The binding specificity of a monoclonal antibody (MAb 12) known to recognize the surface antigen of hepatitis B virus (HBsAg) was studied using a positional scanning synthetic combinatorial library. A hexapeptide library totaling more than 30 x 10(6), sequences, made up of 120 mixtures having a single position defined with individual amino acids and the remaining five positions composed of mixtures of amino acids, was screened by competitive enzyme-linked immunosorbent assay (ELISA). This led to the identification of Ac-STTSMM-NH2 (IC50 = 170 nM), which specifically inhibited the interaction between HBsAg and MAb 12. One of the most active individual mixtures from the library was Ac-XXXPXX-NH2; however, none of the individual peptides synthesized containing proline at the fourth position showed significant activity (IC50 > 100 000 nM). To identify the individual peptide(s) responsible for the activity of Ac-XXXPXX-NH2, a bidirectional iterative synthesis and selection process was carried out. A completely different but active peptide sequence was identified (Ac-SVGPPH-NH2, IC50 = 165 nM). The two different hexapeptide sequences were prepared as linear homo- and heterodimer peptides in an attempt to improve the antigenicity. Of the four different sequences prepared, one heterodimer (Ac-STTSMMGGSVGPPH-NH2) was found by ELISA to have a 10-fold improvement in activity over the two individual hexapeptides, and was equal to the inhibitory activity of the protein antigen. The equilibrium affinity constant of the MAb 12 toward this heterodimer sequence was 50-fold higher than the protein antigen when measured in a biosensor system. Since motifs from these two hexapeptides, namely, -STTS- and -GP-, were located in the primary sequence of the protein (residues 114-120, subtype ad), overlapping hexapeptides of this region were synthesized and assayed. The most active hexapeptide, namely, Ac-TTSTGP-NH2 (IC50 = 2.3 mu M), was 10-fold less active than either hexapeptide found from the library. Extending the specific motif sequences to eleven residues resulted in an analog (Ac-STTSTGPSRTC-NH2) have an equilibrium affinity constant similar to the heterodimer. The combined used of positional scanning libraries and the iterative synthesis and selection process in this study illustrates the power of these methods for the identification of novel peptides that inhibit anti-protein antibodies. These methods can be directly applied to the development of improved immunodiagnostics.
