An improved selection procedure for the screening of phage display peptide libraries

Abstract

Peptide sequences that bind to a wide range of ligands such as monoclonal antibodies, receptors and carbohydrates have been successfully identified after screening phage display peptide libraries. However, these procedures tend to select mainly medium to low affinity-binding clones. A modified screening procedure has been developed in order to improve the efficiency of this process such that high avidity/affinity binding clones are preferentially selected. Three different solid phase binding surfaces were evaluated for the attachment of antibody during the screening procedure and a stepwise decrease in the pH of the elution buffer introduced during the final round of biopanning. The monoclonal antibody MA 18/7 was used to screen a 15-mer peptide library. This antibody is well-characterised and its binding site has been mapped to residues 28–37 of the pre-S1 protein of the hepatitis B virus. The antibody was either biotinylated and attached to polystyrene plates via a streptavidin–biotin ‘bridge’, or bound directly to 1/4 in. polystyrene beads, or to 11 μm latex beads. A significant enrichment of binding clones was observed when the monoclonal antibody was attached directly to polystyrene or latex beads as compared to the biotinylated antibody. All mimotopes identified after biopanning with the antibody attached to the polystyrene beads possessed a central core motif, identical or similar to the sequence DPAF contained within the epitope binding site of MA 18/7 on the native pre-S molecule. However, this motif was only observed in 30% of clones isolated after biopanning using the 11 μm latex beads and in 2% of clones isolated after biopanning on the streptavidin-coated plates. Immunoblotting with the monoclonal antibody MA 18/7 confirmed binding to clones containing the DPAF sequence or a similar motif. A stepwise reduction in the pH of the elution buffer in the final round of biopanning resulted in the removal of clones that possessed low affinity binding motifs, thereby increasing the percentage of clones containing high affinity binding motifs in the final elution step at pH 2.0. Thus, the combined use of polystyrene beads and a stepwise decrease in the pH of the elution buffer in the final round of biopanning resulted in the elimination of non-binding clones and an increase in the efficiency in isolating high affinity binding clones.