MOLECULAR-CLONING, EXPRESSION, AND CHARACTERIZATION OF A FUNCTIONAL SINGLE-CHAIN FV ANTIBODY TO THE MYCOTOXIN ZEARALENONE

The heavy-chain and kappa light-chain variable region genes of an anti zearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly(4)Ser)(3) as a single-ch ain Fv (scFv) DNA fragment. The scFv DNA fragment was cloned into a ph agemid (pCANTAB5E) and expressed as a fusion protein,vith E tag and ph age M13 p3 in Escherichia coli TG1. In the presence of helper phage M1 3K07, the scFv fusion protein was displayed on the surfaces of recombi nant phages, High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjug ate. The selected recombinant phages were used to infect E. coli HB215 1 for the production of soluble scFv antibodies. One selected clone (p QY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibitio n of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay. However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the pare nt monoclonal antibody did. Nucleotide sequence analyses revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence ident ity of 97% to a mouse germ line gene V(K)23.32 in mouse kappa light-ch ain variable region subgroup V, whereas the heavy-chain nucleotide seq uence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity- determining region sequences. The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts.