Epitope identification by polyclonal antibody from phage-displayed random peptide library

Abstract

Screening of bioactive peptides from random peptide libraries using monoclonal antibodies as ligates is an effective method to define epitopes of protein antigens. However, it is thought that polyclonal antibodies might also serve as promising ligates for screening. We illustrate this approach by using recombinant human lymphotoxin (rhLT) polyclonal antibody as a model. The procedure consists in (a) affinity purification of polyclonal antibody to obtain the 'monospecific' antibody, (b) screening against a phage-displayed random peptide library using the affinity-purified antibody, (c) plating the enriched phage on agar plates, randomly picking clones, and selecting the positive ones by dot blotting, (d) DNA sequencing of the positive clones and conducting a homology search against thc protein sequence databank, and (c) confirming the epitopes by chemical peptide synthesis. By employing this procedure, we identified a dominant epitope ROHPKM, located at residues 15- 20 of the human lymphotoxin amino acid sequence. The usefulness of this general procedure is discussed.