molecules can pass through the dialysis bag, macromolecular proteins cannot pass through the dialysis bag, and by-products can be removed by multiple dialysis) to obtain Biotin- Macromolecular protein-SMCC, the macromolecular protein is HBA, BSA, OVA or casein, and the reaction formula is as follows:

According to the above reaction process, the macromolecular protein, Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylic acid sodium salt (SMCC) can be added when the reactant is added. And succinic acid imide-dodediol-biotin is added to complete the reaction, or the macromolecular protein can be first reacted with succinic acid iodide-dodepolyethylene glycol-biotin, and then SMCC is added. After two steps are completed, the essence of the reaction will not change. In this step reaction, the ellipse represents the amino acid chain of the macromolecular protein, and m represents the number of amino groups on the amino acid chain of the macromolecular protein, y+z≤m, z≥10, y≥1, and y, z and m are integers.

Step 2. The alternate insulin-SH and Biotin-macromolecule protein-SMCC are mixed and reacted at room temperature, and purified by a Sephadex column to obtain Biotin-macromolecule protein-Insulin having the structure shown in Formula 1 as biotinylation. The insulin antigen has the following reaction formula:

In the Biotin-macromolecule protein-Insulin, if z/x is a non-integer, z/x takes an integer and adds 1 to the integer position.

Wherein, the insulin and the S-acetylthioacetic acid succinimide ester are preferably prepared by using a PBS (preferably pH 7.0, 0.01 M PBS, preferably the following PBS is preferred) and dimethyl sulfoxide as a solvent. , ie, insulin in PBS solution, the concentration of insulin is 2-4mg/mL; the solution of S-acetylthioacetic acid succinimidyl ester in dimethyl sulfoxide, the concentration of S-acetylthioacetic acid succinimide ester is 40- 80mmol/L. The volume ratio of the insulin solution of PBS to the solution of S-acetylthioacetic acid succinimidyl ester in dimethyl sulfoxide was 1 mL: 10 μL.

In PBS containing EDTA and hydroxylamine hydrochloride, the concentration of EDTA was 20mmol/L, the concentration of hydroxylamine hydrochloride was 0.5mol/L, and the pH was 7.0; the volume ratio of insulin-SH-P to PBS containing EDTA and hydroxylamine hydrochloride was (6- 10): 1.

HBA and Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylic acid sodium salt are preferably prepared by using PBS as a solvent to prepare a reaction solution, succinic acid iodide-dodecyl The diol-biotin is prepared by using dimethyl sulfoxide as a solvent, that is, human blood albumin (HBA) in PBS, HBA concentration is 10-20 mg/mL; Sulfo-N-succinimidyl 4-( Maleimide methyl) cyclohexane-1-carboxylic acid sodium salt in PBS, Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylic acid The concentration of sodium salt is 10-15 mg/mL; the solution of succinic acid iodide-dodepolyethylene glycol-biotin in dimethyl sulfoxide, the concentration of succinic acid imide-dodediol-biotin is 0.25 -0.4 mol/L. Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylic acid sodium salt in PBS with succinic acid iodide-dodecyl glycol-biotin The volume ratio of the equimolar mixture solution of the methyl sulfoxide solution to the human blood albumin (HBA) PBS solution was (10-20 μL): 1 mL.

In a specific embodiment of the present invention, the above biotinylation method can be specifically as follows:

Prepare a 2-4 mg/mL solution of insulin PBS, and dispose a solution of S-acetylthioacetic acid succinimidyl ester in dimethyl sulfoxide (A solution) at a concentration of about 40-80 mmol/L, which will be 1-10 μl. The solution A was added to 1 ml of insulin PBS solution and mixed at room temperature for 1-4 hours. The reaction product was dialyzed for 3 times in PBS to recover insulin-SH-P, and stored at 4 degrees for temporary storage;

PBS was used as a solvent to prepare 0.5 mol/L hydroxylamine hydrochloride (containing 20 mmol/L EDTA, pH 7.0) as the B solution, and the volume ratio of insulin-SH-P and PBS containing EDTA and hydroxylamine hydrochloride was 6-10. ): 1, mixed room temperature reaction for 1-2 hours to remove the protective group, purified by a Sephadex column to form an active sulfhydryl group-containing insulin-SH;

Prepare 10-20 mg/mL human hemoglobin (HBA) in PBS and configure 10-15 mg/mL Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1 - a sodium carboxylate solution in PBS as a C solution, and a dimethyl sulfoxide solution having a concentration of 0.25-0.4 mol/L succinic acid-dodecyl glycol-biotin as a D solution, and a C solution After mixing with D solution in a ratio of 1:1, 10-20 μl was added to 1 mL of 10-20 mg/mL human serum albumin PBS solution, mixed at room temperature for 1-2 hours, and dialyzed against PBS for 3 times to obtain Biotin- HBA-SMCC;

The purified insulin-SH and Biotin-HBA-SMCC were mixed at room temperature for 10 to 60 minutes at a volume ratio of 10:(1-4) to obtain Biotin-macromolecule protein-Insulin as a biotinylated insulin antigen.

According to the above technical solution, the present invention biotinylates the insulin antigen by an improved biotinylation process, and obtains the biotinylated insulin antigen of the structure shown in Formula 1, and further increases the affinity of the avidin by pre-coating. The coating efficiency of the antigen insulin can significantly improve the sensitivity of the insulin antibody detection reagent or the kit prepared thereby, and can greatly improve the positive rate of insulin antibody detection and reduce the probability of missed detection or false detection.

DRAWINGS

Figure 1 is a schematic view showing the structure of the biotinylated insulin antigen of the present invention.

Detailed ways

The invention discloses a biotinylated insulin antigen and a preparation process thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The biotinylated insulin antigens, preparation processes and related applications of the present invention have been described in terms of preferred embodiments, and it is apparent to those skilled in the art that the biotinylation described herein can be carried out without departing from the spirit, scope and scope of the present invention. The insulin antigen, preparation process and related applications are modified or combined and modified to achieve and apply the techniques of the present invention.

In the structure of the formula 1 of the insulin antigen of the present invention, the macromolecular protein (generally above tens of Kda, such as 66 Kda for HBA) has a large molecule relative to insulin (5.8 Kda), and the number of amino groups on the molecule is far. It is much larger than the number of amino groups on the insulin n; at the same time, because of the large number of amino groups present on the macromolecular protein, SMCC and succinic acid imide-dodediol-biotin can be shared by controlling the amount of reactants. The amino group on the molecular protein is coupled.

For the insulin antigen of the structure of the formula 1 of the present invention, whether x is 1 or the number n of amino groups on the insulin does not affect the effect of connecting a plurality of insulins on the same macromolecular protein, but the number of insulins connected at x=1 most.

The following is a further description of a biotinylated insulin antigen provided by the present invention and a preparation process thereof.

Example 1: Preparation of biotinylated insulin antigen of the present invention

Prepare 2 mg/mL insulin PBS solution, dispose a solution of S-acetylthioacetic acid succinimidyl ester in dimethyl sulfoxide (A solution) at a concentration of about 50 mmol/L, and add 10 μl of A solution to 1 The solution of ML in insulin PBS was mixed for 1 hour at room temperature, and the reaction product was dialyzed in PBS for 3 times to recover insulin-SH-P. The infrared and nuclear magnetic detection showed the same structure as expected, and 4 degrees temporary storage;

PBS was used as a solvent to dispose 0.5 mol/L hydroxylamine hydrochloride (containing 20 mmol/L EDTA, pH 7.0) as a B solution, and 100 μl of B solution was added to the above 1 mL of insulin-SH-P, and mixed at room temperature. Hours, desalting column desalting purification to obtain insulin-SH, infrared and nuclear magnetic detection showed consistent with the expected structure.

Prepare 10 mg/mL human blood albumin (HBA) in PBS and dispose 10 mg/mL of Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate The salt PBS solution is used as the C solution, and the dimethyl sulfoxide solution of 0.25 mol/L succinic acid-dodecyl glycol-biotin is further set as the D solution, and the C solution and the D solution are as follows: After mixing 1 ratio, add 20 μl to 1 mL of 10 mg/mL human serum albumin PBS solution, mix and react at room temperature for 1 hour, and dialyze with PBS for 3 times to obtain Biotin-HBA-SMCC. Infrared and nuclear magnetic detection shows the expected structure. Consistent

The purified insulin-SH and Biotin-HBA-SMCC were mixed at room temperature for 10 minutes at a volume ratio of 10:1 to obtain Biotin-HBA-Insulin, which was used as a biotinylated insulin antigen, and the infrared and nuclear magnetic detection showed the expected structure of Formula 1. Consistent.

Example 2: Preparation of conventional biotinylated insulin antigen

Using the company of Thermo

The Sulfo-NHS-LC-Biotin biotinylation kit biotinylated insulin to obtain a Biotin-Insulinn conjugate.

Example 3: Comparison of the detection effects of the antigens prepared in Example 1 and Example 2 on insulinA

The biotinylated insulin antigens prepared in the above Example 1 and Example 2 were separately coated, and insulin antibodies (insulinA) positive and negative serum were detected, and the detection results were compared. Specific steps are as follows:

1, chip preprocessing:

6K times of streptavidin (0.01 M PBS diluted to pH 7.4) was added, added to a 96-well plate, 50 ul/well, and then allowed to stand in an incubator at 37 ° C for 2 h.

The 96-well plate was taken out, washed 3 times with PBS, and finally washed once with purified water and blotted dry. Washing conditions: 300 ul / well, allowed to stand for 30 sec / time.

Coating: The above two biotinylated insulin antigens were diluted with PH 9.6 CB buffer (5% PEG, 0.05% Proclin 300, and 0.02% Captisol) to a final concentration of 80 ug/mL. They were separately filtered with a 0.22 um filter, and then coated with a BioDot precision spotter in a volume of 10 nL, and then coated at 4 ° C for 24-30 h.

Blocking: Remove the coated chip, wash it 3 times with PBST washing solution of pH 7.4, then add 150 ul of blocking solution (pH 7.4, disodium hydrogen phosphate solution containing 1% BSA, adding 0.8% nectar) to each well. Alcohol, 1% PVA2W, 0.05% sodium azide preservative), sealed at room temperature for 1 h, then patted dry, under 15% humidity, room temperature, dried for 4 h, then sealed and stored at 2-8 °C.

Detection:

4.1 remove the coated plate and the reaction solution, and equilibrate to room temperature;

4.2 Loading: Negative and positive control serum, and the sample to be tested diluted 101 times with the sample dilution, 100 uL per well was added to the reaction well to be tested.

4.3 Incubation: The reaction was allowed to stand at room temperature for 30 min. 300 uL of washing solution (0.02 M Tris, 0.15 M NaCl, 0.05% Tween 20, pH 7.4) was added, washed 3 times, and allowed to stand for 1 min each time.

4.4 Addition of enzyme-labeled antibody: Add 50 uL of enzyme-labeled antibody to each well (4K-fold diluted HRP-labeled rabbit anti-human IgG).

4.5 Incubation: The reaction was allowed to stand at room temperature for 30 min. Add 300 uL of washing solution, wash 3 times, and let stand for 1 min each time.

4.6 Color development: 50 uL of TMB color developer was added to each well, and it was allowed to stand at room temperature for 30 minutes in the dark.

4.7 Measurement: Within 30 min, the signal value of the antibody corresponding to each reaction well was read and judged by a detector.

Test results:

Eight positive samples, eight weak positive samples and five negative samples were selected. The comparison test results are shown in the table below.

Table 1

As can be seen from Table 1, compared with the biotinylated insulin antigen of the present invention, the detection effect of the positive sample by the general biotinylated insulin antigen is inferior to the present invention, and the use of the biotinylated insulin antigen of the present invention can greatly improve the insulinA. The positive rate is significantly better than using a general biotinylation kit.

The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

Claims

A biotinylated insulin antigen characterized by having the structure shown in Formula 1:

Wherein, the circle represents insulin, 1≤x≤n, n represents the number of amino groups on the insulin, x and n are integers; the ellipse represents a macromolecular protein, y+z≤m, z≥10, y≥1,m represents The number of amino groups on the macromolecular protein, y, z, and m are integers. If z/x is a non-integer, then z/x takes an integer and adds 1 to the integer.
The insulin antigen according to claim 1, wherein the macromolecular protein is HBA, BSA, OVA or casein.
Use of the biotinylated insulin antigen of claim 1 in the preparation of an enzyme-linked immunoassay kit.
The use according to claim 3, wherein the enzyme-linked immunoassay kit is a diabetes immunoassay kit.
The process for preparing a biotinylated insulin antigen according to claim 1, which comprises the following steps: Step 1: reacting insulin with S-acetylthioacetic acid succinimidyl ester (SATA) at room temperature, and dialysis and purification to obtain insulin- SH-P: n represents the number of amino groups on the insulator, 1 ≤ x ≤ n, and x and n are integers;

The insulin-SH-P was reacted with hydroxylamine hydrochloride in PBS at room temperature to remove the protecting group, and purified by a Sephadex column to form an active sulfhydryl group-containing insulin-SH, which was used;

Macromolecular protein, Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylic acid sodium salt (SMCC) and succinic acid imide-dodepolyethylene glycol- Biotin was reacted at room temperature and purified by dialysis to obtain Biotin-macromolecule protein-SMCC; m represents the number of amino groups on the macromolecular protein, 2≤y+z≤m, y, z and m are integers

Step 2. The alternate insulin-SH and Biotin-macromolecule protein-SMCC are mixed and reacted at room temperature, and purified by a Sephadex column to obtain Biotin-macromolecule protein-Insulin having the structure shown in Formula 1 as biotinylation. Insulin antigen;

Wherein, the circle represents insulin, 1≤x≤n, n represents the number of amino groups on insulin, x and n are integers; ellipse represents macromolecular protein, 2≤y+z≤m, m represents amino group on macromolecular protein The number, y, z, and m are integers. If z/x is a non-integer, z/x takes an integer and adds 1 to the integer bits.
A diabetes antibody spectrum detecting kit comprising a protein chip coated with a diabetes autoantigen, which is coated with a biotinylated diabetes autoantigen by a protein chip of avidin, The biotinylated diabetes autoantigen comprises the insulin antigen of claim 1 or 2.
The test kit according to claim 6, wherein the diabetes autoantigen is one or more selected from the group consisting of IA-2, GAD, IC, CPH, ZnT8, and insulin.
The kit according to claim 6 or 7, wherein the diabetic autoantigen is coated with an antigen dilution buffer using CB buffer or Tris buffer.
The kit according to any one of claims 6-8, wherein the protein chip further comprises a negative control point, a positive control point, a sample control point, an enzyme standard control point, a reference curve point, and a position. One or more of the reference points.
The kit according to any one of claims 6 to 9, which further comprises an enzyme-labeled antibody, a sample diluent, a washing solution and a color developing solution.