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 109 different clones bearing a 30-residue peptide fused to gene IlI. 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 a-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.Elucidation of epitope structure is an integral part of studying antigen-antibody interactions. Peptide libraries on filamentous phage have recently been used for rapid epitope mapping of antibodies. The procedure entails affinity screening the library against the antibody, isolating the phage that bind, and determining the peptide sequence of the binding phage through DNA sequencing of the relevant portion of the phage genome. By using this procedure, a large number of peptides can be screened for binding to an antibody in a relatively short time (1, 2).A filamentous phage in a typical library carries on its surface 3 to 5 random peptides, 6-38 residues long, fused to the gene III protein. The peptide library used in this paper contains 109 different phages, each displaying a 30-residue peptide fused to the gene III protein (3). The library was originally applied to select peptides that bind to monoclonal antibodies (mAbs) raised against human immunodeficiency virus gpl2O or hepatitis C virus core proteins. In this study, we applied the phage library approach to mapping the surface antigen of the hepatitis B virus (HBV). The antigen is the diagnostic marker for the HBV. The virus causes major endemic illness throughout the world and is associated with a greatly increased frequency of primary hepatoma, a major cancer in the world (4).The envelope of the hepatitis B virion consists of three proteins, termed small, middle, and large HBV surface proteins, and their glycosylated derivatives. The small HBV surface antigen (HBsAg) is the major component of the envelope of the virion and is mainly present in the plasma of HBV-infected individuals as 22-nm spherical particles com-
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