Method for stabilizing microparticles having reactive substance bound thereto, and reagent containing the microparticles

A reactive, material technology, applied in the field of reagents for immunoassays, which can solve the problems of undisclosed gold particle stabilization methods, etc.

Inactive Publication Date: 2011-06-08
ALFRESA PHARMA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these are documents showing a method for producing gold particles in which substances are bonded to the surface of gold particles only for the purpose of improving reactivity, and do not disclose a method for stabilizing gold particles.

Method used

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  • Method for stabilizing microparticles having reactive substance bound thereto, and reagent containing the microparticles
  • Method for stabilizing microparticles having reactive substance bound thereto, and reagent containing the microparticles
  • Method for stabilizing microparticles having reactive substance bound thereto, and reagent containing the microparticles

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0080] Preparation Example 1: Preparation of Colloidal Gold Solution

[0081] While stirring, 2 mL of 10% chloroauric acid aqueous solution was added to 1 L of 95°C distilled water, and after 1 minute, 10 mL of 2% sodium citrate aqueous solution was added, stirred for a further 20 minutes, and then cooled to 30°C. After cooling, adjust the pH to 7.1 with 0.1% potassium carbonate aqueous solution.

preparation example 2

[0082] Preparation Example 2: Preparation of Anti-Cystatin C Antibody Bonded Colloidal Gold Reagent

[0083] Anti-cystatin C antibody (Dako Japan Inc.) was diluted with 10 mM HEPES (pH 7.1) containing 0.05% sodium azide to prepare 50 mug / mL concentration. 100 mL of this anti-cystatin C antibody solution was added to about 1 L of the colloidal gold solution prepared in Preparation Example 1, and stirred for 2 hours under refrigeration. Further, 110 mL of 10 mM HEPES (pH 7.1) containing 5.46% mannitol, 0.5% bovine serum albumin (BSA), and 0.05% sodium azide was added, and stirred at 37° C. for 90 minutes. Then, centrifuge at 8000rpm for 40 minutes, remove the supernatant, then add about 1L of 5mM HEPES (pH7.5) containing 3% mannitol, 0.1%BSA and 0.05% sodium azide (A solution), Antibody-bonded colloidal gold dispersion. Further centrifuge at 8000rpm for 40 minutes, remove the supernatant, disperse the antibody-bonded colloidal gold with A solution containing 1.5% dextran sul...

preparation example 3

[0084] Preparation Example 3: Preparation of Cystatin C Reduced IgG

[0085] Anti-cystatin C antibody (Dako Japan Inc.) was dialyzed against 10 mM HEPES (pH 7.1) (B solution) containing 0.05% sodium azide, 5 mM EDTA·2Na and 150 mM sodium chloride, Then dilute with solution B to make the concentration reach 10mg / mL. 2-Mercaptoethylamine hydrochloride was added to the antibody solution to make the concentration 6 mg / mL, and after dissolution, it was heated at 37° C. for 90 minutes. The 2-mercaptoethylamine hydrochloride was removed using a gel filtration column to obtain an anti-cystatin C reduced IgG.

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Abstract

Disclosed is a method for maintaining the reactivity of microparticles having a reactive substance bound thereto in a dispersion of the microparticles for a long period by stabilizing the microparticles in the dispersion of the microparticles. Specifically disclosed is a method for stabilizing microparticles having a reactive substance bound thereto, which involves a step of allowing a sulfur-containing amino acid or a derivative thereof to co-exist in a dispersion of the microparticles. The sulfur-containing amino acid is preferably cysteine, cystine or methionine. The method is particularly useful when it is intended to coexist a precipitation-inhibiting substance such as a polyanione or dextran in the dispersion.

Description

technical field [0001] The present invention relates to a method for stabilizing microparticles to which a reactive substance such as an antibody or an antigen is bound (hereinafter referred to as reactive substance-bound microparticles) and an immunoassay reagent using the reactive substance-bound microparticles. In particular, it relates to a method for stabilizing reactive substance-bonded microparticles used mainly in clinical examinations for immunological assays utilizing antigen-antibody reactions, and an immunoassay reagent using the reactive substance-bonded microparticles. Background technique [0002] In recent years, automation and shortening of measurement time are being pursued in various examinations such as clinical examinations. For example, methods for the determination of trace substances in biological samples using immune responses are widely used. Examples of the immunoassay method include RIA method, EIA method, immunoturbidimetric method, latex agglut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/543G01N33/553
CPCG01N33/585G01N33/54313
Inventor 田中睦
Owner ALFRESA PHARMA CORP
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