Determination of antigenic binding sites in antibodies directed against a mycobacterial peptide by deletion and substitution of single amino acids.

A component of Mycobacterium bovis (BCG) was previously isolated using a monoclonal antibody to BCG and affinity chromatography. It was designated BCG-a. The sequence of N-terminal residues of BCG-a was used to synthesize a 13 amino acid peptide designated BCG-a-P which elicited immunologic responses. The present study was undertaken to identify which amino acid residues were critical for the immunologic characteristics of BCG-a-P. By virtue of analogues synthesized with either amino acid deletions or substitutions along the BCG-a-P sequence, it was possible to identify the regions of the peptide which were responsible for the recognition and binding by antibodies directed to BCG-a-P. In both direct and competition ELISA systems, the deletion of single amino acids caused a change in the binding of BCG-a-P by an antiserum directed to BCG-a-P. Similar results were evident when the amino acid phenylalanine was substituted for individual residues along the sequence of BCG-a-P. The residues responsible for antibody binding had the highest localized hydrophilic index of any hexapeptide stretch of the BCG-a-P sequence. These findings indicate that the activity expressed by BCG-a-P was due to a defined region of the peptide. The techniques used here may have applications in identifying the regions within other mycobacterial antigens which are involved in antibody binding.