Molecular Cloning and Expression of Recombinant Phage Antibody against Fumonisin B1

Abstract

Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to detection of microbial agents and their toxins in foods. In this study, fumonisin B1 (FB1)-specific antibodies, composed of a single chain containing a variable heavy (VH) and light (VL) chain fragments (ScFv), were cloned using mRNA from either spleen cells of mice immunized with FB1-BSA conjugate or from an existing hybridoma cell line that produces anti-FB1 antibody. The approach consisted of (i) reverse transcription of isolated mRNA, (ii) polymerase chain reaction amplification of VH and VL cDNAs, (iii) ligation of VL and VH chains, and (iv) expression of ScFv proteins on the surface of a filamentous bacteriophage or as a soluble fragment. The efficacy of using the recombinant ScFv proteins for the detection of FB1 were evaluated in a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Compared to polyclonal or monoclonal antibodies for FB1, the recombinant ScFv proteins were 10 to 100 times less sensitive in the CI-ELISA. The secreted ScFv protein did not cross-react with FB2 or FB3. The results indicated that production of a recombinant antibody to a mycotoxin is feasible. However, problems associated with the affinity or avidity of ScFv fragments need to be further addressed before this technology is adaptable to the development of improved immunodiagnostic kits for mycotoxins or other food borne disease hazards.