Development of a second generation monoclonal single chain variable fragment antibody against Venezuelan equine encephalitis virus: Expression and functional analysis

We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V-H) and the light (V-L) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a similar to30 kDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein. The similar to30 kDa A116 antibody displayed weak binding specificity to VEE antigen. Sequence analysis revealed a frame shift in the N-terminal region of the VL domain, upstream to the complementarity-determining region I (CDRI), as the probable cause of reduced activity. The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift.