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Method and kit for measurement of acrolein adduct in sample utilizing agglutination reaction of immunological microparticle

A technology of acrolein and adduct, which is applied in the field of immunological assay and can solve the problems of few binding sites and difficulty in agglutination reaction, etc.

Inactive Publication Date: 2010-09-15
ALFRESA PHARMA CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, especially for pharmaceuticals and chemical substances, which are substances to be measured with a small molecular weight, there are few binding sites between the specific binding substance and the substance to be measured, and the aggregation reaction is difficult to occur, so it is difficult to use a homogeneous measurement system using the aggregation reaction in combination.

Method used

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  • Method and kit for measurement of acrolein adduct in sample utilizing agglutination reaction of immunological microparticle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] (embodiment 1: the preparation of colloidal gold solution)

[0050] 2ml of 10w / v% chloroauric acid solution was added to 1L of distilled water at 95°C while stirring, and 10ml of 2w / v% sodium citrate solution was added after 1 minute, and then cooled to 30°C after further stirring for 20 minutes. After cooling, the pH was adjusted to 7.1 with 0.1 w / v% potassium carbonate.

Embodiment 2

[0051] (Example 2: the preparation of rat anti-mouse IgG monoclonal antibody and bovine serum albumin binding colloidal gold reagent)

[0052] Rat anti-mouse IgG monoclonal antibody (Production of Antibodies, Reagents for Immunology and Services) was diluted with 10 mM HEPES (pH 7.1) containing 0.05 w / v% sodium azide to a concentration of 20 μg / mL. Add 50 mL of this solution into about 1 L of colloidal gold solution prepared in Example 1 above, and stir for 2 hours under refrigerated conditions. Blocking treatment was performed by adding 110 mL of 10 mM HEPES (pH 7.1) containing 0.5 w / v % bovine serum albumin, 5.46 w / v % mannitol, and 0.05 w / v % sodium azide, and stirring at 37° C. for 90 minutes. . Centrifuge at 8000 rpm for 40 minutes, and remove the supernatant. Next, about 1 L of 5 mM HEPES (pH 7.5) (A solution) containing 3w / v% mannitol, 0.1w / v% BSA, and 0.05w / v% sodium azide was added to disperse the antibody-bound colloidal gold, and the Centrifuge at 8000 rpm for 40...

Embodiment 3

[0054] (Example 3: Preparation of the first reagent for measuring acrolein adduct)

[0055] Add anti-acrolein monoclonal antibody (Japanese Oil Co., Ltd.) 1.25 μg / mL and about 1.0 to 2.5 w / v% of polyethylene glycol were added as a reaction accelerator to prepare a first reagent for measuring acrolein adducts.

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Abstract

Disclosed is a method for measuring an acrolein adduct in a sample, which comprises the following steps (a) to (c): (a) mixing the sample containing the acrolein adduct with a solution containing an immunoglobulin capable of recognizing the acrolein adduct specifically; (b) adding a solution that contains a microparticle comprising a substance capable of specifically binding to the immunoglobulin capable of specifically recognizing the acrolein adduct and a blocking agent bound to the substance to the mixed solution produced in the step (a) and agitating the resulting mixture; and (c) measuring the degree of the agglutination reaction of the microparticle in the mixed solution produced in the step (b), wherein the degree of the agglutination reaction is decreased in accordance with the amount of the acrolein adduct in the sample. The method enables to measure an acrolein adduct without the need of any complicated procedure.

Description

technical field [0001] The present invention relates to immunological assay methods utilizing microparticles to which substances are bound. In particular, it relates to an immunological assay method and an immunoassay kit using acrolein adducts of antigen-antibody reactions, mainly in the fields of industrial, environmental, and clinical inspections. Background technique [0002] In recent years, automation and shortening of measurement time have been sought in various tests such as clinical tests. As the inspection method, a measurement method utilizing an immune reaction is widely used to measure a substance in a biological sample. As immunological measurement methods, there are various methods such as RIA method, EIA method, immunoturbidimetric method, latex agglutination method, colloidal gold agglutination method, and immunochromatography. Among them, the latex agglutination method and the colloidal gold agglutination method are suitable for automated or short-term me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/531G01N33/543G01N33/545G01N33/553G01N33/577
CPCG01N33/5308G01N33/54313
Inventor 柳谷真理田中睦小坂美惠子榎本昌泰山田智中岛史雄
Owner ALFRESA PHARMA CORP