Peptide phage libraries can be an efficient tool for identifying antibody ligands for polyclonal antisera

Abstract

SUMMARY We have examined the potential of isolating ligands for polyclonal antibodies from a nanopeptide phage library. The library was screened with a rabbit polyclonal antiserum raised against a synthetic peptide (ALWFRNHFVFGGGTKVT). Following screening, the positive phages were tested in an ELISA for their reactivity with the antiserum. Phages that showed positive reactivity with the antiserum compared with a normal rabbit serum were selected and their displayed peptides were determined. Among the 36 random positive clones, 31 clones carried the sequence AVFGGGTKL, PFFGGGSRA or APTGGSKRT that have a significant homology to the immunizing peptide. Five positive phages displayed the ATNIFIEGT sequence, which has no obvious linear homology with either the other selected peptides or with the peptide used for immunization. In contrast to the control peptide, the immunizing peptide inhibited binding of the antiserum to the peptide-displaying phages in a dose-dependent manner, thus demonstrating the specificity of the interaction. Furthermore, the rabbit B cell response to the peptide was found to be limited and focused on its C-terminal. Taken together, our data demonstrate the potential of random peptide phage libraries for defining epitopes for polyclonal antisera as well as for investigation of the nature of B cell responses to any given antigen.