Elucidation of the core residues of an epitope using membrane-based combinatorial peptide libraries.
Combinatorial peptide libraries have proved to be a valuable tool for the study of the interaction of a functional protein with its ligand. Here, the epitope for a monoclonal antibody 201/9, raised against beta-factor XIIa, has been identified with a two-step approach using peptide libraries attached to a polymer (polyvinylidene difluoride) membrane. First, the octapeptide libraries with two amino acids defined at position 2 and 4, represented by the formula X-O2-X-O4-X-X-X-X, were synthesized on a sheet of polymer membrane in which X represents a mixture of all the natural -amino acids except cysteine, while O2 and O4 each represent a single amino acid. The libraries were probed with the antibody 201/9, and the bound antibody was detected with a sensitive chemiluminescent method. In the first cycle, the peptide mixtures X-Phe-X-Gln-X-X-X-X showed the strongest signal development. In the second cycle Phe and Gln were incorporated into new libraries consisting of sequences O1-Phe-X-Gln-X-X-X-X, X-Phe-O3-Gln-X-X-X-X, X-Phe-X-Gln-O5-X-X-X, X-Phe-X-Gln-X-O6-X-X, X-Phe-X-Gln-X-X-O7-X, and X-Phe-X-Gln-X-X-X-O8. After probing these new peptides, the residues representing the core sequence of the epitope for monoclonal antibody 201/9 were elucidated. The sequence Ser-Phe-Leu-Gln-Glu-Asn, identified as the immunodominant epitope, correlates well with the sequence Ser-Phe-Leu-Gln-Glu-Ala previously identified (Gao, B., and Esnouf, M. P. (1996) J. Immunol. 157, 183-188) in a scan of overlapping peptides based on the sequence of human beta-factor XIIa.
Gao
B
B
Nuffield Department of Clinical Biochemistry, University of Oxford, Radcliffe Infirmary, Oxford OX2 6HE, United Kingdom.
Esnouf
M P
MP
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D005174:Factor XII / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry*; D008969:Molecular Sequence Data; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D016056:Immunodominant Epitopes; D019151:Peptide Library; D005174:Factor XII