A simple solid-phase competition assay with labeled antigen.

Abstract

The determination of epitope specificities of monoclonal antibodies (MAbs) has usually been performed using the competitive solid-phase assay in which the antigen is immobilized, and a radiolabeled antibody and competing unlabeled antibodies are mixed in solution (Fig. 1A) (,). Although this method facilitates separation of free from bound antibody, it possesses the problem of labeling all antibodies to be tested. Since the number of MAbs to be screened is usually large, this method is time consuming and tedious, and the instability of radiolabels represents a significant drawback. In addition, radioactive hazards have to be taken into account. Recently, nonisotopic tracers, such as biotin (,) and fluorescein isothiocyanate (), have been introduced for determination of epitope specificities of MAbs, but these methods still have the problem of labeling all antibodies to be tested. Open image in new window Fig. 1. Diagrammatic representation of a conventional competition immunoassay with labeled antibody (A) and a new type of competition immunoassay with labeled antigen (B) used in epitope mapping. In the competition assay with labeled antibody, the antigen is immobilized, and a radiolabeled antibody as well as competing unlabeled antibodies are mixed in solution. Finally, the radiolabeled antibody bound to the immobilized antigen is detected in a γ-counter. Recently, biotinylated antibody is often used instead of radiolabels. On the other hand, in the competition assay with labeled antigen, a constant amount of biotinylated antigen is incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated antigen bound to the immobilized antibody are then reacted with avidin-peroxidase conjugate and the activity of the bound peroxidase is determined.