A solid-phase mutual inhibition assay with labeled antigen.
A widely applicable method for the determination of the epitope specificities of a large number of monoclonal antibodies (MAbs) is presented. The method is based on the solid-phase mutual inhibition assay using 96-well plates coated with the respective MAbs, competitor MAbs, biotinylated antigen, and avidin-peroxidase conjugate. Using carcinoembryonic antigen (CEA) as a model antigen, the method was applied to determine epitope specificities of anti-CEA MAbs. A constant amount of biotinylated CEA was incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated CEA bound to the immobilized antibody was then reacted with avidin-peroxidase conjugate and the activity of the bound peroxidase was determined by using o-phenylenediamine and hydrogen peroxide. The method used alleviates the laborious procedures of labeling all antibodies to be tested and the confusion caused by differential labeling among different MAbs. It is a convenient method for mapping analysis of many MAbs if the corresponding purified antigen is available.
Department of Biochemistry, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
Masahide
M
Kuroki
D000818:Animals; D055910:Antibodies, Immobilized / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D001360:Avidin / Q000378:metabolism; D001667:Binding, Competitive; D019921:Biotinylation; D002272:Carcinoembryonic Antigen / Q000032:analysis* / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping / Q000191:economics / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007553:Isotope Labeling; D010655:Phenylenediamines / Q000378:metabolism; D011863:Radioimmunoassay
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D010655:Phenylenediamines; C083986:avidin-horseradish peroxidase complex; D001360:Avidin; C034193:1,2-diaminobenzene; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase