Premium
Structural determinants of cobrotoxin for binding to an enzyme‐linked‐immunoassay plate
Author(s) -
LS Chang,
Lin J.,
CC Chang
Publication year - 1996
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1996.tb00391.x
Subject(s) - immunoassay , epitope , chemistry , antibody , biochemistry , biology , immunology
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.