WO2019169798A1 - 一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法 - Google Patents

一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法 Download PDF

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WO2019169798A1
WO2019169798A1 PCT/CN2018/093039 CN2018093039W WO2019169798A1 WO 2019169798 A1 WO2019169798 A1 WO 2019169798A1 CN 2018093039 W CN2018093039 W CN 2018093039W WO 2019169798 A1 WO2019169798 A1 WO 2019169798A1
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enzyme
kit
insulin
diabetes
bsa
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PCT/CN2018/093039
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English (en)
French (fr)
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张大准
张永顶
马伟民
王洪涛
马新民
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深圳市伯劳特生物制品有限公司
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Publication of WO2019169798A1 publication Critical patent/WO2019169798A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the invention relates to the field of enzyme-linked immunoassay, and particularly relates to a composition for an enzyme-linked immunoassay kit, a diabetes antibody spectrum detecting kit and a preparation method thereof.
  • Diabetes is a sugar, protein, caused by genetic factors, immune dysfunction, microbial infections and their toxins, free radical toxins, mental factors and other pathogenic factors that cause the islet dysfunction, insulin resistance, etc.
  • a series of metabolic disorders such as fat, water and electrolytes are clinically characterized by hyperglycemia.
  • polyuria, polydipsia, polyphagia, and weight loss may occur, that is, symptoms of "three more and one less”.
  • Diabetes shows high heterogeneity in terms of etiology, pathogenesis and natural course of disease, so diabetes is a general term for a group of hyperglycemic diseases. Diabetes is classified into type I diabetes, type II diabetes, and other types based on etiology and pathogenesis.
  • Type I diabetes is a disease in which autoimmune destruction of islet ⁇ cells is an important cause.
  • One or more autoantibodies against key enzymes involved in insulin synthesis, transport and secretion of islet ⁇ cells including islet cell antibodies (ICA), insulin autoantibody (IAA), glutamate decarboxylase antibody (GADA), tyrosine phosphate antibody (IA-2A), carboxypeptidase antibody (CPH-A) and zinc transporter 8 antibody (ZnT8-A) ) 6 kinds of autoantibodies.
  • ICA islet cell antibodies
  • IAA insulin autoantibody
  • GADA glutamate decarboxylase antibody
  • IA-2A tyrosine phosphate antibody
  • CPH-A carboxypeptidase antibody
  • ZnT8-A zinc transporter 8 antibody
  • Protein chip is a high-throughput protein function analysis technology. It is a biological detection technology developed in recent years. This technology is to micronize the analysis of proteins onto small chips and detect them by fluorescence or enzymatic color development. Analyze with specific computer software. By relying on ELISA technology, protein chips can quickly, high-throughput, quantitative analysis of a large number of protein samples, as well as the need for fewer reagents and samples, and lower prices after productization, making it a hot research topic. However, the stability of this type of kit is currently not good. In addition, the kit has insufficient positive rate for detecting insulin autoantibodies (IAA), and the positive rate is low, which is easy to miss.
  • IAA insulin autoantibodies
  • an object of the present invention is to provide a composition for an enzyme-linked immunoassay kit, which enables the composition to significantly improve the stability of the kit at low temperature and room temperature when used in the preparation of an enzyme-linked immunoassay kit. , the storage time is extended;
  • Another object of the present invention is to provide an application of the above composition in the preparation of an enzyme-linked immunoassay kit, particularly a kit for detecting diabetes;
  • Another object of the present invention is to provide a diabetes antibody spectrum detecting kit comprising the above composition and a preparation method thereof, which make the kit have long-term stability at low temperature and room temperature, and the detection result is high. Specificity and sensitivity;
  • Another object of the present invention is to provide a diabetes antibody spectrum detecting kit comprising the above composition and a method for preparing the same, which enables the kit to significantly increase the positive rate of detecting insulin autoantibodies (IAA).
  • IAA insulin autoantibodies
  • the present invention provides the following technical solutions:
  • a composition for an enzyme-linked immunoassay kit comprising a blocking solution and an enzyme standard dilution; the blocking solution containing BSA, polyvinyl alcohol, mannitol, sodium azide and disodium hydrogen phosphate, the enzyme standard dilution
  • the solution contains Tris, citric acid, BSA, polyethylene glycol, gum arabic, betaine and Proclin 300.
  • the present invention unexpectedly finds that the blocking solution and the enzyme standard dilution solution (for diluting the enzyme-labeled antigen or antibody) of the preparation kit are used. Selecting the appropriate components to improve the composition of both can significantly improve the stability and storage time of the enzyme-linked immunoassay kit.
  • the blocking solution contains 0.6% to 1% BSA, 0.8% to 1.5% polyvinyl alcohol, 0.8% to 1.5% mannitol, 0.05% sodium azide and 0.01 M disodium hydrogen phosphate, pH 7.4.
  • the balance is water, the percentage is mass percentage (w/v); in a specific embodiment of the invention, the blocking solution contains 1% BSA, 1% polyvinyl alcohol, 0.8% mannitol, 0.05% stack Nitrogen sodium and 0.01 M disodium hydrogen phosphate, pH 7.4, the balance is ultrapure water.
  • the polyvinyl alcohol is preferably polyvinyl alcohol 2W.
  • the enzyme standard dilution contains 0.1 M Tris, 0.05 M citric acid, 2%-3% BSA, 1%-3% polyethylene glycol, 0.01% gum arabic, 0.8%-1.5 % of betaine and 0.05% of Proclin 300, the balance being water, the percentage being the mass percentage (w/v) except Proclin 300 is a percentage by volume; in a particular embodiment of the invention, the enzyme label The dilution contained 0.1 M Tris, 0.05 M citric acid, 2.5% BSA, 2% polyethylene glycol, 0.01% gum arabic, 1% betaine, and 0.05% Proclin 300.
  • the polyethylene glycol is preferably polyethylene glycol 1W.
  • the invention utilizes the above composition to prepare the diabetes antibody enzyme-linked immunoassay kit, and the invention is at a low temperature (2-8 ° C) compared with the conventional blocking solution and the enzyme standard dilution diabetes antibody ELISA kit.
  • the kit was able to maintain stability at the completion of the preparation after 24 months of storage, while the control kit showed a significant degree of instability after 18 months of storage; at room temperature (18-28 ° C),
  • the kit of the present invention can maintain the stability at the completion of preparation after being left for 6 months, and can maintain high stability after being placed for 8 months, while the control kit has appeared very much after being placed for 4 months. A large degree of instability.
  • the present invention proposes the use of the composition in the preparation of an enzyme-linked immunoassay kit, particularly in the preparation of a diabetic enzyme-linked immunoassay kit.
  • the present invention also provides a diabetes antibody spectrum detecting kit comprising the following components:
  • the blocking solution contains BSA, polyvinyl alcohol, mannitol, sodium azide and disodium hydrogen phosphate, and the diluted diluent contains Tris, citric acid, BSA, polyethylene glycol, gum arabic, betaine and Proclin 300.
  • the blocking solution and the enzyme standard dilution are the same as in the previous composition scheme.
  • the envelope is coated with a biotinylated diabetes autoantigen by avidinized protein chip.
  • the avidin is preferably streptavidin.
  • the diabetes autoantigen is selected from one or more of IA-2, GAD, IC, CPH, ZnT8, IA; in a specific embodiment of the present invention, the diabetes autoantigen is IA-2, GAD, IC, CPH, ZnT8 and IA (IA and insulin mentioned in the present invention all represent insulin).
  • the protein chip of the kit of the present invention comprises at least biotinylated IA as a diabetes autoantigen, and the present invention improves the biotinylation method of IA in order to increase the positive rate of the IA antibody IAA.
  • Multiple insulin molecules are coupled to the macromolecular amino group via SATA and SMCC, while the macromolecular protein is coupled to biotin to form a Biotin-macromolecule protein-insulin conjugate that binds multiple insulin molecules and biotin molecules.
  • 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:
  • insulin means insulin
  • SH means an active thiol group
  • P means a protective group
  • 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:
  • the macromolecular protein Sulfo-N-succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylic acid sodium salt (SMCC) can be added when the reactant is added.
  • 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.
  • the ellipse represents the amino acid chain of the macromolecular protein
  • 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, if z/x is a non-integer, then z/x takes an integer and adds 1 to the integer bits.
  • 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:
  • z/x 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.
  • IA and S-acetylthioacetic acid succinimide ester are preferably prepared by using PBS (preferably pH 7.0, 0.01 M PBS, preferably PBS is preferred) and dimethyl sulfoxide as a solvent.
  • PBS preferably pH 7.0, 0.01 M PBS, preferably PBS is preferred
  • dimethyl sulfoxide preferably dimethyl sulfoxide as a solvent.
  • IA PBS solution IA concentration of 2-4mg / mL
  • S-acetylthioacetic acid succinimide ester dimethyl sulfoxide solution S-acetylthioacetic acid succinimide ester concentration of 40- 80mmol/L.
  • the volume ratio of the IA solution of PBS to the dimethyl sulfoxide solution of S-acetylthioacetic acid succinimide ester was 1 mL: 10 ⁇ L.
  • the concentration of EDTA was 20mmol/L
  • the concentration of hydroxylamine hydrochloride was 0.5mol/L
  • 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;
  • the concentration of sodium salt is 10-15 mg/mL; the solution of succinic acid iodide-dodepolyethylene glycol-biotin in dimethyl sulfoxide, the
  • the above biotinylation method can be specifically as follows:
  • 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;
  • HBA human hemoglobin
  • 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.
  • biotinylated IA obtained by the conventional method and the IA obtained by the biotinylation method according to the present invention can obtain a higher IAA positive rate when used for detection, and avoid the sample being lower in concentration. Have been missed or misjudged.
  • 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- ⁇ -cyclodextrin or carboxymethyl- ⁇ -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.
  • 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, IA, and ZnT8 was pH 9.6 CB buffer, and the final concentrations were 10 ug/ml, 10 ug/ml, 80 ug/ml, and 15 ug/ml, respectively.
  • 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 quality 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.
  • NC negative property control point
  • PC positive nature control point
  • SC sample point quality control point
  • EC enzyme standard control point
  • S1-S3 enzyme standard control point
  • 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.
  • NC negative property control point
  • PC positive nature control point
  • SC sample point quality control point
  • EC enzyme standard control point
  • S1-S3 position reference point coated by the chip itself.
  • 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.
  • 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.
  • 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.
  • the invention also provides a method for preparing the kit, which comprises:
  • the diabetes autoantigen is coated on a protein chip, washed after coating, and then blocked by adding a blocking solution to obtain a protein chip coated with diabetes autoantigen, which contains BSA, polyvinyl alcohol, mannitol, azide Sodium and disodium hydrogen phosphate;
  • the serum samples were detected by the diabetes antibody spectrum detection kit of the present invention, and the positive rate of total antibody in T1MD was 98.21%, the positive rate of T2DM was 10.58%, and the positive rate of normal people was only 2.56%, and the difference was significant (P ⁇ 0.01). .
  • the present invention starts from the blocking solution and the enzyme standard dilution of the enzyme-linked immunoassay kit, and the enzyme-linked immunoassay kit can maintain the stability of the detection for a long time by selecting appropriate components.
  • the diabetes test kit prepared by the composition can accurately diagnose and classify diabetes on the basis of better stability, especially for the detection of IAA, and has a high positive rate.
  • Figure 1 is a schematic view showing the structure of the biotinylated insulin antigen of the present invention.
  • the invention discloses a composition for an enzyme-linked immunoassay kit and a diabetes antibody spectrum detecting kit and a preparation method 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 compositions, kits and applications of the present invention have been described in terms of preferred embodiments, and it will be apparent to those skilled in the art that the compositions, kits and applications described herein can be modified without departing from the scope, spirit and scope of the invention. The invention may be implemented and applied by appropriate modifications and combinations.
  • composition for an enzyme-linked immunoassay kit and a diabetes antibody spectrum detecting kit and a preparation method thereof according to the present invention are further described.
  • 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.
  • the 37 ° C constant temperature blast drying oven is dry for use.
  • the biotinylation of insulin (IA) antigens uses improved techniques, as follows:
  • 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 ⁇ l 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.
  • 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-macromolecule protein-Insulin, which has the structure of formula 1, as biotinylated insulin antigen, infrared and nuclear magnetic
  • the test display is consistent with the expected structure.
  • PC, NC, S1, S2, S3, and EC in the protein chip array were biotinylated at 2 ug/ml, 0.01 ug/ml, 0.5 ug/ml, 2 ug/ml, and 4 ug/ml, respectively.
  • the ELISA kit the same as human IgG, can be diluted with C9.6 buffer of pH 9.6 (containing 5% PEG, 0.05% Proclin 300, and 0.02% Captisol).
  • the SC point used was 2ug/ml biotinylated goat anti-human IgG antibody, and the dilution buffer was pH 9.6 CB buffer.
  • the Loc site used a 2 ug/ml biotinylated human IgG solution, and the dilution buffer was pH 9.6 CB buffer.
  • the dilution buffer for GAD and CPH was 20 mM Tris buffer (5% PEG, 0.05% Proclin 300, 0.02% Captisol, and 15% glycerol) at pH 8.5, with final concentrations of 8 ug/ml and 30 ug/ml, respectively. .
  • the dilution of IA-2, IC, IA, and ZnT8 was pH 9.6 CB buffer, and the final concentrations were 10 ug/ml, 10 ug/ml, 80 ug/ml, and 15 ug/ml, respectively.
  • the diluted antigens were separately filtered through a 0.22 um filter, and then coated with an array using a BioDot precision spotter. After all the arrays were spotted, the chip was placed at 2-8 ° C and overnight coated for 24-30 h.
  • the array of protein chips can be referred to the array presented in the following table, or can be adjusted according to actual needs, without limitation:
  • the coated chips were removed, washed 3 times with PBST washing solution of pH 7.4, and then 150 ul of blocking solution (pH 7.4, disodium hydrogen phosphate solution containing 1% BSA, to which 0.8% mannitol was added, was added to each well.
  • blocking solution pH 7.4, disodium hydrogen phosphate solution containing 1% BSA, to which 0.8% mannitol was added, was added to each well.
  • 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.
  • Enzyme dilution containing 100 mM Tris, 50 mM citric acid, 2.5% BSA, 2% PEG1W, 0.01% gum arabic, 1% betaine, and 0.05% Proclin 300;
  • Enzyme-labeled antibody horseradish peroxidase-labeled rabbit anti-human IgG antibody
  • the horseradish peroxidase-labeled rabbit anti-human IgG antibody was diluted to 4K times (enzyme-labeled antibody concentration) with an enzyme standard dilution.
  • Color developing solution Settling type TMB.
  • the above-mentioned diabetic antibody spectrum detection kit of the present invention is composed of the above-mentioned protein chip having diabetes autoantigen, the enzyme-labeled antibody diluted with the enzyme standard dilution solution, and the color developing solution, and the sample diluent and the 10-fold concentrated washing solution.
  • Sample source Diabetes patients were all from newly diagnosed patients, in line with the 1999 diagnostic criteria for diabetes, including 56 patients with T1DM, aged 6-52 years, 104 patients with T2DM, aged 20-56 years, and 78 normal subjects. There were no family history of diabetes from healthy subjects, aged 15-55 years. Venous blood was taken separately, serum was separated, and after storage, it was frozen at -20 ° C for examination.
  • Type 1 diabetes is caused by autoimmune antibody-induced destruction of islet cells, which is an absolute deficiency of insulin.
  • Type 2 diabetes T2DM is caused by insulin resistance and is generally associated with lifestyle-related diets, among which autoantibodies can be detected. less. The positive rate of autoantibodies in diabetes in these three populations should be significantly different.
  • the serum samples were tested with the kit of the present invention, and the total antibody titer was 98.21% in the T1MD, the T2DM positive rate was 10.58%, and the normal human positive rate was only 2.56%. The difference between the three groups was significant. (P ⁇ 0.01), in line with the rules and standards of clinical testing.
  • Example 3 Stability test of the kit of the present invention
  • Control kit prepared according to the method of Example 1, except that the blocking solution and the enzyme standard dilution were both conventional 0.01 M PBS (pH 7.4) + 10% BSA;
  • Test kit the kit of Example 1;
  • Detection method the two kits were placed at room temperature (18-28 ° C) and low temperature (2-8 ° C) for a period of time, and then using the same serum according to the detection method of Example 1, statistical instrument signal values, the results see Table 3-6.
  • kits of the present invention respectively detect the detection signal values at 0, 6, 12, 18, and 24 months at a low temperature, and simultaneously count the ratios of the respective signal values, and the results show that 24 cells are placed.
  • the detection signal value is almost over 90% compared with the detection signal values placed at 0, 6, 12, and 18 months, which proves that the kit of the present invention is placed under low temperature conditions for 24 months. High detection stability.
  • the kit of the present invention detects the detection signal values of 0, 2, 4, 6, and 8 months at normal temperature, and simultaneously counts the ratio of each signal value, and the results show that 8 are placed.
  • the kit of the month has a detection signal value of more than 80% compared with the detection signal value of 0, 2, 4, and 6 months, which proves that the kit of the present invention is still placed under normal temperature for 8 months. Higher detection stability.
  • the kit of the present invention and the control kit respectively detect the detection signal values placed at low temperature for 6, 12, 18, and 24 months, and simultaneously count the signal values of the two kits at the same time.
  • the ratio of the results showed that the 18-month control kit showed a significant decrease in the detection signal value.
  • the stability of the control kit is not as good as the conclusion of the kit of the present invention. The only difference is the blocking solution and the enzyme standard dilution.
  • the kit of the present invention and the control kit respectively detect the detection signal values placed at normal temperature for 3, 6, 9, and 12 months, and simultaneously count the signal values of the two kits at the same time.
  • the ratio of the results showed that the control kits that were placed for 4 months showed a significant decrease in the detection signal values of some of the indicators.
  • the data in Table 4 clearly showed that the stability of the control kit was not as good as that of the kit of the present invention. In conclusion, the only difference between the two is in the blocking solution and the enzyme standard dilution.
  • Example 4 Test kit for positive rate of IAA in the kit of the present invention
  • Control kit Prepared according to the method of Example 1, except that the biotinylation of IA was also used.
  • Sulfo-NHS-LC-Biotin biotinylation kit
  • Test kit the kit of Example 1;
  • Detection method 8 positive samples, 8 weak positive samples and 5 negative samples were selected, and the above two kits were used to detect according to the detection method of Example 1, and the signal values of the instruments were statistically analyzed. The results are shown in Table 7.
  • the biotinylation of IA by a general biotinylation kit is less effective than the method used in the present invention.
  • the biotinylation method of IA of the present invention can greatly increase the positive rate of IAA, and the effect is obviously better than using a general biotinylation kit.

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Abstract

一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法。所述组合物包括封闭液和酶标稀释液；所述封闭液含有BSA、聚乙烯醇、甘露醇、叠氮钠和磷酸氢二钠，所述酶标稀释液含有Tris、柠檬酸、BSA、聚乙二醇、阿拉伯树胶、甜菜碱和Proclin300。该酶联免疫试剂盒能够较长时间保持检测的稳定性。同时，以所述组合物制备的糖尿病检测试剂盒在具有较佳稳定性的基础上，还能够对糖尿病进行准确诊断和分型，特别是针对IAA的检测上，具备较高的阳性率。

Description

一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法
本申请要求于2018年03月07日提交中国专利局、申请号为201810188203.6、发明名称为“一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及酶联免疫技术领域，具体涉及一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法。
背景技术
糖尿病(diabetes)是由遗传因素、免疫功能紊乱、微生物感染及其毒素、自由基毒素、精神因素等等各种致病因子作用于机体导致胰岛功能减退、胰岛素抵抗等而引发的糖、蛋白质、脂肪、水和电解质等一系列代谢紊乱综合征，临床上以高血糖为主要特点，典型病例可出现多尿、多饮、多食、消瘦等表现，即“三多一少”症状。糖尿病在病因，发病机制和自然病程等方面显示高度异质性，所以糖尿病是一组高血糖疾病的总称。糖尿病基于病因和发病机理分为Ⅰ型糖尿病、Ⅱ型糖尿病及其它类型。Ⅰ型糖尿病是以胰岛β细胞自身免疫破坏为重要病因的一种疾病，患者体内存在一种或多种针对胰岛β细胞胰岛素合成、转运和分泌的关键酶的自身抗体，主要包括胰岛细胞抗体(ICA)、胰岛素自身抗体(IAA)、谷氨酸脱羧酶抗体(GADA)、酪氨酸磷酸抗体(IA-2A)、羧基肽酶抗体(CPH-A)和锌转运蛋白8抗体(ZnT8-A)等6种自身抗体。85％～90％患者在首次检出空腹高血糖时，通常存在一种或多种上述抗体。
蛋白质芯片是一种高通量的蛋白功能分析技术，是近年来发展起来的一项生物检测技术，这项技术是将蛋白质的分析微缩到小型芯片上，利用荧光或酶显色进行探测，最后用特定的计算机软件加以分析。依靠ELISA技术，蛋白质芯片能够快速、高通量、定量分析大量的蛋白样品，以及所 需试剂和样品较少，产品化后价格更低廉等优势，使其迅速成为研究热点。但是，目前该类试剂盒的稳定性并不好。此外，该类试剂盒在检测糖尿病胰岛素自身抗体(IAA)的阳性率上存在不足，阳性率较低，容易漏检。
发明内容
有鉴于此，本发明的目的在于提供一种用于酶联免疫试剂盒的组合物，使得所述组合物用于制备酶联免疫试剂盒时能够显著提高试剂盒在低温和室温下的稳定性，保存时间延长；
本发明的另外一个目的在于提供上述组合物在制备酶联免疫试剂盒中的应用，特别是用于检测糖尿病的相关试剂盒；
本发明的另外一个目的在于提供一种包含上述组合物的糖尿病抗体谱检测试剂盒及其制备方法，使得所述试剂盒在低温和室温下具有较长时间的稳定性，同时检测结果具有较高的特异性和敏感性；
本发明的另外一个目的在于提供一种包含上述组合物的糖尿病抗体谱检测试剂盒及其制备方法，使得所述试剂盒能够显著提高检测糖尿病胰岛素自身抗体(IAA)的阳性率。
为了实现上述目的，本发明提供如下技术方案：
一种用于酶联免疫试剂盒的组合物，包括封闭液和酶标稀释液；所述封闭液含有BSA、聚乙烯醇、甘露醇、叠氮钠和磷酸氢二钠，所述酶标稀释液含有Tris、柠檬酸、BSA、聚乙二醇、阿拉伯树胶、甜菜碱和Proclin300。
针对现有酶联免疫试剂盒稳定较差、保存时间较短的缺陷，本发明意外发现了从制备试剂盒的封闭液和酶标稀释液(用于稀释酶标抗原或抗体用)入手，通过选择合适的组分完善两者的组成，能够显著提高酶联免疫试剂盒的稳定性和保存时间。
作为优选，所述封闭液含有0.6％-1％BSA，0.8％-1.5％的聚乙烯醇、0.8％-1.5％甘露醇、0.05％叠氮钠和0.01M磷酸氢二钠，pH值为7.4，余量为水，所述百分比为质量百分比(w/v)；在本发明具体实施方式中，所述封闭液含有1％BSA，1％的聚乙烯醇、0.8％甘露醇、0.05％叠氮钠和0.01M磷酸氢二钠，pH值为7.4，余量为超纯水。其中，聚乙烯醇优选为 聚乙烯醇2W。
作为优选，所述酶标稀释液含有0.1M的Tris、0.05M的柠檬酸、2％-3％的BSA、1％-3％的聚乙二醇、0.01％的阿拉伯树胶、0.8％-1.5％的甜菜碱以及0.05％的Proclin300，余量为水，所述百分比中除Proclin300是体积百分比之外，其余都是质量百分比(w/v)；在本发明具体实施方式中，所述酶标稀释液含有0.1M的Tris、0.05M的柠檬酸、2.5％的BSA、2％的聚乙二醇、0.01％的阿拉伯树胶、1％的甜菜碱以及0.05％的Proclin300。其中，聚乙二醇优选为聚乙二醇1W。
本发明利用上述组合物进行糖尿病抗体酶联免疫试剂盒的制备，与采用常规的封闭液和酶标稀释液糖尿病抗体酶联免疫试剂盒相比，在低温(2-8℃)下，本发明试剂盒在放置24个月后仍然能够保持制备完成时的稳定性，而对照的试剂盒在放置18个月后就已经出现很大程度的不稳定性；在室温(18-28℃)下，本发明试剂盒在放置6个月后仍然能够保持制备完成时的稳定性，在放置8个月后仍然能够保持较高的稳定性，而对照的试剂盒在放置4个月后就已经出现很大程度的不稳定性。
基于上述优异的技术效果，本发明提出了所述组合物在制备酶联免疫试剂盒中的应用，特别是在制备糖尿病酶联免疫检测试剂盒中的应用。
同时，本发明还提供一种糖尿病抗体谱检测试剂盒，包括以下组分：
包被有糖尿病自身抗原的蛋白芯片、用酶标稀释液稀释的酶标抗体、样品稀释液、洗涤液和显色液；其中，所述蛋白芯片包被糖尿病自身抗原后采用封闭液封闭，所述封闭液含有BSA、聚乙烯醇、甘露醇、叠氮钠和磷酸氢二钠，所述酶标稀释液含有Tris、柠檬酸、BSA、聚乙二醇、阿拉伯树胶、甜菜碱和Proclin300，所述封闭液和酶标稀释液与前述组合物方案相同。
作为优选，所属包被通过亲和素化的蛋白芯片与生物素化的糖尿病自身抗原相互结合包被。其中，亲和素优选为链霉亲和素。
作为优选，所述糖尿病自身抗原选自IA-2、GAD、IC、CPH、ZnT8、IA中的一种或两种以上；在本发明具体实施方式中，所述糖尿病自身抗原为IA-2、GAD、IC、CPH、ZnT8和IA(在本发明中提到的IA和insulin 均表示胰岛素)。
在优选的方案中，本发明所述试剂盒的蛋白芯片至少包含生物素化的IA作为糖尿病自身抗原，为了能够提高IA抗体IAA的阳性率，本发明对IA的生物素化方法进行了改进，将多个胰岛素分子通过SATA以及SMCC偶联到大分子蛋白氨基上，同时大分子蛋白偶联上生物素，形成的Biotin-大分子蛋白-insulin偶联物，结合多个insulin分子和生物素分子(示意图见图1)，再经预先包被亲和素放大作用，大大提高了抗原insulin的包被效率，从而能显著提高以其制备的insulin抗体检测试剂或试剂盒的灵敏度，可大大提高insulin抗体检测的阳性率，减少漏检或误检的几率。其具体步骤如下：
步骤1、将insulin与S-乙酰硫基乙酸琥珀酰亚胺酯(SATA，购自Thermo)室温反应，透析纯化(副产物是小分子可以透过透析袋，大分子insulin-SH-P不能透过透析袋，通过多次透析的方式可以除去副产物)后获得insulin-SH-P，反应式如下：
Figure PCTCN2018093039-appb-000001
insulin-SH-P insulin-SH-P中，insulin表示胰岛素，SH表示活性巯基基团，P表示保护基团，即
Figure PCTCN2018093039-appb-000002
insulin结构式中，圆形表示胰岛素氨基酸链，n表示胰岛素氨基酸链上氨基的数目，1≤x≤n，x和n均为整数，x优选为1。
insulin-SH-P与含EDTA(螯合溶液中的金属离子，除去溶液中金属离子的干扰)和盐酸羟胺的PBS室温反应去掉保护基团，经葡聚糖凝胶柱纯化后形成含活性巯基基团的insulin-SH，备用，反应式如下：
Figure PCTCN2018093039-appb-000003
大分子蛋白、Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐(SMCC，购自Thermo)和琥珀酸亚酰胺-十二聚乙二醇-生物素室温反应，经过PBS透析3次纯化(副产物是小分子可以透过透析袋，大分子蛋白不能透过透析袋，通过多次透析的方式可以除去副产物)获得Biotin-大分子蛋白-SMCC，所述大分子蛋白为HBA、BSA、OVA或酪蛋白，反应式如下：
Figure PCTCN2018093039-appb-000004
Figure PCTCN2018093039-appb-000005
根据上述反应过程，在添加反应物时，既可以将大分子蛋白、Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐(SMCC)和琥珀酸亚酰胺-十二聚乙二醇-生物素一同添加完成反应，也可以先将大分子蛋白和琥珀酸亚酰胺-十二聚乙二醇-生物素反应，然后再添加SMCC分两步完成，反应实质不会发生改变。在该步反应中，椭圆代表大分子蛋白氨基酸链，m表示大分子蛋白氨基酸链上的氨基数目，y+z≤m，z≥10，y≥1，y、z和m均为整数，若z/x为非整数，则z/x取整数且在整数位上加1。
步骤2、将备用的insulin-SH和Biotin-大分子蛋白-SMCC混合室温反应，经过葡聚糖凝胶柱纯化后获得Biotin-大分子蛋白-Insulin，具有式1所示结构，作为生物素化的insulin抗原，反应式如下：
Figure PCTCN2018093039-appb-000006
Biotin-大分子蛋白-Insulin中，若z/x为非整数，则z/x取整数且在整数位上加1。对于本发明特殊生物素化的insulin抗原，x无论为1还是为胰岛素上的氨基数目n，都不影响在同一个大分子蛋白上连接多个胰岛素的效果，但x＝1时所连接的胰岛素数量最多。
其中，IA与S-乙酰硫基乙酸琥珀酰亚胺酯分别优选以PBS(优选为pH值为7.0、0.01M的PBS，以下PBS均优选于此)和二甲基亚砜为溶剂配制反应溶液，即IA的PBS溶液，IA浓度为2-4mg/mL；S-乙酰硫基乙酸琥珀酰亚胺酯的二甲基亚砜溶液，S-乙酰硫基乙酸琥珀酰亚胺酯浓度为40-80mmol/L。IA的PBS溶液与S-乙酰硫基乙酸琥珀酰亚胺酯的二甲基亚砜溶液的体积比为1mL：10μL。
含EDTA和盐酸羟胺的PBS中，EDTA浓度为20mmol/L，盐酸羟胺浓度为0.5mol/L，pH值为7.0；insulin-SH-P和含EDTA和盐酸羟胺的PBS的体积比为(6-10):1。
HBA和Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐优选采用PBS作为溶剂配制反应溶液，琥珀酸亚酰胺-十二聚乙二醇-生物素以二甲基亚砜为溶剂配制反应溶液，即人血白蛋白(HBA)的PBS溶液，HBA浓度为10-20mg/mL；Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐的PBS溶液，Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐浓度为10-15mg/mL；琥珀酸亚酰胺-十二聚乙二醇-生物素的二甲基亚砜溶液，琥珀酸亚酰胺-十二聚乙二醇-生物素的浓度为0.25-0.4mol/L。Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐的PBS溶液与琥珀酸亚酰胺-十二聚乙二醇-生物素的二甲基亚砜溶液等比例混合溶液与人血白蛋白(HBA)的PBS溶液的体积比为(10-20μL)：1mL。
在本发明具体实施方式中，上述生物素化的方法可具体如下：
准备2-4mg/mL的胰岛素PBS溶液，配置浓度约为40-80mmol/L的S-乙酰硫基乙酸琥珀酰亚胺酯的二甲基亚砜溶液(A溶液)，将10微升的A溶液加入到1毫升的胰岛素PBS溶液中混匀室温反应1-4小时，反应产物在PBS中透析3次后回收得到insulin-SH-P,4度暂存备用；
以PBS为溶剂配置0.5mol/L的盐酸羟胺(含20mmol/L的EDTA，pH7.0)作为B溶液，按insulin-SH-P和含EDTA和盐酸羟胺的PBS的体积比为(6-10):1，混合室温反应1-2小时脱去保护基团，经葡聚糖凝胶柱纯化后形成含活性巯基基团的insulin-SH；
准备10-20mg/mL的人血白蛋白(HBA)的PBS溶液，配置10-15mg/mL的Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐的PBS溶液作C溶液，另配置浓度为0.25-0.4mol/L琥珀酸亚酰胺-十二聚乙二醇-生物素的二甲基亚砜溶液作D溶液，将C溶液与D溶液按1：1比例混合后取10-20微升加入到1mL 10-20mg/mL的人血白蛋白PBS溶液中混匀室温反应1-2小时，用PBS透析3次纯化得到Biotin-HBA-SMCC；
将上述纯化的insulin-SH与Biotin-HBA-SMCC按体积比10:(1-4)混合室温反应30-60分钟后得到Biotin-大分子蛋白-Insulin，作为生物素化的insulin抗原。
经过试验验证，采用常规方法获得的生物素化的IA与按照本发明生物素化方法获得的IA，在用于检测时，本发明能够获得更高的IAA阳性率，避免样本因较低浓度而被漏检或错判。
在包被中，所述糖尿病自身抗原采用CB缓冲液或Tris缓冲液为抗原稀释缓冲液进行包被；作为优选，所述缓冲液选自PH9.6的CB缓冲液或PH8.5的Tris缓冲液，更优选地，在缓冲液中添加PEG或者PVP，Proclin300，同时添加了水溶性的环糊精，可使包被更稳定、抗原包被点更规则、更圆，CV更小。
其中，水溶性环糊精可以是Captisol、2-羟基-β-环糊精或羧甲基-β-环糊精等，浓度为0.02％；PEG或者PVP的浓度为5％，Proclin300浓度为0.05％，所述百分比中除Proclin300是体积百分比之外，其余都是质量百分比(w/v)。
在本发明具体实施方式中，GAD和CPH的稀释缓冲液为PH8.5的0.02M Tris缓冲液(含5％的PEG、0.05％的Proclin300，0.02％Captisol，以及15％的甘油)，终浓度分别为8ug/ml、30ug/ml。
IA-2、IC、IA、ZnT8的稀释缓冲液为PH9.6的CB缓冲液，终浓度分别为10ug/ml、10ug/ml、80ug/ml、15ug/ml。
此外，本发明试剂盒中的蛋白芯片还包括阴性质控点、阳性质控点、样品质控点、酶标质控点、参考曲线点以及位置参考点中的一个或两个以上；更为具体地，至少有一个阴性质控点(NC)和一个阳性质控点(PC)； 至少一个样品点质控点(SC)和一个酶标质控点(EC)；至少3个参考曲线点(S1-S3)以及一个芯片本身包被的位置参考点(Loc)。
在具体实施方式中，本发明蛋白芯片上还包含一个阴性质控点(NC)和一个阳性质控点(PC)；一个样品点质控点(SC)和一个酶标质控点(EC)；3个参考曲线点(S1-S3)以及一个芯片本身包被的位置参考点(Loc)。
其中，阳性质控点可以是人IgG，则对应使用的酶标抗体就是抗人IgG的酶标。阳性质控点也可以是包被BSA偶联的DNP，则对应使用的酶标抗体就是抗人IgG的酶标以及抗DNP的酶标的混合液。
而阴性质控点可以是低于反应信号值的微量浓度的人IgG，或采用其他的无关蛋白来替代；样品质控点可以是羊抗人的IgG或其他的抗人IgG；酶标质控点可以是人IgG，或其他的抗兔的抗体(酶标用的是兔抗人IgG)，例如羊抗兔IgG抗体。所述参考曲线点在具体实施过程中是低、中、高三种浓度的人IgG。
蛋白芯片本身的位置参考点是人IgG溶液，主要对蛋白芯片上阵列取值时的定位作用。
优选地，上述阴性质控点、阳性质控点、样品质控点、酶标质控点、参考曲线点以及位置参考点也通过预先生物素化与亲和素化的底板包被。
本发明所述酶标抗体中酶标记物可选择常规的酶以及对应的显色液，如辣根过氧化物酶和TMB显色剂。
本发明还对应提供了所述试剂盒的制备方法，包括：
将糖尿病自身抗原在蛋白芯片上进行包被，包被后洗涤，然后加入封闭液封闭，获得包被有糖尿病自身抗原的蛋白芯片，所述封闭液含有BSA、聚乙烯醇、甘露醇、叠氮钠和磷酸氢二钠；
配制含有Tris、柠檬酸、BSA、聚乙二醇、阿拉伯树胶、甜菜碱和Proclin300的酶标稀释液并稀释酶标抗体，获得用酶标稀释液稀释的酶标抗体，然后配制样品稀释液、洗涤液和显色液，获得糖尿病抗体谱检测试剂盒。
采用本发明糖尿病抗体谱检测试剂盒检测血清样本，其总抗体在T1MD阳性率为98.21％，T2DM阳性率为10.58％，正常人阳性率仅2.56％，其差别具有显著学意义(P<0.01)。
由以上技术方案可知，本发明从酶联免疫试剂盒的封闭液和酶标稀释液入手，通过选择适宜成分，使得酶联免疫试剂盒能够较长时间保持检测的稳定性。同时，以所述组合物制备的糖尿病检测试剂盒在具有较佳稳定性的基础上，还能够对糖尿病进行准确诊断和分型，特别是针对IAA的检测上，具备较高的阳性率。
附图说明
图1所示为本发明所述生物素化的insulin抗原的结构示意图。
具体实施方式
本发明公开了一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述组合物、试剂盒和应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的组合物、试剂盒和应用进行改动或适当变更与组合，来实现和应用本发明技术。
以下就本发明所提供的一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法做进一步说明。
实施例1：本发明所述糖尿病抗体谱检测试剂盒的制备
1、芯片底板和糖尿病自身抗原的预处理
(1)芯片的预处理：
将稀释了6K倍的链霉亲和素(稀释液为PH7.4的0.01M的PBS)，加入96孔板中，50ul/孔，然后于37℃恒温箱中静置2h。
取出96孔板，用PBS洗涤3次，最后用纯化水洗涤一次，吸干。洗涤条件：300ul/孔，静置30sec/次。
37℃恒温鼓风干燥箱干燥备用。
(2)糖尿病自身抗原的预处理：
用Thermo的
Figure PCTCN2018093039-appb-000007
Sulfo-NHS-LC-Biotin生物素化试剂盒对IA-2、GAD、IC、CPH、ZnT8分别进行生物素化。
胰岛素(IA)抗原的生物素化采用改良的技术，具体方案如下：
准备2mg/mL的胰岛素PBS溶液，配置浓度约为50mmol/L的S-乙酰硫基乙酸琥珀酰亚胺酯的二甲基亚砜溶液(A溶液)，将10微升的A溶液加入到1毫升的胰岛素PBS溶液中混匀室温反应1小时，反应产物在PBS中透析3次后回收得到insulin-SH-P,红外和核磁检测显示与预期结构一致，4度暂存备用；
以PBS为溶剂配置0.5mol/L的盐酸羟胺(含20mmol/L的EDTA，pH7.0)作为B溶液，在上述1mL的insulin-SH-P中加入100微升的B溶液，混合室温反应1小时，脱盐柱脱盐纯化得到insulin-SH，红外和核磁检测显示与预期结构一致。
准备10mg/mL的人血白蛋白(HBA)的PBS溶液，配置10mg/mL的Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐的PBS溶液作C溶液，另配置浓度为0.25mol/L琥珀酸亚酰胺-十二聚乙二醇-生物素的二甲基亚砜溶液作D溶液，将C溶液与D溶液按1：1比例混合后取20微升加入到1mL 10mg/mL的人血白蛋白PBS溶液中混匀室温反应1小时，用PBS透析3次纯化得到Biotin-HBA-SMCC，红外和核磁检测显示与预期结构一致；
将上述纯化的insulin-SH与Biotin-HBA-SMCC按体积比10:1混合室温反应30分钟后得到Biotin-大分子蛋白-Insulin，具备式1结构，作为生物素化的insulin抗原，红外和核磁检测显示与预期结构一致。
2、糖尿病自身抗原和相关蛋白的包被
蛋白芯片阵列中的PC、NC、S1、S2、S3、EC分别包被的是2ug/ml、0.01ug/ml、0.5ug/ml、2ug/ml、4ug/ml的生物素化(采用前述生物素化试剂盒，下同)人IgG，可用PH9.6的CB缓冲液(含5％的PEG、0.05％的Proclin300，以及0.02％的Captisol)进行稀释。
SC点使用的是2ug/ml生物素化羊抗人IgG抗体，稀释缓冲液为PH9.6的CB缓冲液。
Loc点使用的是2ug/ml的生物素化人IgG溶液，稀释缓冲液为PH9.6的CB缓冲液。
GAD和CPH的稀释缓冲液为PH8.5的20mM Tris缓冲液(含5％的PEG、0.05％的Proclin300，0.02％Captisol，以及15％的甘油)，终浓度分别为8ug/ml、30ug/ml。
IA-2、IC、IA、ZnT8的稀释液为PH9.6的CB缓冲液，终浓度分别为10ug/ml、10ug/ml、80ug/ml、15ug/ml。
将稀释好的抗原分别用0.22um的滤膜过滤，然后用BioDot精密点样仪进行阵列的包被。全部阵列完成点样之后，将芯片置于2-8℃，过夜包被24-30h。蛋白芯片阵列可参照如下表呈现的阵列，也可根据实际需要调整，不做限制：
表1蛋白芯片阵列
PC NC IC NC
S1 GAD IA SC
S2 CPH ZnT8 EC
S3 IA-2 PC Loc
3、封闭
取出包被的芯片，用PH7.4的PBST洗涤液清洗3次,然后每孔加入150ul的封闭液(PH7.4，含有1％BSA的磷酸氢二钠溶液，其中添加了0.8％甘露醇，1％的PVA2W、0.05％的叠氮钠防腐剂)，室温封闭1h，然后拍干，于湿度15％以下，室温放置，干燥4h，后密封、2-8℃保存。
4、配制酶标稀释液、酶标抗体、显色液、样品稀释液和浓缩洗涤液
酶标稀释液：含有100mM的Tris、50mM的柠檬酸、2.5％的BSA、2％的PEG1W、0.01％的阿拉伯树胶、1％的甜菜碱、以及0.05％的Proclin300；
酶标抗体：辣根过氧化物酶标记的兔抗人IgG抗体；
使用时，用酶标稀释液将辣根过氧化物酶标记的兔抗人IgG抗体稀释至4K倍(酶标抗体浓度)。
显色液：沉降型TMB。
样品稀释液：0.02M Tris，0.15M NaCl，0.05％Tween20，0.01％酪蛋 白，pH7.4。
10倍浓缩洗涤液：0.2M Tris，1.5M NaCl，0.5％Tween20，pH7.4。
上述包被有糖尿病自身抗原的蛋白芯片、酶标稀释液稀释的酶标抗体和显色液、以及样品稀释液和10倍浓缩洗涤液组成本发明糖尿病抗体谱检测试剂盒。
5、检测方法
(1)取出蛋白芯片，平衡至室温；
(2)加样：将阴性和阳性对照血清、以及用样品稀释液稀释了101倍的待测样品，每孔100uL加入待测芯片孔中反应。
(3)温育：室温静置反应30min。加300uL洗涤液(用超纯水稀释10倍后使用)，洗涤3次，每次静置1min。
(4)加酶标抗体：每孔加入50uL酶标抗体。
(5)温育：室温静置反应30min。加300uL洗涤液，洗涤3次，每次静置1min。
(6)显色：每孔加入TMB显色剂50uL，室温静置，避光反应30min。
(7)测定：30min内，用检测仪读取并计算每个反应孔对应抗体的信号值及浓度；其中，信号检测体系也可以是化学发光的方式来实现，可用化学发光底物，如鲁米诺等，然后通过荧光检测装置来进行结果的读取。
实施例2：临床样本的检测
样本来源：糖尿病患者均来自新近确诊的病人，符合1999年糖尿病诊断标准，其中T1DM患者56例，年龄在6-52岁，T2DM患者104例，年龄在20-56岁，正常人78例，均来自健康体检者，均无糖尿病家族史，年龄在15-55岁。分别抽取静脉血，分离血清，分装后于-20℃冻存，待检。
具体的实验结果和数据如下表：
表2
组别 n IA-2A GADA ICA CPH-A ZnT8-A IAA 总阳性率
T1DM 56 26 42 17 3 15 14 55/56(98.21％)
T2DM 104 2 10 1 3 0 2 11/104(10.58％)
正常组 78 0 0 0 2 0 0 2/78(2.56％)
1型糖尿病(T1DM)由自身抗体免疫导致胰岛细胞受破坏，属于胰岛素的绝对不足，而2型糖尿病(T2DM)是由胰岛素抵抗产生的，一般跟生活习惯饮食有关，其中能检测到的自身抗体较少。糖尿病的自身抗体在这三种人群的阳性率应该有显著的差异。
从本发明实验结果看，用本发明的试剂盒检测血清样本，其总抗体在T1MD阳性率为98.21％，T2DM阳性率为10.58％，正常人阳性率仅2.56％，三组差别具有显著学意义(P<0.01)，符合临床上检测的规律和标准。
实施例3：本发明所述试剂盒稳定性检测
对照试剂盒：按照实施例1的方法进行制备，区别在于封闭液和酶标稀释液均采用常规的0.01M PBS(PH7.4)+10％BSA；
试验试剂盒：实施例1试剂盒；
检测方法：将两种试剂盒分别置于室温(18-28℃)和低温(2-8℃)下放置一段时间，然后采用相同血清按照实施例1检测方法检测，统计仪器信号值，结果见表3-6。
1、实施例1试剂盒低温下的稳定性数据
表3
Figure PCTCN2018093039-appb-000008
Figure PCTCN2018093039-appb-000009
由表3可以看出，本发明试剂盒分别检测了在低温下放置0、6、12、18、24个月的检测信号值，同时统计了各个信号值的比值，结果显示，放置了24个月的试剂盒，其检测信号值与放置0、6、12、18个月的检测信号值相比，几乎均处于90％以上，证明本发明所述试剂盒在低温条件下放置24个月仍然具备较高的检测稳定性。
2、实施例1试剂盒室温下的稳定性数据
表4
Figure PCTCN2018093039-appb-000010
由表4可以看出，本发明试剂盒分别检测了在常温下放置0、2、4、6、8个月的检测信号值，同时统计了各个信号值的比值，结果显示，放置了8个月的试剂盒，其检测信号值与放置0、2、4、6个月的检测信号值相比，均处于80％以上，证明本发明所述试剂盒在常温条件下放置8个月仍然具备较高的检测稳定性。
3、实施例1试剂盒和对照试剂盒低温下的稳定性数据对比
表5
Figure PCTCN2018093039-appb-000011
由表5可以看出，本发明试剂盒和对照试剂盒分别检测了在低温下放置6、12、18、24个月的检测信号值，同时统计了相同时间下，两个试剂盒的信号值的比值，结果显示，放置了18个月的对照试剂盒，其检测信号值开始出现显著的下降，结合表3数据可以明显得出对照试剂盒的稳定性不如本发明试剂盒的结论，而两者的差别仅在于封闭液和酶标稀释液。
4、实施例1试剂盒和对照试剂盒常温下的稳定性数据对比
表6
Figure PCTCN2018093039-appb-000012
Figure PCTCN2018093039-appb-000013
由表6可以看出，本发明试剂盒和对照试剂盒分别检测了在常温下放置3、6、9、12个月的检测信号值，同时统计了相同时间下，两个试剂盒的信号值的比值，结果显示，放置了4个月的对照试剂盒，其某些指标的检测信号值开始出现显著的下降，结合表4数据可以明显得出对照试剂盒的稳定性不如本发明试剂盒的结论，而两者的差别仅在于封闭液和酶标稀释液。
实施例4：本发明所述试剂盒对IAA的阳性率检测试验
对照试剂盒：按照实施例1的方法进行制备，区别在于IA的生物素化同样采用
Figure PCTCN2018093039-appb-000014
Sulfo-NHS-LC-Biotin生物素化试剂盒；
试验试剂盒：实施例1试剂盒；
检测方法：选取8个阳性样本，8个弱阳性样本以及5个阴性样本，分别采用上述两种试剂盒按照实施例1检测方法检测，统计仪器信号值，结果见表7。
表7
Figure PCTCN2018093039-appb-000015
Figure PCTCN2018093039-appb-000016
由表7可以看出，与本发明中对IA的生物素化的方法比较，采用一般的生物素化试剂盒对IA进行生物素化对阳性样本的检测效果不如本发明中采用的方法，使用本发明这种对IA的生物素化方法可以大幅提高IAA的阳性率，效果比使用一般的生物素化试剂盒明显要好。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (14)

  1. 一种用于酶联免疫试剂盒的组合物，其特征在于，包括封闭液和酶标稀释液；所述封闭液含有BSA、聚乙烯醇、甘露醇、叠氮钠和磷酸氢二钠，所述酶标稀释液含有Tris、柠檬酸、BSA、聚乙二醇、阿拉伯树胶、甜菜碱和Proclin300。
  2. 根据权利要求1所述组合物，其特征在于，所述封闭液含有0.6％-1％BSA，0.8％-1.5％的聚乙烯醇、0.8％-1.5％甘露醇、0.05％叠氮钠和0.01M磷酸氢二钠，pH值为7.4，余量为水。
  3. 根据权利要求1所述组合物，其特征在于，所述酶标稀释液含有0.1M的Tris、0.05M的柠檬酸、2％-3％的BSA、1％-3％的聚乙二醇、0.01％的阿拉伯树胶、0.8％-1.5％的甜菜碱以及0.05％的Proclin300，余量为水。
  4. 权利要求1-3任意一项所述组合物在制备酶联免疫试剂盒中的应用。
  5. 根据权利要求4所述应用，其特征在于，所述酶联免疫试剂盒为糖尿病免疫检测试剂盒。
  6. 一种糖尿病抗体谱检测试剂盒，其特征在于，包括以下组分：
    包被有糖尿病自身抗原的蛋白芯片、用酶标稀释液稀释的酶标抗体、样品稀释液、洗涤液和显色液；其中，所述蛋白芯片包被糖尿病自身抗原后采用封闭液封闭，所述封闭液含有BSA、聚乙烯醇、甘露醇、叠氮钠和磷酸氢二钠，所述酶标稀释液含有Tris、柠檬酸、BSA、聚乙二醇、阿拉伯树胶、甜菜碱和Proclin300。
  7. 根据权利要求6所述试剂盒，其特征在于，所述封闭液含有0.6％-1％BSA，0.8％-1.5％的聚乙烯醇、0.8％-1.5％甘露醇、0.05％叠氮钠和0.01M磷酸氢二钠，pH值为7.4，余量为水。
  8. 根据权利要求6所述试剂盒，其特征在于，所述酶标稀释液含有0.1M的Tris、0.05M的柠檬酸、2％-3％的BSA、1％-3％的聚乙二醇、0.01％的阿拉伯树胶、0.8％-1.5％的甜菜碱以及0.05％的Proclin300，余量为水。
  9. 根据权利要求6所述试剂盒，其特征在于，所述包被通过亲和素化的蛋白芯片与生物素化的糖尿病自身抗原相互结合包被。
  10. 根据权利要求6或9所述试剂盒，其特征在于，所述糖尿病自身抗原选自IA-2、GAD、IC、CPH、ZnT8、IA中的一种或两种以上。
  11. 根据权利要求9所述试剂盒，其特征在于，所述生物素化的糖尿病自身抗原包含生物素化的IA，其生物素化过程如下：
    步骤1、将insulin与S-乙酰硫基乙酸琥珀酰亚胺酯(SATA)室温反应，透析纯化后获得insulin-SH-P：n表示insulin上氨基数目，1≤x≤n，x和n均为整数；
    Figure PCTCN2018093039-appb-100001
    insulin-SH-P与盐酸羟胺的PBS室温反应去掉保护基团，经葡聚糖凝胶柱纯化后形成含活性巯基基团的insulin-SH，备用；
    Figure PCTCN2018093039-appb-100002
    大分子蛋白、Sulfo-N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷-1-羧酸钠盐(SMCC)和琥珀酸亚酰胺-十二聚乙二醇-生物素室温反应，经过PBS透析纯化获得Biotin-大分子蛋白-SMCC；m表示大分子蛋白上的氨基数目，2≤y+z≤m，y、z和m均为整数；
    Figure PCTCN2018093039-appb-100003
    Figure PCTCN2018093039-appb-100004
    步骤2、将备用的insulin-SH和Biotin-大分子蛋白-SMCC混合室温反应，经过葡聚糖凝胶柱纯化后获得Biotin-大分子蛋白-Insulin，具有式1所示结构，作为生物素化的insulin抗原；
    Figure PCTCN2018093039-appb-100005
    其中，圆形表示胰岛素，1≤x≤n，n表示胰岛素上氨基的数目，x和n均为整数；椭圆代表大分子蛋白，y+z≤m，z≥10，y≥1，m表示大分子蛋白上的氨基数目，y、z和m均为整数，若z/x为非整数，则z/x取整数且在整数位上加1。
  12. 根据权利要求6或9所述试剂盒，其特征在于，所述糖尿病自身抗原采用CB缓冲液或Tris缓冲液为抗原稀释缓冲液进行包被。
  13. 根据权利要求6-12任意一项所述试剂盒，其特征在于，所述蛋白芯片还包括阴性质控点、阳性质控点、样品质控点、酶标质控点、参考曲线点以及位置参考点中的一个或两个以上。
  14. 权利要求6所述试剂盒的制备方法，其特征在于，包括：
    将糖尿病自身抗原在蛋白芯片上进行包被，包被后洗涤，然后加入封闭液封闭，获得包被有糖尿病自身抗原的蛋白芯片，所述封闭液含有BSA、聚乙烯醇、甘露醇、叠氮钠和磷酸氢二钠；
    配制含有Tris、柠檬酸、BSA、聚乙二醇、阿拉伯树胶、甜菜碱和Proclin300的酶标稀释液并稀释酶标抗体，获得用酶标稀释液稀释的酶标抗体，然后配制样品稀释液、洗涤液和显色液，获得糖尿病抗体谱检测试剂盒。
PCT/CN2018/093039 2018-03-07 2018-06-27 一种用于酶联免疫试剂盒的组合物以及糖尿病抗体谱检测试剂盒及其制备方法 WO2019169798A1 (zh)

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