CN111077323A - Insulin determination kit for eliminating insulin autoantibody interference - Google Patents

Insulin determination kit for eliminating insulin autoantibody interference Download PDF

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Publication number
CN111077323A
CN111077323A CN202010022770.1A CN202010022770A CN111077323A CN 111077323 A CN111077323 A CN 111077323A CN 202010022770 A CN202010022770 A CN 202010022770A CN 111077323 A CN111077323 A CN 111077323A
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China
Prior art keywords
insulin
autoantibody
interference
eliminating
determination
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CN202010022770.1A
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Inventor
李立和
苗振伟
李明卫
孙国忠
丁弘
崔喆
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Tianjin Baodi Hospital
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Tianjin Baodi Hospital
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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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins
    • 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

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  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
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  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
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  • Cell Biology (AREA)
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  • Medicinal Chemistry (AREA)
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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Endocrinology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses an insulin determination kit for eliminating insulin autoantibody interference, and belongs to the technical field of immunoassay. The technical scheme of the invention is as follows: the method is not influenced by the insulin autoantibody when detecting the insulin, and the using method is the same as the original chemiluminescence method, so that the method is economical, convenient and easy to implement and is an insulin detection method with higher accuracy.

Description

Insulin determination kit for eliminating insulin autoantibody interference
Technical Field
The invention belongs to the technical field of immunoassay; or a method of testing a material by measuring a color change resulting from a reaction using visible light.
Background
Normally, insulin is mainly secreted and released into blood from human islet cells, during a series of processes for producing insulin, the islet cells first synthesize proinsulin, which is a long protein chain, and the proinsulin is decomposed into three segments under the action of enzyme, the front and rear segments are reconnected to form insulin consisting of an A chain and a B chain, and the middle segment is separated and called C peptide.
In the β cell secretion of the pancreatic islet, the total gram molecular weight of the insulin and the C peptide is equal, in the clinical laboratory measurement, the common unit of the insulin is uIU/ml, the unit of the C peptide is ng/ml, the half-life of the insulin is 4.8min, and the C peptide is 11 min proinsulin is 17.5 min.
Type 1 diabetes mellitus is caused by the destruction of β cells mediated by T cells and has a genetic predisposition, characterized by the presence in the serum of patients of circulating autoantibodies including Islet Cell Antibodies (ICA), glutamate decarboxylase antibodies (GADA), tyrosine phosphatase antibodies (IA-2A), insulin antibodies (IAA) and zinc transporter 8 antibodies (ZnT8A), IAA, an insulin autoantibody, which is 20-30% positive in pre-diabetic and type 1 diabetes mellitus diabetic patients treated with xenogenic insulin, the serum of which often has an anti-insulin anti-antibody (IAB) present, which binds to insulin to form a complex, inactivating insulin, one of the major causes of insulin resistance.
The invention content is as follows:
in order to solve the problem that the determination of serum insulin is interfered by an endogenous insulin autoantibody in the prior art, the invention provides the chemiluminescence determination method which is economic, convenient and easy to implement, has higher accuracy and can eliminate the influence of the endogenous insulin autoantibody.
The technical scheme is as follows: when the serum is added into the sample diluent, the insulin autoantibody secondary antibody and the insulin autoantibody generate antigen-antibody reaction, so that competitive reaction between the insulin autoantibody and the insulin antibody and insulin in the determination reagent is sealed, and the occurrence of reaction interference in insulin determination is avoided.
The specific implementation mode is as follows:
the present invention is further illustrated by the following example of serum insulin measurement using the Ampere A2000PLUS chemiluminescence analyzer.
Example 1
The composition of the reagent is as follows:
a. the insulin reagent is a reagent matched with Zhengzhou Antu bioengineering GmbH, adopts a double-antigen sandwich method principle to detect, uses insulin antibody to coat magnetic particles, uses horseradish peroxidase to mark the insulin antibody to prepare an enzyme conjugate, forms an antibody-antigen-antibody-enzyme labeled compound through immunoreaction, and the compound catalyzes a luminescent substrate to emit photons, wherein the luminescent intensity is in direct proportion to the content of insulin.
b. The sample diluent is the diluent prepared by the invention: 0.15M KH2PO40.2 g, Na2HPO4·12H2O2.9 g, NaCl 8.0 g, KCl 0.2 g, Tween-200.5 ml distilled water to 1000ml, and insulin autoantibody secondary antibody with 20IU/ml concentration.
c. The insulin autoantibody determination kit (chemiluminescence method) is produced by Shenzhen Yahulong Biotech limited.
Example 2
The composition of the reagent is as follows:
a. the insulin reagent is a matching reagent of Zhengzhou Antu bioengineering GmbH.
b. The sample diluent is a reagent matched with Zhengzhou Antu bioengineering GmbH.
c. The insulin autoantibody determination kit (chemiluminescence method) is produced by Shenzhen Yahulong Biotech limited.
Example 3
1. Detecting an object: 128 hospital examiners, of which there are 60 insulin autoantibody positives, 32 males, with a mean age of 48.5 years; 28 women, mean age 46.5 years; of these, 64 insulin autoantibody negative cases, male, 34 cases, mean age 49.5 years; 30 women, average age 48.5 years old.
2. The method comprises the following steps: 128 subjects were tested for insulin, C-peptide, and insulin autoantibodies in example 1 and example 2, respectively, and the insulin and C-peptide changes in the insulin autoantibody positive group and the insulin autoantibody negative group were compared.
3. The instrument comprises the following steps: an AutoLumo A2000PLUS chemiluminescence detector, and an iFlash 3000 chemiluminescence immunoassay analyzer.
4. Results
4.1 insulin, C-peptide, of the insulin autoantibody negative group was determined using examples 1 and 2, comparing as shown in Table 1:
TABLE 1. example 1 and example assay 2 determination of insulin autoantibody (COI) negative group insulin (uIU/ml), C peptide (ng/ml) comparison
Figure RE-DEST_PATH_IMAGE002AAA
4.2 insulin, C-peptide, positive groups of insulin autoantibodies were determined using examples 1 and 2, comparing as shown in Table 2:
TABLE 2 comparison of insulin (uIU/ml) and C-peptide (ng/ml) in insulin autoantibody (COI) positive groups determined in example 1 and example assay 2
Project number IAA insulin C peptide insulin/C peptide
Example 16055.6. + -. 38.712.47. + -. 9.983.14. + -. 2.463.97
Example 26055.6. + -. 38.73.06. + -. 3.663.16. + -. 2.420.97
As can be seen from the above experiments, in the negative group for measuring insulin autoantibodies, there was no significant difference in the insulin measurement values of the measurement methods of the present invention in example 1 and example 2, t =1.01,P=0.6224, n =64, insulin/C-peptide ratio of around 4.0, insulin levels higher than C-peptide levels; in the positive group for determination of insulin autoantibodiesThe insulin measurement values of the measurement methods of the present invention in example 1 and example 2 were highly significantly different from each other, t =64.39,P=0.0051, n =60, insulin/C-peptide is 1.0 or less, and insulin levels are lower than C-peptide levels. The method of the invention can ensure the accuracy of the insulin determination result by adding the insulin autoantibody secondary antibody.
The comparison shows that the insulin autoantibody is sealed by the insulin autoantibody secondary antibody, so that the competitive reaction of the insulin autoantibody and the insulin antibody and insulin in the determination reagent is eliminated, the occurrence of the interference of the insulin determination reaction is avoided, and the method has popularization and application prospects.

Claims (3)

1. The invention discloses an insulin determination kit for eliminating insulin autoantibody interference, which is characterized in that a sample diluent contains an insulin autoantibody secondary antibody, the insulin autoantibody secondary antibody in the sample diluent is combined with an insulin autoantibody in serum, and competitive reaction between the insulin autoantibody and the insulin antibody and insulin in an insulin determination reagent is eliminated.
2. The kit for measuring insulin for eliminating interference of insulin autoantibody according to claim 1, wherein the dilution of the sample by the chemiluminescence method comprises per liter: 0.15M KH2PO40.2 g, Na2HPO4·12H2O2.9 g, NaCl 8.0 g, KCl 0.2 g, Tween-200.5 ml, and the concentration of the insulin autoantibody secondary antibody is 10000-30000 IU.
3. The insulin assay kit for eliminating interference of insulin autoantibodies according to claim 1, wherein the concentration of phosphate buffer in the sample diluent is 150mmol/L and the pH value is 7.2 ± 0.2.
CN202010022770.1A 2020-01-12 2020-01-12 Insulin determination kit for eliminating insulin autoantibody interference Pending CN111077323A (en)

Priority Applications (1)

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Application Number Priority Date Filing Date Title
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Citations (10)

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US5804391A (en) * 1994-11-04 1998-09-08 Boehringer Mannheim Gmbh Elimination of rheumatoid factor interference using anti-FD antibodies
US6632682B1 (en) * 1987-05-23 2003-10-14 Dade Behring Marburg Gmbh One-step immunoassay for the determination of antigen-specific antibodies of one of the immunoglobulin classes A, M, D, or E, and an agent suitable for this purpose
CN1888904A (en) * 2005-06-27 2007-01-03 上海透景生命科技有限公司 Method for indicating xenotropic antibody interference in immune reaction
US20090176252A1 (en) * 2005-06-29 2009-07-09 Shibayagi Co., Ltd. Method for the Measurement of Endocrine Substances In an Analyte
US20090246800A1 (en) * 2006-10-26 2009-10-01 Abbott Laboratories Immunoassay of analytes in samples containing endogenous anti-analyte antibodies
US20110136141A1 (en) * 2009-12-03 2011-06-09 Abbott Laboratories Peptide reagents and method for inhibiting autoantibody antigen binding
US20110136103A1 (en) * 2009-12-03 2011-06-09 Abbott Laboratories Autoantibody enhanced immunoassays and kits
CN203385722U (en) * 2013-10-11 2014-01-08 重庆奥创生物技术开发有限责任公司 IgM (Immunoglobulin M) detection test strip capable of eliminating IgG (Immunoglobulin G) interference
CN107286239A (en) * 2016-03-30 2017-10-24 复旦大学 Compound of antibody with autoantibody or similar anti self antibody and preparation method thereof
CN109239329A (en) * 2018-10-18 2019-01-18 郑州安图生物工程股份有限公司 A kind of insulin immue quantitative detection reagent box

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6632682B1 (en) * 1987-05-23 2003-10-14 Dade Behring Marburg Gmbh One-step immunoassay for the determination of antigen-specific antibodies of one of the immunoglobulin classes A, M, D, or E, and an agent suitable for this purpose
US5804391A (en) * 1994-11-04 1998-09-08 Boehringer Mannheim Gmbh Elimination of rheumatoid factor interference using anti-FD antibodies
CN1888904A (en) * 2005-06-27 2007-01-03 上海透景生命科技有限公司 Method for indicating xenotropic antibody interference in immune reaction
US20090176252A1 (en) * 2005-06-29 2009-07-09 Shibayagi Co., Ltd. Method for the Measurement of Endocrine Substances In an Analyte
US20090246800A1 (en) * 2006-10-26 2009-10-01 Abbott Laboratories Immunoassay of analytes in samples containing endogenous anti-analyte antibodies
US20110136141A1 (en) * 2009-12-03 2011-06-09 Abbott Laboratories Peptide reagents and method for inhibiting autoantibody antigen binding
US20110136103A1 (en) * 2009-12-03 2011-06-09 Abbott Laboratories Autoantibody enhanced immunoassays and kits
CN203385722U (en) * 2013-10-11 2014-01-08 重庆奥创生物技术开发有限责任公司 IgM (Immunoglobulin M) detection test strip capable of eliminating IgG (Immunoglobulin G) interference
CN107286239A (en) * 2016-03-30 2017-10-24 复旦大学 Compound of antibody with autoantibody or similar anti self antibody and preparation method thereof
CN109239329A (en) * 2018-10-18 2019-01-18 郑州安图生物工程股份有限公司 A kind of insulin immue quantitative detection reagent box

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Title
N. NISHII, ET AL.: "Presence of anti-insulin natural autoantibodies in healthy cats and its interference with immunoassay for serum insulin concentrations", 《DOMESTIC ANIMAL ENDOCRINOLOGY》 *
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