Long Zou1, Yang Xu, Yanping Li, Qinghua He, Bo Chen, Dan Wang. 1. State Key Laboratory of Food Science and Technology, Sino-Germany Joint Research Institute, Nanchang University, Nanchang, 330047, China.
Abstract
BACKGROUND: Fumonisin B1 (FB1 ) is a cancer-promoting mycotoxin produced by Fusarium species and one of the major food-borne toxins in corn and corn products. The objective of this study was to produce a single-chain variable fragment (scFv) antibody for determination of FB1 in corn samples. RESULTS: Anti-FB1 monoclonal antibodies were obtained via the hybridoma technique. Specific heavy- and light-chain variable fragments were amplified with degenerate primers and constructed into scFv antibody fragments by splice overlap extension polymerase chain reaction with linker sequences. The resulting scFv DNA fragments were cloned into the phagemid pHEN1for selection and identification of functional scFv fragment by phage display. Prokaryotic expression vector pET22b-scFv was constructed to prepare anti-FB1 scFv antibody for establishment of indirect competitive ELISA. The detection capability (CCβ) of the scFv-based ELISA was 15.00 µg kg(-1), and the limit of detection was 8.32 µg kg(-1). The recoveries and coefficients of variation were 86.74-107.34% and 9.72-14.03%, respectively. In addition, the determined results of 30 naturally contaminated corn samples by the scFv-based ELISA are in agreement with the findings of high-performance liquid chromatography (R(2) = 0.97). CONCLUSION: This scFv-based ELISA could be used as an efficient screening method for routine monitoring the residues FB1 in corn samples.
BACKGROUND: Fumonisin B1 (FB1 ) is a cancer-promoting mycotoxin produced by Fusarium species and one of the major food-borne toxins in corn and corn products. The objective of this study was to produce a single-chain variable fragment (scFv) antibody for determination of FB1 in corn samples. RESULTS: Anti-FB1 monoclonal antibodies were obtained via the hybridoma technique. Specific heavy- and light-chain variable fragments were amplified with degenerate primers and constructed into scFv antibody fragments by splice overlap extension polymerase chain reaction with linker sequences. The resulting scFv DNA fragments were cloned into the phagemid pHEN1for selection and identification of functional scFv fragment by phage display. Prokaryotic expression vector pET22b-scFv was constructed to prepare anti-FB1 scFv antibody for establishment of indirect competitive ELISA. The detection capability (CCβ) of the scFv-based ELISA was 15.00 µg kg(-1), and the limit of detection was 8.32 µg kg(-1). The recoveries and coefficients of variation were 86.74-107.34% and 9.72-14.03%, respectively. In addition, the determined results of 30 naturally contaminated corn samples by the scFv-based ELISA are in agreement with the findings of high-performance liquid chromatography (R(2) = 0.97). CONCLUSION: This scFv-based ELISA could be used as an efficient screening method for routine monitoring the residues FB1 in corn samples.