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Development of a single‐chain variable fragment antibody‐based enzyme‐linked immunosorbent assay for determination of fumonisin B₁ in corn samples

Author:
Zou, Long, Xu, Yang, Li, Yanping, He, Qinghua, Chen, Bo, Wang, Dan
Source:
Journal of the science of food and agriculture 2014 v.94 no.9 pp. 1865-1871
ISSN:
0022-5142
Subject:
DNA, Fusarium, bacteriophages, corn, corn products, detection limit, enzyme-linked immunosorbent assay, hybridomas, liquid chromatography, monoclonal antibodies, mycotoxins, polymerase chain reaction
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 (R² = 0.97). CONCLUSION: This scFv‐based ELISA could be used as an efficient screening method for routine monitoring the residues FB₁ in corn samples. © 2013 Society of Chemical Industry
Agid:
392406