Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-08-03
1997-06-17
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 79, 435 794, 436518, 436536, G01N 3353, G01N 33543, G01N 33536
Patent
active
056396279
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for high sensitivity assay of an antibody specific to a certain antigen.
BACKGROUND ART
There have been reported various methods for quantitative assay of antibody, which are based on the principles of immunoassay. Typical techniques include, for example, agglutination, competitive assay and sandwich method. From the aspect of sensitivity, it is well known that assays based on the principles of the sandwich method can achieve their highest.
The sandwich method for specific antibody includes, for example, a method comprising bringing a carrier bound with an antigen into contact with a liquid sample containing the antibody to be assayed, reacting the carrier with a labeled anti-Ig antibody (secondary antibody) and assaying the antibody level from the amount of the label bound to the carrier. This method is defective in that the non-specific antibodies contained in the liquid sample in a large amount are also adsorbed to the carrier to increase the background, thus making higher detection sensitivity difficult to achieve.
Another method includes binding an anti-Ig antibody to a carrier, bringing the carrier into contact with a liquid sample and allowing a (labeled) antigen specific to the antibody to be assayed, to react with the antibody. In this method, the defect lies in the fact that binding the object antibody to the carrier is prevented by non-specific antibodies contained in greater amounts in the liquid sample and signals cannot be obtained in sufficient amounts. Consequently, high sensitive assay is difficult like the above-mentioned method.
Another method includes bringing a carrier bound with an antigen into contact with a liquid sample to allow reaction of a labeled antigen. This method is defective in that signals cannot be obtained in sufficient amounts in a short time due to the low reactivity of the labeled antigen to the antibody bound to the carrier. Again, high sensitivity assay by this method is associated with difficulty.
As a method for highly sensitive and easy assay of an antibody, a method has been recently reported which includes reacting a labeled antigen and an antigen bound to hapten with the object antibody in a liquid sample in a liquid phase, trapping the resultant complex on a carrier using an anti-hapten antibody, and assaying the complex [Ishikawa et al., Analytical Letters, vol. 21, p. 2033 (1988), DE 3705686 to Thomas Geiger et al] This method is expected to achieve high sensitive assay. However, the method is defective in that the assay components are complicated and it rather gives lower assay values (prozoning phenomenon) when the antibody to be assayed is present in considerably great amounts in the liquid sample.
DISCLOSURE OF THE INVENTION
An object of the present invention is to develop a method for assaying a specific antibody, which can be performed as easily as the conventional sandwich method, has high sensitivity and is free of the prozoning phenomenon.
The present inventors have found that the object specific antibody can be assayed with high sensitivity and the prozoning phenomenon can be eliminated by, in a reaction system wherein a carrier-bound antigen, a labeled antigen, and a liquid sample containing the specific antibody to be assayed are simultaneously mixed to react the above-mentioned two antigens with the above-mentioned specific antibody, forming a complex of the carrier-bound antigen-specific antibody-labeled antigen, separating said carrier after the reaction from the aforementioned reaction system, and again mixing the aforementioned separated carrier and the aforementioned labeled antigen, which resulted in the completion of the present invention.
That is, the present invention provides a method for assaying a specific antibody, comprising the following steps (A), (B) and (C):
(A): forming a complex of a labeled antigen-a specific antibody-a carrier-bound antigen in a first reaction system comprising a mixture of the antigen (labeled antigen) against the specific antibody to be as
REFERENCES:
patent: 4945042 (1990-07-01), Geiger et al.
Analytical Letters, vol. 21, (11), pp. 2033-2048, Takeyuki Kohno et al, "Novel and Sensitive Enzyme Immunoassay for Anti-Thyroglobulin Antibodies in Serum Using . . . Conjugate".
Kohno et al., 1988, Analytical Letters, 21: 2033-2048.
Derwent Abstract (Accession Number: 90-198361; Ishikawa et al).
Housel James C.
Shaver Jennifer
Sumitomo Pharmaceuticals Co. Ltd.
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