Highly reproducible agglutination immunoassay method and reagents

An analytical method and reagent technology, applied in the field of immunoassay reagents of the method, can solve the problems of difficult to produce a reaction with good reproducibility, uneven coagulation and the like

Inactive Publication Date: 2003-10-15
KYOWA MEDEX CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, when an aggregation reaction occurs between insoluble carrier particles and an antigen or antibody, it is difficult to generate a reproducible reaction due to the heterogeneity of the aggregation.
In particular, insoluble carrier particles that are not actually bound to antigens or antibodies are allowed to bind to antigenic substances in the test sample, bind to antibodies or antibody complexes that specifically react to the antigenic substances, and selectively aggregate In the agglutination immunoassay of insoluble carrier particles, there are problems such as direct binding of antibodies to insoluble carrier particles, so it is necessary to improve the reproducibility of the reaction

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Reagents of the following compositions were prepared. [R1 reagent] HEPES buffer (made by Dojin Chemical Co., Ltd., pH 7.8) 4.77 g / L latex particles (particle size 0.0775 μm, made by Sekisui Chemical Co., Ltd.) 0.033 wt% / LNaN 3 (manufactured by Kanto Chemical Co., Ltd.) 0.1 g / L [R2 reagent] HEPES buffer (manufactured by Dojin Chemical Co., Ltd., pH 7.0) 4.77 g / L sodium chloride (manufactured by Wako Pure Chemical Industries) 15 g / L polymer 2 (reference example) 2 manufacturing) 2g / LNaN 3 (manufactured by Kanto Chemical Co., Ltd.) 0.1 g / L anti-human HbA1c mouse monoclonal antibody (Production Example 1) 0.025 g (converted to IgG) / L anti-mouse IgG goat polyclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) 0.025 g ( Converted to IgG) / L Example 2

Embodiment 2

[0035] Reagents of the following compositions were prepared. [R1 reagent] HEPES buffer (made by Dojin Chemical Co., Ltd., pH 7.8) 4.77 g / L latex particles (particle size 0.0775 μm, made by Sekisui Chemical Co., Ltd.) 0.033 wt% / LNaN 3 (manufactured by Kanto Chemical Co., Ltd.) 0.1 g / L [R2 reagent] HEPES buffer (manufactured by Dojin Chemical Co., Ltd., pH 7.0) 4.77 g / L sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) 15 g / L polymer 3 (reference example) 3 manufacturing) 2g / LNaN 3 (manufactured by Kanto Chemical Co., Ltd.) 0.1g / L anti-human HbA1c mouse monoclonal antibody 0.025g (converted to IgG) / L anti-mouse IgG goat polyclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) 0.025g (converted to IgG) / LComparative Example 1

[0036] Polymer 2 in Example 1 and Polymer 3 in Example 2 were replaced with Tween 20, and the following compositions were prepared. [R1 reagent] HEPES buffer (made by Dojin Chemical Co., Ltd., pH 7.8) 4.77 g / L latex ...

Embodiment 3

[0039] After collecting human blood with an EDTA blood collection tube (VENOJECT vacuum blood collection tube, manufactured by Terumo Corporation), it was left to stand for 2 hours, and 10 μl of the deposited blood cell layer was collected and diluted with 1 ml of purified water. Store frozen at -20°C before use, dissolve immediately before quantification and use as a sample. The concentration of HbA1c was determined with R1 reagent and R2 reagent prepared in Example 1, Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively. The HbA1c value was measured with an automatic glycohemoglobin analyzer HLC-723GHbV (manufactured by Tosoh Corporation), and a standard curve was drawn using the reagents immediately after opening of the standard samples of 0.0%, 4.2%, 7.7%, 11.3%, and 14.8%, respectively. For the determination of HbA1c in the sample, add 8.0 μl of the sample prepared by the above method to 240 μl of R1 reagent, react at 37°C for 5 ...

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Abstract

The present invention provides an agglutination immunoassay, wherein the agglutination of insoluble carrier particles such as latex are stabilized and uniformized to give good reproducibility, and a reagent therefor. In the agglutination immunoassay which comprises allowing an antigenic substance in a sample to bind to insoluble carrier particles carrying substantially neither antigens nor antibodies thereon, and allowing an antibody or an antibody complex which reacts specifically to the antigenic substance to bind to the antigenic substance to give a selective agglutination of the insoluble carrier particles, a homopolymer prepared by polymerization of a monomer such as 2-methacryloyloxyethyl phosphorylcholine having a phosphorylcholine group and a vinyl group, or a copolymer prepared by polymerization of a monomer having a phosphorylcholine group and a vinyl group, with a monomer having a vinyl group such as n-butyl methacrylate is used.

Description

technical field [0001] The present invention provides an agglutination immunoassay method for immunological measurement using antigenic substances and aggregation reactions in water-soluble media such as body samples, and an immunoassay reagent used for the method. Background technique [0002] In recent years, in hospitals and inspection centers, due to problems such as manpower shortages, cost reductions, or handling of a large number of specimens, it is necessary to automate various inspections such as clinical inspections and shorten the measurement time. In this way, the present method suitable for automation has attracted much attention by an agglomeration immunoassay for quantifying antigenic substances by agglomeration reaction of insoluble carrier particles. For example, Japanese Patent Laid-Open No. 7-35752 describes that insoluble carrier particles that do not substantially bind to antigens or antibodies are bound to an antigenic substance in a test sample, and an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08F220/60C08F230/02G01N33/543G01N33/545G01N33/72
CPCY10S435/81C08F230/02C08F220/60G01N33/721Y10S435/961G01N33/545G01N33/54313G01N33/543
Inventor 重信香代子首藤健志郎榊秀次郎
Owner KYOWA MEDEX CO LTD
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