Competitive ELISA employing monoclonal antibodies specific for coronafacoyl amino acid conjugates

Coronatine (COR) is composed of two structural components, coronafacic acid (CFA) and the amino acid coronamic acid (CMA), linked by an amide bond. Monoclonal antibodies (MAbs) were prepared against COR and incorporated into competitive ELISAs. MAbs were secreted by hybridoma cells which had been prepared from mice immunized with COR and conjugated, through the free carboxyl group on CMA, to ovalbumin, the available amino groups of which had been increased by derivatization with propyl diamine via free carboxyl groups. COR and coronafacoyl valine could be quantified at 6.800 ng ml^‐1(0.34–40 ng/assay) using peroxidase‐labelled MAb 8H3G2 in an indirect competitive ELISA. The corresponding limit of detection was 1 ng ml～’. Reduction and subsequent acetylation of the ketone oxygen of the CFA moiety of COR reduced the affinity of MAb 8H3G2 some 250 times, whereas methylation of the free carboxyl group on COR slightly enhanced the affinity four‐fold. These and other results suggest that CFA and the amide bond are important structural features of the epitope recognized by MAb 8H3G2.