A monoclonal antibody epitope on keratin 8 identified using a phage peptide library

A bacteriophage random hexapeptide library was used to define the epitope of a monoclonal anti-keratin antibody. Phage selected by the keratin 8-specific antibody LE41 displayed highly related sequences on their pIII coat protein. The consensus sequence S(X)LNP allowed the precise localization of an LE41 epitope (SLLSP) within the head domain (H1 subdomain) of human keratin 8, known to be important for correct filament polymerisation. By sequencing the immunizing antigen, keratin 8 from Potorous tridactylis, it was shown that the natural epitope of LE41 is the pentapeptide SLLNP, which confirmed predictions from the phage library results. An SLL(X)P motif is found in the H1 region of all type II keratins (keratins 1 to 8) in different species, but mutational analysis revealed that LE41 can only bind to keratin 8 when Asn (N) or Ser (S) is found in the (X) position. Thus the monoclonal antibody LE41 retains its specificity for keratin 8, dependent on a single amino acid residue, even though it recognizes an epitope within the highly conserved H1 subdomain of the head region. Six other monoclonal antibodies tested on the phage library failed to select motifs.