A SCFV-ALKALINE PHOSPHATASE FUSION PROTEIN WHICH DETECTS POTATO LEAFROLL LUTEOVIRUS IN PLANT-EXTRACTS BY ELISA

A single chain Fv antibody fragment (scFv) was obtained from a synthet ic phage-antibody library after four rounds of selection against purif ied preparations of potato leafroll luteovirus (PLRV). Nucleotide sequ ence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkal ine phosphatase fusion protein was produced by transformed bacteria Fo llowing induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). Th e fusion protein was obtained at concentrations of 10 mg/l of Escheric hia coli culture medium and these fusion protein preparations were use d directly in ELISA to detect PLRV in sap extracts from infected plant s. Our work is the first report of the selection of a scFv specific fo r a luteovirus from a synthetic phage-display library and the producti on of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus in fection. Copyright (C) 1997 Elsevier Science B.V.