[A simple method of determination of antigenic determinants in proteins with known primary structure]

A simple and rapid method is proposed for the localization of antigenic determinants in proteins of known primary structure exemplified by human myoglobin. The polypeptide chain of myoglobin was cleaved with BrCN (at Met residues) or with bromosuccinimide (at Trp and Tyr residues) under conditions which on average gave less than one scission per myoglobin molecule. The "single-hit" cleavage products were separated by gel electrophoresis and transferred to nitrocellulose by electroblotting. The peptides containing intact antigenic determinants were vizualized by immuno-peroxidase staining with four monoclonal anti-myoglobin antibodies. Comparison of the lengths of the immuno-reactive peptides with the known positions of methionine, tryptophan and tyrosine residues suggested that the four monoclonal antibodies were bound by myoglobin over the region Trp-14 to Met-55. As compared with other methods of localization, the method proposed is much faster and takes much lesser amount of protein.