Selection of single chain fragments against the phytopathogen Xanthomonas axonopodis pv. citri by ribosome display

Abstract

A ribosome display technique was applied to a mouse single chain variable fragment (ScFv) library to select ScFvs specific for Xanthomonas axonopodis pv. citri (Xac). The ScFv DNA library was transcribed in vitro to mRNA for ribosome display and antibody–ribosome–mRNA (ARM) complexes were produced by a rabbit reticulocyte lysate system. The O-specific lippolysaccharide (LPS) of Xac was used as an antigen to pan ARM complexes and putative ScFv-encoding genes were recovered by RT-PCR following each panning. After three rounds of ribosome display, the ScFv DNA was cloned into Escherichia coli TG1 for expression and the expressed products of 180 clones were analyzed by indirect ELISA. Sixty clones of those showed an antibody activity to the O-specific LPS of Xac, and three (GX13, GX44 and GX95) exhibited higher activity and specificity to Xac and no cross-reactions were observed with 10 saprophytic xanthomonads from citrus or other bacteria. The equilibrium constant ( K A) determined by BIAcore analysis for ScFvs GX13, GX44 and GX95 were 1.98 × 10 10 M −1, 1.89 × 10 10 M −1 and 3.43 × 10 10 M −1, respectively.