Two neutralizing monoclonal antibodies specific for Naja nigricollis cardiotoxin: Preparation, characterization and localization of the epitopes

Abstract

Two monoclonal antibodies have been raised against the native form of the potent cardiotoxin isolated from the venom of Naja nigricollis. The toxic action to mice as well as the depolarizing effect on muscle fibres in culture of the cardiotoxin are neutralized by the two immunoglobulins. Binding studies revealed that the radiolabelled toxin has a high affinity for both antibodies, the equilibrium dissociation constant values being equal to 0.2 and 0.4 n M. The epitopes that are recognized by the antibodies have been localized on the basis of competition experiments between the labelled toxin and a series of variants or a Trp-11 modified derivative, toward both antibodies. The data obtained indicate that the antibodies bind at topographically different antigenic sites. Knowing that the toxin is a single polypeptide chain folded in a structure that contains three adjacent loops emerging from a small globular region, it appears that one of the two antibodies binds on loop I, at a site which involves Trp-11 whereas the other binds at a site which involves one or both of loops II and III. Possible mechanisms of neutralization of the toxin by the antibodies are discussed.