Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

TLDR

The phage peptide library contains ~10^9 clones each displaying a 30‑residue peptide fused to gene III. The study aimed to map the structure of the small hepatitis B virus surface antigen (HBsAg) by epitope mapping of four anti‑HBsAg monoclonal antibodies. Epitope sequences were identified by biopanning the phage library against the antibodies, and sequence homology with native HBsAg defined precise binding regions. Three antibodies bound distinct discontinuous epitopes spanning residues 101–207, a fourth bound residues 121–124, and the data enabled a topological model of the 101–207 region, demonstrating the method’s utility for proteins lacking crystal structures.

Abstract

The structure of the small hepatitis B virus surface antigen (HBsAg) was investigated by epitope mapping of four anti-HBsAg monoclonal antibodies (mAbs). Amino acid sequences of epitopes were derived from affinity-enrichment experiments (biopanning) using a filamentous phage peptide library. The library consists of 10(9) different clones bearing a 30-residue peptide fused to gene III. Sequence homologies between peptides obtained from panning the library against the antibodies and the native HBsAg sequence allowed for precise description of the binding regions. Three of four mAbs were found to bind to distinct discontinuous epitopes between amino acid residues 101 and 207 of HBsAg. The fourth mAb was demonstrated to bind to residues 121-124. The sequence data are supported by ELISA assays demonstrating the binding of the HBsAg-specific peptides on filamentous phage to mAbs. The sequence data were used to map the surface of HBsAg and to derive a topological model for the alpha-carbon trace of the 101-207 region of HBsAg. The approach should be useful for other proteins for which the crystal structure is not available but a representative set of mAbs can be obtained.