EPITOPE IDENTIFICATION BY POLYCLONAL ANTIBODY FROM PHAGE-DISPLAYED RANDOM PEPTIDE LIBRARY

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