Structural determinants of cobrotoxin for binding to an enzyme-linked-immunoassay plate

In order to assess the manner in which the structural state of a protein affects the results of an ELISA, the antigenic reactivities of cobrotoxin and reduced and S-carboxymethylated (RCM-) cobrotoxin with anti-RCM-cobrotoxin antibodies were studied. The results of competitive enzyme-linked immunoassay showed that the affinity of RCM-cobrotoxin for anti-RCM-cobrotoxin antibodies was higher than that of cobrotoxin. However, the cobrotoxin-coated wells had a greater reactivity towards anti-RCM-cobrotoxin antibodies than against RCM-cobrotoxin-coated wells. The lower reactivity observed with RCM-cobrotoxin-coated plates could be improved by adding 0.01% glutaraldehyde during the coating procedure. Studies on the antigenic structures of RCM-cobrotoxin showed that it contained an immunodominant epitope at residues 22-38. Moreover, the N-terminal and C-terminal regions of RCM-cobrotoxin encompassed other antigenic determinants which exhibited low reactivities towards anti-RCM-cobrotoxin antibodies. After removal of the antibodies against residues 22-38 of cobrotoxin from anti-RCM-cobrotoxin antibodies by passage through an affinity column, the remaining antibodies exhibited a similar reactivity towards cobrotoxin and RCM-cobrotoxin. The antibodies against residues 22-38 retained a little reactivity with the RCM-cobrotoxin-coated wells. These results suggest that the structural determinants of cobrotoxin and RCM-cobrotoxin for binding to the microtitre plates differ. Unlike RCM-cobrotoxin, the loop II structure of cobrotoxin encompassing residues 22-38 is not exclusively involved in the binding of cobrotoxin to microtitre plates.