Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).