Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 113-120 (antigenic site 4) of myoglobin.

Immunochemical cross-reactivity of protein variants has been very frequently used to map protein antigenic sites. The approach is based on the assumption that amino acid substitutions affecting the binding of a protein to its antibody, particularly when monoclonal antibodies (mAbs) are used, must be part of the antigenic site and not far from it. The assumption was investigated in this study by determining the effects of amino acid substitutions outside the antigenic site on the reactivity of six myoglobin (Mb) variants with three mAbs of predetermined specificity prepared by immunization with a free synthetic peptide representing region 113-120 (antigenic site 4) of Mb. Two of the Mb variants used had no substitutions within residues 113-120 (the region to which the specificity of the mAbs is directed) and yet exhibited markedly decreased cross-reactions and binding affinities, relative to the reference antigen, sperm-whale Mb. The other three Mb variants possessed substitutions within, as well as outside, region 113-120 and showed very little cross-reactivities. The results of this study, particularly with the Mbs that have no substitutions within the indicated antigenic site, clearly show that substitutions outside the site, and which by design are not part of the site, can influence very markedly the reactivity of the protein variant with the anti-site mAbs. The approach can, therefore, lead to serious errors if used to identify residues of protein antigenic sites.