SINGLE-CHAIN FV FUSION PROTEINS SUITABLE AS COATING AND DETECTING REAGENTS IN A DOUBLE ANTIBODY SANDWICH ENZYME-LINKED-IMMUNOSORBENT-ASSAY

A recombinant enzyme-linked immunosorbent assay (ELISA) system, entire ly based on antibody fragments, is described here as an attractive alt ernative to assays using polyclonal antisera or monoclonal antibodies. Two expression vectors have been developed for cloning and production of single chain Fv (scFv) fusion proteins suitable as coating and det ecting reagents, respectively, in a highly sensitive double antibody s andwich ELISA. The coating reagent is produced from the vector pZIP1, as a bivalent miniantibody with a leucine zipper for dimerization, The detection reagent is a fusion protein, in which a modified Escherichi a coli alkaline phosphatase with increased specific activity is attach ed to the carboxy-terminus of the scFv. This conjugate is produced usi ng pDAP2/S as cloning and expression vector. Optimized bacteria expres sion and simple one-step purification by hexa-histidine tag-mediated m etal chelate affinity chromatography yielded milligrams of ELISA reage nt per liter of bacterial culture in both cases. A double antibody san dwich ELISA for the detection of beet necrotic yellow vein virus, the causal agent of sugarbeet rhizomania, was developed using fusion prote ins obtained by means of pZIP1 and pDAP2/S. The plant pathogen was det ected with a sensitivity higher than that reached in a conventional EL ISA employing polyclonal antisera. Both plasmid vectors are compatible to phage display vectors such as pHEN1, pCOCK, and the pCANTAB series , allowing simple subcloning after isolation of scFv from phage displa y libraries. It is therefore easy to develop and produce an ELISA enti rely by using bacterial recombination and expression techniques. (C) 1 997 Academic Press.