CN108469519B

CN108469519B - Composition for enzyme-linked immunosorbent assay kit, diabetes antibody spectrum detection kit and preparation method thereof

Info

Publication number: CN108469519B
Application number: CN201810188203.6A
Authority: CN
Inventors: 张大准; 张永顶; 马伟民; 王洪涛; 马新民
Original assignee: SHENZHEN CITY BOLAOTE BIOLOGICAL PRODUCTS CO Ltd
Current assignee: SHENZHEN CITY BOLAOTE BIOLOGICAL PRODUCTS CO Ltd
Priority date: 2018-03-07
Filing date: 2018-03-07
Publication date: 2020-03-24
Anticipated expiration: 2038-03-07
Also published as: WO2019169798A1; CN108469519A

Abstract

The invention relates to the technical field of enzyme-linked immunosorbent assay, and discloses a composition for an enzyme-linked immunosorbent assay kit, a diabetes antibody spectrum detection kit and a preparation method thereof. The composition comprises a confining liquid and an enzyme-labeled diluent; the blocking solution contains BSA, polyvinyl alcohol, mannitol, sodium azide and disodium hydrogen phosphate, and the enzyme-labeled diluent contains Tris, citric acid, BSA, polyethylene glycol, gum arabic, betaine and Proclin 300. The enzyme-linked immunoassay kit starts with the confining liquid and the enzyme-labeled diluent of the enzyme-linked immunoassay kit, and can keep the stability of detection for a long time by selecting proper components. Meanwhile, the diabetes detection kit prepared from the composition can be used for accurately diagnosing and parting diabetes on the basis of better stability, and particularly has higher positive rate on the detection of IAA.

Description

Composition for enzyme-linked immunosorbent assay kit, diabetes antibody spectrum detection kit and preparation method thereof

Technical Field

The invention relates to the technical field of enzyme-linked immunosorbent assay, and in particular relates to a composition for an enzyme-linked immunosorbent assay kit, a diabetes antibody spectrum detection kit and a preparation method thereof.

Background

Diabetes (diabetes) is a series of metabolic disorder syndromes of sugar, protein, fat, water, electrolyte and the like caused by hypofunction of pancreatic islets, insulin resistance and the like due to actions of various pathogenic factors such as genetic factors, immune dysfunction, microbial infection and toxins thereof, radical toxins, mental factors and the like on organisms, and is clinically mainly characterized by hyperglycemia, and typical cases can show symptoms of polyuria, polydipsia, polyphagia, emaciation and the like, namely, three more or one less, diabetes shows high heterogeneity in etiology, pathogenesis, natural course and the like, so diabetes is a general term for a group of hyperglycemia diseases, and the diabetes is divided into type I diabetes, type II diabetes and other types based on etiology and pathogenesis, type I diabetes is a disease taking autoimmune destruction of pancreatic islet β cells as an important etiology, and one or more autoantibodies aiming at key enzymes of synthesis, transportation and secretion of insulin of pancreatic islet β cells exist in patients, and mainly comprise pancreatic Islet Cell Antibodies (ICA), insulin autoantibodies (IAA), glutamic acid antibodies (GADA), tyrosine-2A antibody (CPA-2A), carboxypeptidase (antibodies) and antibodies (antibodies) to protein decarboxylase, zinc-pyruvate decarboxylase and the like, and the antibodies (antibodies) of fasting glucose, and the antibodies).

The protein chip is a high-flux protein function analysis technology, and is a biological detection technology developed in recent years, and the technology is to shrink the analysis of protein onto a small chip, detect by fluorescence or enzyme color development, and finally analyze by using specific computer software. By means of the ELISA technology, the protein chip can rapidly analyze a large amount of protein samples in a high-throughput and quantitative manner, and has the advantages of less required reagents and samples, lower price after product production and the like, so that the protein chip can rapidly become a research hotspot. However, the stability of such kits is not good at present. In addition, the kit has insufficient positive rate for detecting the diabetic insulin autoantibody (IAA), and the kit has low positive rate and is easy to miss detection.

Disclosure of Invention

In view of the above, the present invention aims to provide a composition for an enzyme linked immunosorbent assay kit, so that when the composition is used for preparing the enzyme linked immunosorbent assay kit, the stability of the kit at low temperature and room temperature can be significantly improved, and the storage time is prolonged;

the invention also aims to provide the application of the composition in preparing an enzyme linked immunosorbent assay kit, in particular to a related kit for detecting diabetes;

another object of the present invention is to provide a kit for detecting a diabetes antibody profile comprising the above composition and a preparation method thereof, such that the kit has long-term stability at low temperature and room temperature, and simultaneously has high specificity and sensitivity of detection results;

another object of the present invention is to provide a kit for detecting a diabetic antibody profile comprising the above composition and a method for preparing the same, such that the kit can significantly improve the positive rate of detecting diabetic insulin autoantibodies (IAA).

In order to achieve the above purpose, the invention provides the following technical scheme:

a composition for an enzyme-linked immunoassay kit comprises a confining liquid and an enzyme-labeled diluent; the blocking solution contains BSA, polyvinyl alcohol, mannitol, sodium azide and disodium hydrogen phosphate, and the enzyme-labeled diluent contains Tris, citric acid, BSA, polyethylene glycol, gum arabic, betaine and Proclin 300.

Aiming at the defects of poor stability and short preservation time of the conventional enzyme-linked immunoassay kit, the invention unexpectedly discovers that the stability and the preservation time of the enzyme-linked immunoassay kit can be obviously improved by starting from a confining liquid for preparing the kit and an enzyme-labeled diluent (for diluting an enzyme-labeled antigen or antibody) and perfecting the composition of the confining liquid and the enzyme-labeled diluent by selecting proper components.

Preferably, the confining liquid contains 0.6% -1% BSA, 0.8% -1.5% polyvinyl alcohol, 0.8% -1.5% mannitol, 0.05% sodium azide and 0.01M disodium hydrogen phosphate, the pH value is 7.4, and the balance is water, wherein the percentages are mass percentages (w/v); in a specific embodiment of the invention, the blocking solution contains 1% BSA, 1% polyvinyl alcohol, 0.8% mannitol, 0.05% sodium azide and 0.01M disodium hydrogen phosphate, the pH is 7.4, and the balance is ultrapure water. Among them, polyvinyl alcohol is preferably polyvinyl alcohol 2W.

Preferably, the enzyme labeling diluent contains 0.1M Tris, 0.05M citric acid, 2% -3% BSA, 1% -3% polyethylene glycol, 0.01% Arabic gum, 0.8% -1.5% betaine and 0.05% Proclin300, and the balance of water, wherein the percentages are mass percent (w/v) except Proclin 300; in a specific embodiment of the invention, the enzyme-labeled diluent contains 0.1M Tris, 0.05M citric acid, 2.5% BSA, 2% polyethylene glycol, 0.01% gum arabic, 1% betaine, and 0.05% Proclin 300. Among them, polyethylene glycol 1W is preferable.

Compared with the conventional diabetes antibody enzyme-linked immunosorbent assay kit adopting a confining liquid and an enzyme-labeled diluent, the preparation of the diabetes antibody enzyme-linked immunosorbent assay kit adopting the composition can still keep the stability of the prepared kit after being placed for 24 months at low temperature (2-8 ℃), and the comparative kit has great instability after being placed for 18 months; at room temperature (18-28 ℃), the inventive kit still maintained stability at the completion of preparation after 6 months of storage, and maintained higher stability after 8 months of storage, whereas the control kit was already largely unstable after 4 months of storage.

Based on the excellent technical effects, the invention provides the application of the composition in preparing an enzyme-linked immunoassay kit, in particular to the application in preparing a diabetes enzyme-linked immunoassay kit.

Meanwhile, the invention also provides a diabetes antibody spectrum detection kit, which comprises the following components:

the kit comprises a protein chip coated with diabetes autoantigen, an enzyme-labeled antibody diluted by enzyme-labeled diluent, sample diluent, washing liquid and developing liquid; the protein chip is sealed by adopting a sealing solution after being coated with the diabetes autoantigen, the sealing solution contains BSA, polyvinyl alcohol, mannitol, sodium azide and disodium hydrogen phosphate, the enzyme-labeled diluent contains Tris, citric acid, BSA, polyethylene glycol, gum arabic, betaine and Proclin300, and the scheme of the sealing solution and the enzyme-labeled diluent is the same as that of the composition.

Preferably, the coating is coated by the binding of an avidin-linked protein chip to a biotinylated diabetes autoantigen. Among them, streptavidin is preferable.

Preferably, the diabetes autoantigen is one or more than two selected from IA-2, GAD, IC, CPH, ZnT8 and IA; in a particular embodiment of the invention, the diabetic autoantigen is IA-2, GAD, IC, CPH, ZnT8 and IA (both IA and insulin referred to herein are insulin).

In a preferred scheme, a protein chip of the kit at least comprises biotinylated IA as a diabetes autoantigen, and in order to improve the positive rate of an IA antibody IAA, the invention improves the biotinylated method of IA, couples a plurality of insulin molecules to macromolecular protein amino through SATA and SMCC, couples the macromolecular protein with Biotin at the same time, forms a Biotin-macromolecular protein-insulin conjugate, combines a plurality of insulin molecules and Biotin molecules (shown in a schematic diagram in figure 1), and greatly improves the coating efficiency of antigen insulin through the pre-coating avidin amplification effect, thereby obviously improving the sensitivity of the prepared insulin antibody detection reagent or kit, greatly improving the positive rate of detection of the insulin antibody, and reducing the probability of missed detection or false detection. The method comprises the following specific steps:

step 1, carrying out room temperature reaction on insulin and S-acetylthio acetic acid succinimide ester (SATA, purchased from Thermo), and dialyzing and purifying (by-products are small molecules which can permeate a dialysis bag, and large molecules of insulin-SH-P which cannot permeate the dialysis bag, and by-products can be removed by means of multiple dialysis) to obtain insulin-SH-P, wherein the reaction formula is as follows:

in insulin-SH-P, insulin represents insulin, SH represents an active sulfhydryl group, and P represents a protecting group, i.e.

in the structural formula of the insulin, a circle represents an insulin amino acid chain, n represents the number of amino groups on the insulin amino acid chain, x is more than or equal to 1 and less than or equal to n, x and n are integers, and x is preferably 1.

The insulin-SH-P reacts with PBS containing EDTA (chelating metal ions in solution to remove the interference of the metal ions in the solution) and hydroxylamine hydrochloride at room temperature to remove a protective group, and is purified by a sephadex column to form the insulin-SH containing an active sulfhydryl group for later use, wherein the reaction formula is as follows:

macromolecular protein, Sulfo-N-succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylic acid sodium salt (SMCC, from Thermo) and succinimidyl-dodecapolyethylene glycol-Biotin react at room temperature, and are dialyzed by PBS for 3 times for purification (by-products are small molecules which can permeate a dialysis bag, macromolecular protein which can not permeate the dialysis bag, and by-products can be removed by means of multiple dialysis), so that the Biotin-macromolecular protein-SMCC is obtained, wherein the macromolecular protein is HBA, BSA, OVA or casein, and the reaction formula is as follows:

according to the above reaction process, when the reactants are added, the macromolecular protein, Sulfo-N-succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylic acid sodium Salt (SMCC) and succinimidyl-dodecapolyethylene glycol-biotin may be added together to complete the reaction, or the macromolecular protein and succinimidyl-dodecapolyethylene glycol-biotin may be reacted first and then SMCC may be added to complete the reaction in two steps, and the reaction is not substantially changed. In the reaction of the step, an ellipse represents a macromolecular protein amino acid chain, m represents the number of amino groups on the macromolecular protein amino acid chain, y + z is less than or equal to m, z is more than or equal to 10, y is more than or equal to 1, y, z and m are integers, if z/x is a non-integer, z/x is an integer and 1 is added to an integer number.

Step 2, mixing the prepared Insulin-SH and Biotin-macromolecular protein-SMCC for reaction at room temperature, purifying by a sephadex column to obtain Biotin-macromolecular protein-Insulin which has a structure shown in a formula 1 and is used as a biotinylated Insulin antigen, wherein the reaction formula is as follows:

in Biotin-macromolecular protein-Insulin, if z/x is a non-integer, then z/x is an integer and 1 is added to the integer number. For the specific biotinylated insulin antigen of the present invention, the number of amino groups n on x, whether 1 or insulin, does not affect the effect of linking multiple insulins to the same macromolecular protein, but the number of linked insulins is the largest when x is 1.

Wherein, the IA and S-acetylsulfanylacetic acid succinimide ester are respectively preferably prepared by using PBS (preferably PBS with pH values of 7.0 and 0.01M, and the PBS is preferably selected below) and dimethyl sulfoxide as solvents to prepare a reaction solution, namely a PBS solution of IA, and the concentration of IA is 2-4 mg/mL; the concentration of S-acetylsulfanylacetic acid succinimide ester in dimethyl sulfoxide solution is 40-80 mmol/L. The volume ratio of the PBS solution of IA to the dimethyl sulfoxide solution of S-acetylsulfanylacetic acid succinimide ester is 1 mL: 10 μ L.

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

Preferably, PBS is used as a solvent to prepare a reaction solution by HBA and Sulfo-N-succinimidyl 4- (maleimide methyl) cyclohexane-1-carboxylic acid sodium salt, and dimethyl sulfoxide is used as a solvent to prepare a reaction solution by succinimidyl-dodecapolyethylene glycol-biotin, namely a PBS solution of human serum albumin (HBA), wherein the concentration of HBA is 10-20 mg/mL; a PBS solution of Sulfo-N-succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylic acid sodium salt, wherein the concentration of the Sulfo-N-succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylic acid sodium salt is 10-15 mg/mL; the concentration of the succinic acid imide-dodecapolyethylene glycol-biotin in the dimethyl sulfoxide solution is 0.25-0.4 mol/L. The volume ratio of the mixed solution of the PBS solution of Sulfo-N-succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylic acid sodium salt and the dimethyl sulfoxide solution of succinimidyl-dodecapolyethylene glycol-biotin in equal proportion to the PBS solution of human serum albumin (HBA) is (10-20 muL): 1 mL.

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

preparing 2-4mg/mL insulin PBS solution, preparing a dimethyl sulfoxide solution (solution A) of S-acetylthio acetic acid succinimidyl ester with the concentration of about 40-80mmol/L, adding 10 microliters of the solution A into 1 milliliter insulin PBS solution, uniformly mixing, reacting at room temperature for 1-4 hours, dialyzing the reaction product in PBS for 3 times, recovering to obtain insulin-SH-P, and temporarily storing at 4 ℃ for later use;

preparing 0.5mol/L hydroxylamine hydrochloride (containing 20mmol/L EDTA, pH7.0) as a B solution by using PBS as a solvent, mixing the solution and reacting at room temperature for 1-2 hours according to the volume ratio of the solution to the PBS containing EDTA and hydroxylamine hydrochloride (6-10) to 1, removing a protecting group, and purifying by using a sephadex column to form the solution containing active sulfhydryl groups;

preparing 10-20mg/mL PBS solution of human serum albumin (HBA), preparing 10-15mg/mL PBS solution of Sulfo-N-succinimidyl 4- (maleimide methyl) cyclohexane-1-carboxylic acid sodium salt as C solution, preparing 0.25-0.4mol/L dimethyl sulfoxide solution of succinimidyl-dodecapolyethylene glycol-biotin as D solution, mixing the C solution and the D solution according to the proportion of 1: 1 proportion, adding 10-20 microliter into 1mL of 10-20mg/mL human serum albumin PBS solution, mixing uniformly, reacting at room temperature for 1-2 hours, dialyzing with PBS for 3 times, and purifying to obtain Biotin-HBA-SMCC;

mixing the purified Insulin-SH and Biotin-HBA-SMCC according to a volume ratio of 10 (1-4) and reacting at room temperature for 30-60 minutes to obtain Biotin-macromolecular protein-Insulin which is used as a biotinylated Insulin antigen.

Tests prove that when the biotinylated IA obtained by the conventional method and the IA obtained by the biotinylation method are used for detection, the method can obtain higher IAA positive rate and avoid the condition that a sample is missed to be detected or misjudged due to lower concentration.

In coating, the diabetes autoantigen is coated by using CB buffer solution or Tris buffer solution as antigen dilution buffer solution; preferably, the buffer is selected from CB buffer at pH9.6 or Tris buffer at pH8.5, more preferably, PEG or PVP, Proclin300 is added in the buffer, and water-soluble cyclodextrin is added, so that the coating is more stable, the antigen coating points are more regular and round, and the CV is smaller.

Wherein the water-soluble cyclodextrin can be Captisol, 2-hydroxy- β -cyclodextrin or carboxymethyl- β -cyclodextrin and the like, the concentration is 0.02 percent, the concentration of PEG or PVP is 5 percent, the concentration of Proclin300 is 0.05 percent, and the rest percentages are mass percentages (w/v) except that Proclin300 is volume percentage.

In a specific embodiment of the invention, the GAD and CPH dilution buffer is 0.02M Tris buffer (containing 5% PEG, 0.05% Proclin300, 0.02% Captisol, and 15% glycerol) at pH8.5 to final concentrations of 8ug/ml and 30ug/ml, respectively.

The dilution buffer of IA-2, IC, IA and ZnT8 is CB buffer with pH9.6, and the final concentration is 10ug/ml, 80ug/ml and 15ug/ml respectively.

In addition, the protein chip in the kit also comprises one or more than two of a negative quality control point, a positive quality control point, a sample quality control point, an enzyme-labeled quality control point, a reference curve point and a position reference point; more specifically, there is at least one negative quality control point (NC) and one positive quality control Point (PC); at least one sample spot quality control Spot (SC) and one enzyme-labeled quality control spot (EC); at least 3 reference curve points (S1-S3) and a chip-self-coated location reference point (Loc).

In specific embodiments, the protein chip of the present invention further comprises a negative quality control point (NC) and a positive quality control Point (PC); a sample point quality control point (SC) and an enzyme-labeled quality control point (EC); 3 reference curve points (S1-S3) and one chip-itself-coated location reference point (Loc).

Wherein, the positive quality control point can be human IgG, and the corresponding enzyme-labeled antibody is enzyme-labeled anti-human IgG. The positive control point can also be coated with DNP coupled by BSA, and the correspondingly used enzyme-labeled antibody is a mixed solution of enzyme-labeled anti-human IgG and enzyme-labeled anti-DNP.

The negative quality control point can be human IgG with trace concentration lower than the reaction signal value or other unrelated proteins are adopted for replacing; the sample quality control point can be goat anti-human IgG or other anti-human IgG; the enzyme-labeled quality control point can be human IgG or other anti-rabbit antibodies (the enzyme-labeled enzyme is rabbit anti-human IgG), such as goat anti-rabbit IgG antibodies. The reference curve points are human IgG at three concentrations, low, medium, and high, during the implementation.

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

Preferably, the negative quality control point, the positive quality control point, the sample quality control point, the enzyme-labeled quality control point, the reference curve point and the position reference point are also coated by a base plate which is biotinylated and avidin-treated in advance.

The enzyme label in the enzyme-labeled antibody can select conventional enzyme and corresponding color development liquid, such as horseradish peroxidase and TMB color development agent.

The invention also correspondingly provides a preparation method of the kit, which comprises the following steps:

coating the diabetes autoantigen on a protein chip, washing after coating, and then adding a confining liquid for sealing to obtain the protein chip coated with the diabetes autoantigen, wherein the confining liquid contains BSA, polyvinyl alcohol, mannitol, sodium azide and disodium hydrogen phosphate;

preparing an enzyme-labeled diluent containing Tris, citric acid, BSA, polyethylene glycol, gum arabic, betaine and Proclin300, diluting an enzyme-labeled antibody to obtain the enzyme-labeled antibody diluted by the enzyme-labeled diluent, and then preparing a sample diluent, a washing solution and a developing solution to obtain the diabetes antibody spectrum detection kit.

When the diabetes antibody spectrum detection kit is used for detecting a serum sample, the positive rate of total antibodies in T1MD is 98.21%, the positive rate in T2DM is 10.58%, the positive rate in normal people is only 2.56%, and the difference has significant meaning (P is less than 0.01).

According to the technical scheme, the enzyme-linked immunoassay kit starts from the confining liquid and the enzyme-labeled diluent of the enzyme-linked immunoassay kit, and can keep the stability of detection for a long time by selecting proper components. Meanwhile, the diabetes detection kit prepared from the composition can be used for accurately diagnosing and parting diabetes on the basis of better stability, and particularly has higher positive rate on the detection of IAA.

Drawings

FIG. 1 is a schematic diagram showing the structure of the biotinylated insulin antigen of the present invention.

Detailed Description

The invention discloses a composition for an enzyme-linked immunosorbent assay kit, a diabetes antibody spectrum detection kit and a preparation method thereof, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the compositions, kits and uses of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the compositions, kits and uses described herein may be made and employed without departing from the spirit, scope and spirit of the invention.

The composition for the enzyme linked immunosorbent assay kit, the diabetes antibody spectrum detection kit and the preparation method thereof provided by the invention are further described below.

Example 1: preparation of diabetes antibody spectrum detection kit

1. Chip substrate and pretreatment of diabetes autoantigen

(1) Chip pretreatment:

streptavidin diluted 6K-fold (0.01M PBS at pH7.4) was added to 96-well plates at 50 ul/well, and then allowed to stand in a 37 ℃ incubator for 2 hours.

The 96-well plate was removed, washed 3 times with PBS and finally once with purified water, and blotted dry. Washing conditions are as follows: 300 ul/hole, standing for 30 sec/time.

Drying in a constant-temperature air drying oven at 37 ℃ for later use.

(2) Pretreatment of diabetes autoantigen:

with Thermo

The Sulfo-NHS-LC-Biotin biotinylation kit biotinylates IA-2, GAD, IC, CPH, ZnT8, respectively.

The biotinylation of the Insulin (IA) antigen employs an improved technique, the specific scheme is as follows:

preparing 2mg/mL insulin PBS solution, preparing a dimethyl sulfoxide solution (solution A) of S-acetylthio acetic acid succinimide ester with the concentration of about 50mmol/L, adding 10 microliters of the solution A into 1 milliliter insulin PBS solution, uniformly mixing, reacting at room temperature for 1 hour, dialyzing the reaction product in the PBS for 3 times, and recovering to obtain insulin-SH-P, wherein infrared and nuclear magnetic detection shows that the structure is consistent with the expected structure, and storing at 4 ℃ for later use;

0.5mol/L hydroxylamine hydrochloride (containing 20mmol/L EDTA, pH7.0) is prepared by taking PBS as a solvent to be used as a B solution, 100 microliter of the B solution is added into 1mL of the solution of the insulin-SH-P, the solution of the B solution is mixed and reacted for 1 hour at room temperature, the solution of the insulin-SH is obtained by desalting and purifying a desalting column, and the infrared and nuclear magnetic detection shows that the solution of the insulin-SH is consistent with the expected structure.

Preparing 10mg/mL PBS solution of human serum albumin (HBA), preparing 10mg/mL PBS solution of Sulfo-N-succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylic acid sodium salt as C solution, preparing 0.25mol/L dimethyl sulfoxide solution of succinimidyl-dodecapolyethylene glycol-biotin as D solution, and mixing the C solution and the D solution according to the ratio of 1: mixing at a ratio of 1, adding 20 microliters of the mixture into 1mL of 10mg/mL human serum albumin PBS solution, uniformly mixing, reacting at room temperature for 1 hour, dialyzing with PBS for 3 times, and purifying to obtain Biotin-HBA-SMCC, wherein infrared and nuclear magnetic detection shows that the structure of the Biotin-HBA-SMCC is consistent with the expected structure;

mixing the purified Insulin-SH and Biotin-HBA-SMCC according to the volume ratio of 10:1, reacting at room temperature for 30 minutes to obtain Biotin-macromolecular protein-Insulin, wherein the Biotin-macromolecular protein-Insulin has a structure shown in a formula 1 and is used as a biotinylated Insulin antigen, and infrared and nuclear magnetic detection shows that the structure is consistent with an expected structure.

2. Coating of diabetes autoantigen and related proteins

The PC, NC, S1, S2, S3 and EC in the protein chip array were coated with 2ug/ml, 0.01ug/ml, 0.5ug/ml, 2ug/ml and 4ug/ml of biotinylated (using the aforementioned biotinylation kit, the same applies below) human IgG, respectively, and diluted with CB buffer (5% PEG, 0.05% Proclin300, and 0.02% Captisol) at pH 9.6.

For SC dot, 2ug/ml of biotinylated goat anti-human IgG antibody was used, and the dilution buffer was CB buffer at pH 9.6.

The Loc point used a 2ug/ml solution of biotinylated human IgG in CB buffer at pH 9.6.

The dilution buffer for GAD and CPH was 20mM Tris buffer (containing 5% PEG, 0.05% Proclin300, 0.02% Captisol, and 15% glycerol) at pH8.5 to final concentrations of 8ug/ml and 30ug/ml, respectively.

The dilution of IA-2, IC, IA and ZnT8 is CB buffer solution with pH9.6, and the final concentration is 10ug/ml, 80ug/ml and 15ug/ml respectively.

The diluted antigens were filtered through 0.22um filters and coated onto arrays using a BioDot precision printer. After spotting of all arrays was complete, the chips were placed at 2-8 ℃ overnight for 24-30 h. The protein chip array can refer to the array presented in the following table, and can also be adjusted according to actual needs without limitation:

TABLE 1 protein chip array

PC	NC	IC	NC
				S1	GAD	IA	SC
S2	CPH	ZnT8	EC
				S3	IA-2	PC	Loc

3. Sealing of

The coated chip was removed, washed 3 times with PBST (pH7.4) wash solution, 150ul of blocking solution (pH7.4, 1% BSA in disodium hydrogen phosphate solution with 0.8% mannitol, 1% PVA2W, 0.05% sodium azide preservative) was added to each well, blocked at room temperature for 1 hour, patted dry, allowed to stand at a humidity of 15% or less, dried at room temperature for 4 hours, and sealed and stored at 2-8 ℃.

4. Preparing enzyme-labeled diluent, enzyme-labeled antibody, developing solution, sample diluent and concentrated washing solution

Enzyme-labeled diluent: containing 100mM Tris, 50mM citric acid, 2.5% BSA, 2% PEG1W, 0.01% acacia, 1% betaine, and 0.05% Proclin 300;

enzyme-labeled antibody: rabbit anti-human IgG antibody marked by horseradish peroxidase;

when in use, the rabbit anti-human IgG antibody marked by horseradish peroxidase is diluted to 4K times (the concentration of the enzyme-labeled antibody) by using an enzyme-labeled diluent.

Color development liquid: (iii) a sinker type TMB.

Sample diluent: 0.02M Tris, 0.15M NaCl, 0.05% Tween20, 0.01% casein, pH7.4.

10 times of concentrated washing solution: 0.2M Tris, 1.5M NaCl, 0.5% Tween20, pH7.4.

The protein chip coated with the diabetes autoantigen, the enzyme-labeled antibody and the developing solution diluted by the enzyme-labeled diluent, the sample diluent and the 10-fold concentrated washing solution form the diabetes antibody spectrum detection kit.

5. Detection method

(1) Taking out the protein chip, and balancing to room temperature;

(2) sample adding: negative and positive control serum and a sample to be tested diluted by 101 times by using a sample diluent are added into each hole by 100uL for reaction.

(3) And (3) incubation: standing and reacting for 30min at room temperature. 300uL of washing solution (diluted 10 times with ultrapure water) was added thereto, and the mixture was washed 3 times, each time for 1 min.

(4) Adding an enzyme-labeled antibody: 50uL of enzyme-labeled antibody was added to each well.

(5) And (3) incubation: standing and reacting for 30min at room temperature. Adding 300uL of washing solution, washing for 3 times, and standing for 1min each time.

(6) Color development: adding 50uL of TMB color developing agent into each hole, standing at room temperature, and reacting for 30min in a dark place.

(7) And (3) determination: reading and calculating the signal value and the concentration of the antibody corresponding to each reaction hole by using a detector within 30 min; the signal detection system can also be realized by chemiluminescence, and a chemiluminescent substrate such as luminol can be used for reading the result by a fluorescence detection device.

Example 2: detection of clinical samples

Sample source: the diabetic patients were all from newly diagnosed patients, and met the 1999 diabetes diagnosis criteria, wherein 56 patients with T1DM, aged 6-52 years, 104 patients with T2DM, aged 20-56 years, 78 normal people, all from healthy physical examiners, had no family history of diabetes, and aged 15-55 years. Respectively extracting venous blood, separating serum, subpackaging, and freezing at-20 deg.C for inspection.

Specific experimental results and data are given in the following table:

TABLE 2

Group of	n	IA-2A	GADA	ICA	CPH-A	ZnT8-A	IAA	Total positive rate
									T1DM	56	26	42	17	3	15	14	55/56(98.21％)
T2DM	104	2	10	1	3	0	2	11/104(10.58％)
									Normal group	78	0	0	0	2	0	0	2/78(2.56％)

Type 1 diabetes (T1DM) is an absolute deficiency of insulin due to destruction of islet cells by autoantibody immunity, whereas type 2 diabetes (T2DM) is produced by insulin resistance, generally associated with a lifestyle-related diet, in which fewer autoantibodies are detectable. The positive rates of diabetic autoantibodies in these three populations should be significantly different.

According to the experimental results, when the kit is used for detecting serum samples, the positive rate of total antibodies in T1MD is 98.21%, the positive rate of T2DM is 10.58%, the positive rate of normal people is only 2.56%, three groups of differences have significant meaning (P is less than 0.01), and the kit conforms to the rules and standards of clinical detection.

Example 3: the stability detection of the kit of the invention

Control kit: the preparation was carried out as described in example 1, except that the blocking solution and the enzyme-labeled diluent were each prepared using conventional 0.01MPBS (pH7.4) + 10% BSA;

test kit: example 1 kit;

the detection method comprises the following steps: the two kits were placed at room temperature (18-28 ℃) and low temperature (2-8 ℃) respectively for a period of time, and then the same serum was used for detection according to the detection method of example 1, and the signal values of the instrument were counted, and the results are shown in tables 3-6.

1. Example 1 stability data of the kit at Low temperatures

TABLE 3

As can be seen from Table 3, the detection signal values of the kit of the invention after being placed for 0, 6, 12, 18 and 24 months at low temperature are respectively detected, and the ratio of the signal values is counted, and the results show that the detection signal values of the kit of the invention after being placed for 24 months are almost all over 90% compared with the detection signal values of the kit of the invention after being placed for 0, 6, 12 and 18 months, thus proving that the kit of the invention still has higher detection stability after being placed for 24 months at low temperature.

2. Example 1 stability data of the kit at room temperature

TABLE 4

As can be seen from Table 4, the detection signal values of the kit of the invention after being placed for 0, 2, 4, 6 and 8 months at normal temperature are respectively detected, and meanwhile, the ratio of the signal values is counted, and the result shows that the detection signal values of the kit of the invention after being placed for 8 months are all more than 80% compared with the detection signal values after being placed for 0, 2, 4 and 6 months, thus proving that the kit of the invention still has higher detection stability after being placed for 8 months at normal temperature.

3. Example 1 comparison of stability data at Low temperatures for kits and control kits

TABLE 5

As can be seen from Table 5, the detection signal values of the kit and the control kit which are placed at low temperature for 6 months, 12 months, 18 months and 24 months are respectively detected by the kit and the control kit, and the ratio of the signal values of the two kits at the same time is counted, and the result shows that the detection signal value of the control kit which is placed for 18 months starts to be remarkably reduced, and the data in the table 3 can obviously draw the conclusion that the stability of the control kit is not as good as that of the kit, but the difference between the stability of the control kit and the stability of the enzyme-labeled diluent is only in the presence of the confining liquid and the.

4. EXAMPLE 1 comparison of stability data between kits and control kits at Normal temperature

TABLE 6

As can be seen from Table 6, the detection signal values of the kit and the control kit which are placed at normal temperature for 3 months, 6 months, 9 months and 12 months are respectively detected by the kit and the control kit, and meanwhile, the ratio of the signal values of the two kits at the same time is counted, the result shows that the detection signal values of some indexes of the control kit which is placed for 4 months begin to obviously decrease, the data in the table 4 can obviously show that the stability of the control kit is not as the conclusion of the kit, and the difference between the two is only in the confining liquid and the enzyme-labeled diluent.

Example 4: detection test of positive rate of IAA by using kit provided by the invention

Control kit: preparation is carried out as in example 1, with the difference that biotinylation of IA is likewise carried out

Sulfo-NHS-LC-Biotin biotinylation kit;

test kit: example 1 kit;

the detection method comprises the following steps: 8 positive samples, 8 weak positive samples and 5 negative samples are selected, the two kits are respectively adopted for detection according to the detection method of the embodiment 1, the signal value of the instrument is counted, and the result is shown in a table 7.

TABLE 7

As can be seen from table 7, compared with the method for biotinylating IA in the present invention, the detection effect of biotinylating IA with a general biotinylation kit on a positive sample is not as good as that of the method in the present invention.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The kit for detecting the antibody spectrum of the diabetes is characterized by comprising the following components:

the kit comprises a protein chip coated with diabetes autoantigen, an enzyme-labeled antibody diluted by enzyme-labeled diluent, sample diluent, washing liquid and developing liquid; wherein, the protein chip is sealed by a sealing liquid after being coated with the diabetes autoantigen, the sealing liquid contains 1% BSA disodium hydrogen phosphate solution, 0.8% mannitol, 1% PVA2W, 0.05% sodium azide preservative, pH value of 7.4 and the balance of water, the enzyme labeling diluent contains 100mM Tris, 50mM citric acid, 2.5% BSA, 2% PEG1W, 0.01% acacia, 1% betaine, 0.05% Proclin300 and the balance of water;

the coating is coated by the mutual combination of an avidin protein chip and biotinylated diabetes autoantigen;

the biotinylated diabetes autoantigen comprises biotinylated insulin, and the biotinylation process is as follows:

preparing 2mg/mL insulin PBS solution, preparing dimethyl sulfoxide solution of S-acetylsulfanylacetic acid succinimidyl ester with the concentration of about 50mmol/L, namely solution A, adding 10 microliter of solution A into 1 milliliter insulin PBS solution, uniformly mixing, reacting at room temperature for 1 hour, dialyzing the reaction product in PBS for 3 times, recovering to obtain insulin-SH-P, and temporarily storing at 4 ℃ for later use;

preparing 0.5mol/L hydroxylamine hydrochloride by using PBS as a solvent, wherein the hydroxylamine hydrochloride contains 20mmol/L EDTA and has pH of 7.0, taking the solution B as a solution B, adding 100 microliters of the solution B into 1mL of the solution B, mixing the solution B and the solution B for reaction at room temperature for 1 hour, and desalting and purifying by using a desalting column to obtain the solution B;

preparing 10mg/mL human serum albumin PBS solution, preparing 10mg/mL sulfoo-N-succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylic acid sodium salt PBS solution as C solution, preparing 0.25mol/L succinic acid imide-dodecapolyethylene glycol-biotin dimethyl sulfoxide solution as D solution, and mixing the C solution and the D solution according to the ratio of 1: mixing at a ratio of 1, adding 20 microliters of the mixture into 1mL of 10mg/mL human serum albumin PBS solution, uniformly mixing, reacting at room temperature for 1 hour, dialyzing with PBS for 3 times, and purifying to obtain Biotin-HBA-SMCC;

mixing the purified Insulin-SH and Biotin-HBA-SMCC according to the volume ratio of 10:1, reacting at room temperature for 30 minutes to obtain Biotin-macromolecular protein-Insulin which has a structure shown in a formula 1 and is used as biotinylated Insulin antigen;

wherein, the circle represents insulin, x is more than or equal to 1 and less than or equal to n, n represents the number of amino groups on the insulin, and x and n are integers; the ellipse represents the macromolecular protein, y + z is less than or equal to m, z is more than or equal to 10, y is more than or equal to 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, z/x is an integer and 1 is added to an integer number.

2. The kit of claim 1, wherein the diabetic autoantigen is coated with CB buffer or Tris buffer as an antigen dilution buffer.

3. The kit of claim 1 or 2, wherein the protein chip further comprises one or more of a negative quality control point, a positive quality control point, a sample quality control point, an enzyme-labeled quality control point, a reference curve point and a position reference point.

4. The method for preparing the kit according to claim 1, comprising:

coating the diabetes autoantigen on a protein chip, washing after coating, and then adding a confining liquid for sealing to obtain the protein chip coated with the diabetes autoantigen, wherein the confining liquid contains a 1% BSA disodium hydrogen phosphate solution, 0.8% mannitol, 1% PVA2W and 0.05% sodium azide preservative are added, the pH value is 7.4, and the balance is water;

preparing enzyme-labeled diluent containing 100mM Tris, 50mM citric acid, 2.5% BSA, 2% PEG1W, 0.01% gum arabic, 1% betaine, 0.05% Proclin300 and the balance of water, diluting the enzyme-labeled antibody to obtain the enzyme-labeled antibody diluted by the enzyme-labeled diluent, and then preparing sample diluent, washing solution and developing solution to obtain the diabetes antibody spectrum detection kit.