Production and Characterization of Monoclonal Antibodies against Fumonisin B1

Abstract

Monoclonal antibodies (mAb) against fumonisin B1 (FmB1) were produced from a stable hybridoma cell line, 9C11E6, generated by the fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with FmB1-keyhole limpet hemocyanin. The 9C11E6 mAb belong to the immunoglobulin G1 (kappa chain) isotype with the highest specificity for FmB3. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were established for antibody characterization and toxin analysis. The concentrations causing 50% inhibition (IC 50) of binding of FmB1horseradish peroxidase with the antibodies by FmB1, FmB2, and FmB3 in the dc-ELISA were found to be 29.1, 17.5, and 4.1 ng ml-1 , respectively. The IC 50 values of the binding of 9C11E6 mAb to the solid-phase FmB1-ovalbumin by free FmB1, FmB2, and FmB3 in the idc-ELISA were found to be 20.1, 13.2, and 16.8 ng ml-1 , respectively. The average recovery of FmB1 (50-2000 ng g-1) added to cornmeal and subsequently extracted with phosphatebuffered saline in the dc-ELISA was found to be 71.3%. The mAb did not react with the hydrolyzed FmB1, sphingosine, sphinganine, or tricarballylic acid, but showed weak crossreactivity (about 6.8% of FmB1) with the Alternaria alternata f. sp. lycopersici toxin TA.

Production of a Single-Chain Variable Fragment Antibody against Fumonisin B1

“…The vast majority of antibodies produced against mycotoxins were obtained by immunization of animals with the molecule coupled to a carrier protein (fumonisin B1 (24), aflatoxin B1 (25), or diacetylnivalenol (26)). All but one scFvs directed against mycotoxins, described up to now, originated from cloning of monoclonal mouse hybridoma cell lines or from phage display (24,25,27,28). Only one report described the production of scFvs against the mycotoxin aflatoxin B1 starting from a naı ¨ve scFv library (12).…”

Visual Non-Instrumental On-Site Detection of Fumonisin B1, B2, and B3 in Cereal Samples Using a Clean-Up Combined with Gel-Based Immunoaffinity Test Column Assay

“…According to the periodate method [ 29 ], the enzyme conjugate FB 1 -HRP was synthesized as follows: 2 mg HRP in 500 μL sodium periodate solution (1 mol L −1 ) was activated for 20 min at room temperature; then, 1 mL of FB 1 solution (1 mg of FB 1 was dissolved in 1 mL of 0.05 mol L −1 carbonated buffer, pH 9.5) was dropwise added into the solution described above, and the mixture was allowed to react for 2 h at room temperature under magnetic stirring. Then, 100 μL sodium borohydride solutions (1 mol L −1 ) were added, and the mixture was incubated at 4 °C for 1 h to terminate the reaction.…”

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

“…FB 1 –GA–KLH, as the artificial immunogen, was synthesised by conjugation of FB 1 to KLH using the modified glutaraldehyde (GA) method previously described . Briefly, 1 mg of FB 1 in 2 mL of phosphate‐buffered saline (PBS, 0.01 mol L −1 , pH 7.4) was mixed with 5 mg of KLH, to which 500 µL of a 2% GA solution (a 25% GA solution was diluted with PBS, previously) was added drop‐wise with continuous stirring.…”