Identification of Binding Epitope for Anti-Rabies Virus Glycoprotein Single-Chain Fv Fragment FV57

Single-chain Fv fragment (scFv) of anti-rabies glycoprotein (G protein) has been recommended as a new agent for detecting and neutralizing lethal rabies virus. In this study, we constructed scFv that corresponded to the FV fragment of CR57, a monoclonal antibody against rabies virus, and called it FV57. Despite its virus neutralization activity, FV57 may or may not recognize the same epitope as that recognized by CR57. To resolve this issue, the binding epitope of rabies virus G protein recognized by FV57 was identified. A recombinant rabies virus G protein fragment (RVG179; residues 179-281) comprising several epitopes was expressed in E.coli, purified, and the specificity of its binding with FV57 was determined. In addition, a peptide (abbreviated as EP, residues 224-236) comprising the known epitope of G protein to which CR57 binds was synthesized and the potency of its binding with FV57 was also determined. The results showed that FV57 could specifically bind to RVG179 and EP. Competitive ELISA experiments indicated that RVG179 and EP were able to compete with the rabies virus G protein for binding with FV57. Since no other epitope within residues 224- 236 has been reported, except for the epitope to which CR57 binds (residues 226-231), the epitope recognized by FV57 was the same as its intact antibody CR57. This demonstrated that the complementarity-determining regions (CDRs) of the heavy and light chains of FV57 have folded into the correct conformation as those of CR57.