Liposome reagent for immunoagglutination and immunoanalytical me

Details Liposome reagent for immunoagglutination and immunoanalytical me Liposome reagent for immunoagglutination and immunoanalytical me

1995-07-14

1998-05-26

Stucker, Jeffrey

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

Carrier is organic

436518, 436529, 436530, 436531, 436532, 436533, 436534, 436829, 435 5, 435 71, G01N 33544

Patent

active

057563632

DESCRIPTION:

BRIEF SUMMARY
This application is a 371 continuation of PCT/JP94/00136.


BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to an immunoagglutination reagent. More specifically, the present invention relates to an immunoagglutination reagent capable of assaying a wide concentration range of a substance present in a sample, with high sensitivity, by a simple procedure or by using an automatic analyzer. The present invention also relates to an immunoanalytical method using the same.
2. Background Art
Immunoanalytical methods using an antigen-antibody reaction are extremely important in clinical diagnosis of endocrine diseases and the like. A variety of immunoanalytical methods have become conventional.
Among these conventional methods is an immunoanalytical method comprising reacting an antibody or an antigenic substance in a sample with an immunoagglutination reagent comprising an antigenic substance or an antibody bound to a carrier, measuring the agglutination level caused by the antigen-antibody reaction using the naked eye or as turbidity using a spectrophotometer, comparing the results with a standard curve and thereby obtaining a value for the antibody or the antigenic substance contained in the sample. Synthetic latex particles such as polystyrene have primarily been used as the carrier in such immunoanalytical methods.
Because the surface of the immunoagglutination reagent using such conventional synthetic latex particles as the carrier is hydrophobic, agglutination via hydrophobic binding readily occurs in an aqueous solution of pH 7 to 7.5, suitable for antigen-antibody reaction. Thus, such a reagent tends to induce non-specific agglutination. Therefore, the assay precision is likely to vary. Thus, patent applications (for example, Japanese Patent Publication No. Hei 3-142360 and Japanese Patent Publication No. Hei 3-76425) have been directed to techniques for adding additives to immunoagglutination reagents bound to synthetic latex particles as the carrier so as to suppress such non-specific agglutination.
Because an antigen or an antibody is immobilized via physical adsorption onto synthetic latex particles, the antigen or the antibody, in long-term storage, dissociates from the synthetic latex particles, causing a decrease in assay sensitivity.
More recently, such latex agglutination reagents have been used in general purpose biochemical automatic analyzers, to assay an antigen or an antibody in a sample. However, such use encounters the problem that an immunoagglutination reagent using the synthetic latex particles as a carrier adheres to the reaction cells of the automatic analyzer, thereby adversely affecting the assay precision.
So as to overcome these problems, the present inventors have developed a liposome agglutination reagent using a liposome as the carrier. The liposome is covalently coated with an antigen or an antibody. See Japanese Patent Application No. Hei 4-164783. Because an antigen or an antibody is covalently bound onto the surface of the liposome in the liposome agglutination reagent, the antigen or the antibody does not dissociate from the liposomes even after a long-term storage. Because the surface of liposome of itself is hydrophilic, agglutination via hydrophobic binding and, therefore, non-specific agglutination hardly occurs.
The reagent also offers the advantage that the reaction cells of an automatic analyzer do not become contaminated, which advantage has never been offered by a conventional latex agglutination reagent.
Because latex agglutination reagents themselves have high turbidities, i.e., because the blank reading for the reagent itself is high, such reagents should be used for assay with a spectrophotometer in the long wave region due to the smaller change in turbidity. Hence, improvement in the sensitivity thereof has been very difficult. Alternatively, due to the lower refractive index, the liposome agglutination reagent can be used for assay in the short wave region, i.e., about 34 nm, where the change in turbidity is observed as a l

REFERENCES:
patent: 4598051 (1986-07-01), Papahadjopoulos et al.
patent: 4762915 (1988-08-01), Kung et al.
patent: 5000960 (1991-03-01), Wallach
"Large Liposome Agglutination Technqiue for the Serological Detection of Syphilis," Viola T. Kung et al., Journal of Immunological Methods vol. 90 1986, pp. 189-196.
"Antibody Induced Agglutination of Galactocerebroside Liposomes," Debi P. Sarkar et al., Indian Journal of Experimental Biology vol. 20 Jul. 1982, pp. 522-524.

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