Determination of the epitope specificities of monoclonal antibodies using unprocessed supernatants of hybridoma cultures

Abstract

A double monoclonal antibody (mAb) ELISA has been developed to determine the epitope specificites of murine monoclonal (mAbs). It permits the mAbs which bind to the same or adjacent epitopes to be distinguished from those which bind to separate epitopes on soluble monomeric proteins. The assay is designed for the early evaluation of mAbs in hybridoma culture supernatants when purified or labeled mAbs are not available. It does not rely on chemical or enzymatic fragmentation of the antigen and does not generate results which may ne due to differences in the affinities of the mAbs. Moreover the characteristic multiple binding of polychonal antibodies to the same epitope is also avoided. A further advantage is the accessibility of epitopes on a given antigen since the antigen is presented by a solid-phase mAb, in contrast to assays in which the antigen itself is adsorbed onto the solid phase. The test was evaluated using the culture supernatants from hybridomas which produced mAbs against the complement proteins C3a, C6 or C7.