Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1

Abstract

Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb.

Affinity maturation of single-chain variable fragment specific for aflatoxin B1 using yeast surface display

“…Indeed, a considerable portion of the scFv variant, expressed at 20°C (over 90% of total scFv protein) was aggregated as the inclusion body in the cytoplasm and periplasm (Min et al, 2010). Fig.…”

Production and characterization of a single chain variable fragment (scFv) against the mycotoxin deoxynivalenol

“…Specific scFv have been developed for a number of mycotoxins including zearalenone (Chang, Choi, & Chun, 2008;Wang, Du, Lin, Huang, & Wang, 2008;Yuan et al, 1997), aflatoxin B 1 (Daly et al, 2002;Moghaddam, Løbersli, Gebhardt, Braunagel, & Marvik, 2001) and fumonisin B 1 (Lauer, Ottleben, Jacobsen, & Reinard, 2005;Min et al, 2010). There are also two reports of scFv specific for DON (Choi et al, 2004;Wang et al, 2007) and a report of a single-domain antibody fragment with an affinity to a related congener, 15-acetyl-deoxynivalenol (Doyle et al, 2008).…”

Development of a single-chain variable fragment antibody-based enzyme-linked immunosorbent assay for determination of fumonisin B1in corn samples

“…The presence of non‐functional variable gene fragments in the hybridoma is a recongnised phenomenon that can cause considerable problems with the selective amplification of the functional fragments . In general, specific PCR primers designed from N‐terminal amino acid sequences of the V H and V L of McAb was a good choice to avoid the above problem, but the N‐terminal amino acid sequence was very expensive. Here, we developed a universal method for cloning and identifying functional scFv antibody fragment by combining degenerate PCR with phage display, which was effectively remove non‐specific amplification fragment and suitable for great majority hybridoma.…”

“…Single‐chain variable fragment (scFv), the smallest functional unit of antibody, offers several significant advantages over parent antibody, including large‐scale production in bacteria and yeast. An increasing number of scFv against mycotoxins, including aflatoxin B 1 (AFB 1 ), zearalenone (ZEN), deoxynivalenol (DON), and FB 1 , have been successfully produced. Furthermore, we are interested in determination of FB 1 in food matrices using sensitive and specific scFv‐based ELISA.…”