WO2019169799A1 - Antigène d'insuline biotinylé et procédé de biotinylation associé - Google Patents

Antigène d'insuline biotinylé et procédé de biotinylation associé Download PDF

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Publication number
WO2019169799A1
WO2019169799A1 PCT/CN2018/093058 CN2018093058W WO2019169799A1 WO 2019169799 A1 WO2019169799 A1 WO 2019169799A1 CN 2018093058 W CN2018093058 W CN 2018093058W WO 2019169799 A1 WO2019169799 A1 WO 2019169799A1
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insulin
antigen
protein
biotinylated
biotin
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PCT/CN2018/093058
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English (en)
Chinese (zh)
Inventor
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 WO2019169799A1 publication Critical patent/WO2019169799A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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, in particular to a biotinylated insulin antigen and a biotinylation process thereof.
  • the detection of insulin autoantibodies can be used as a marker of autoimmune ⁇ -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.
  • affinity constant k 1015L/mol, ratio
  • the protein can be biotinylated and then specifically bound to avidin/streptavidin to achieve signal Amplify and improve the sensitivity of detection.
  • biotinylation reagents 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.
  • 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.
  • 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;
  • the present invention provides the following technical solutions:
  • 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.
  • 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.
  • the macromolecular protein is HBA, BSA, OVA or casein; the x is preferably 1.
  • 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.
  • 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.
  • 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.
  • the avidin is preferably streptavidin.
  • 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.
  • 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.
  • 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, 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.
  • 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.
  • NC negative property control point
  • PC positive nature control point
  • SC sample point 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 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:
  • 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
  • x and n are integers
  • 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.
  • 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:
  • 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.
  • a PBS preferably pH 7.0, 0.01 M PBS, preferably the following PBS is preferred
  • dimethyl sulfoxide a solvent.
  • 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 ⁇ 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.
  • 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.
  • Figure 1 is a schematic view showing the structure of the biotinylated insulin antigen of the present invention.
  • 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.
  • 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.
  • biotinylated insulin antigen provided by the present invention and a preparation process thereof.
  • 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-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 3 Comparison of the detection effects of the antigens prepared in Example 1 and Example 2 on insulinA
  • 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:
  • 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.
  • 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.
  • blocking solution pH 7.4, disodium hydrogen phosphate solution containing 1% BSA, adding 0.8% nectar
  • 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.

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Abstract

L'invention concerne un antigène d'insuline biotinylé ayant une structure telle que représentée par la formule 1 et un procédé de biotinylation associé. L'antigène d'insuline biotinylé selon la présente invention améliore l'efficacité de revêtement d'un antigène d'insuline parce qu'il est amplifié par l'avidine pré-revêtue, peut améliorer de manière significative la sensibilité d'un réactif ou d'un kit de détection d'anticorps d'insuline préparé par celui-ci, améliore la vitesse positive de détection d'anticorps d'insuline, et réduit la probabilité de détection manquée ou de fausse détection.
PCT/CN2018/093058 2018-03-07 2018-06-27 Antigène d'insuline biotinylé et procédé de biotinylation associé WO2019169799A1 (fr)

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CN201810186407.6A CN108440666B (zh) 2018-03-07 2018-03-07 一种生物素化的insulin抗原及其生物素化工艺
CN201810186407.6 2018-03-07

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101377514A (zh) * 2008-03-25 2009-03-04 北京科美东雅生物技术有限公司 胰岛素自身抗体化学发光免疫分析测定试剂盒及其制备方法
CN101609090A (zh) * 2008-06-19 2009-12-23 北京华科泰生物技术有限公司 一种采用吖啶酯标记抗原或抗体分析方法
CN107548461A (zh) * 2015-03-30 2018-01-05 Hycor生物医学有限责任公司 用于执行自身免疫性疾病和感染性疾病的诊断性测定的自动化的免疫分析仪系统

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110207916A1 (en) * 2007-11-12 2011-08-25 Novozymes A/S Dual Affinity Polypeptides for Purification
WO2013071055A1 (fr) * 2011-11-10 2013-05-16 Wellstat Diagnostics, Llc Dosage pour autoanticorps associés au diabète

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101377514A (zh) * 2008-03-25 2009-03-04 北京科美东雅生物技术有限公司 胰岛素自身抗体化学发光免疫分析测定试剂盒及其制备方法
CN101609090A (zh) * 2008-06-19 2009-12-23 北京华科泰生物技术有限公司 一种采用吖啶酯标记抗原或抗体分析方法
CN107548461A (zh) * 2015-03-30 2018-01-05 Hycor生物医学有限责任公司 用于执行自身免疫性疾病和感染性疾病的诊断性测定的自动化的免疫分析仪系统

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