Linear epitopes of sperm whale myoglobin identified by polyclonal antibody screening of random peptide library

Distinct enhancement of antibody-specific clones was apparent during the screening against random peptide libraries with antigen-specific polyclonal antibodies. Several sequence motifs obtained from these screenings were homologous with the primary sequence of myoglobin. Two of these motifs have been confirmed as antigenic determinants by competitive inhibition tests using eight-branched synthetic peptides. One of the peptides has a sequence that corresponds to amino acid residues 42-50, KFDRFKHLK, of the myoglobin sequence. This is a new epitope of myoglobin that is reported for the first time. The epitope is located precisely in the "turn' or "loop' region between helices C and D of the crystal structure of myoglobin. The second antibody binding site has a sequence of DIAAKYKELGYQG, and this is located between residues 141-153, which is the C-terminus of myoglobin. This epitope encompassed two linear epitopes of myoglobin, amino acid residues 145-151 and 147-153, that have been reported earlier based on immunochemical characterisation of cleavage fragments of the protein. These results clearly indicate that epitope mapping using polyclonal antibodies against random peptide libraries can identify new epitopes precisely, as well as confirm epitopes of myoglobin obtained earlier using established methodologies.