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.