Abstract
In this study, a fluorescence polarization immunoassay (FPIA) was developed based on the single-chain variable fragments (scFvs) for fumonisin Bs (FBs). The scFvs were prepared from FBs-specific monoclonal antibody secreting hybridomas (4F5 and 4B9). The established FPIA could determine the sum of fumonisin B1 (FB1) and fumonisin B2 (FB2) within a short time. The IC50 of FPIA for the detection of FB1 and FB2 were 29.36 ng/ml and 1,477.82 ng/ml with 4F5 scFv, and 125.16 ng/ml and 30.44 ng/ml with 4B9 scFv, so the 4B9 scFv was selected for detection of FB1 and FB2 in maize samples with a limit of detection of 441.54 μg/kg and 344.933 μg/kg. The recoveries ranged from 84.7 to 104.1% with a coefficient of variation less than 14.1% in spiked samples, and the result of the FPIA method was in good consistency with that of HPLC-MS/MS. To supply a better understanding of the immunoassay results, the interactions mechanism of scFvs-FBs was further revealed by the homology modelling, molecular docking, and molecular dynamic simulation. It was indicated that six complementarity-determining regions (CDRs) were involved in 4B9 scFv recognition, forming a narrow binding cavity, and FB1/FB2 could be inserted into this binding cavity stably through strong hydrogen bonds and other interactions. While in 4F5 scFv, only the FB1 stably inserted in the binding pocket formed by four CDRs through strong hydrogen bonds, and FB2 did not fit the binding cavity due to the lack of hydroxyl at C10, which is the key recognition site of 4F5 scFv. Also, the binding energy of FB2-4B9 scFv complex is higher than the FB2-4F5 scFv complex. This study established a FPIA method with scFv for the detection of FB1 and FB1 in maize, and systematically predicted recognition mechanism of FBs and scFvs, which provided a reference for the better understanding of the immunoassay mechanism.
Highlights
Fumonisins (FBs) produced by Fusarium pathogens, are mycotoxins present in maize and other grains during storage and pose a serious threat to humans and domestic animals worldwide
Total RNA was extracted from the hybridoma cell lines (Figures 1A,B), and the VH and VL genes were cloned from RT-PCR cDNA with specific primers (Supplementary Table S1)
The VH and VL genes were linked by a peptide to construct the scFv gene (VH(Gly4Ser)3-VL) with the gene splicing performed by overlap extension PCR (SOE-PCR), which was expected to be approximately 700–750 bp
Summary
Fumonisins (FBs) produced by Fusarium pathogens, are mycotoxins present in maize and other grains during storage and pose a serious threat to humans and domestic animals worldwide. The Food and Drug Administration stated the maximum levels of total FB1, FB2, and fumonisin B3 (FB3) were 2–4 mg/kg in human food, and the European Union has established a maximum residue limit in human maize of 1 mg/kg FB1 + FB2 (Weidenbörner, 2001). Immunoassays, due to the advantage of high specificity, high efficiency, and low cost, become more and more popular in residue detection. Enzyme-linked immunosorbent assay (ELISA) as the most used immunoassay is a heterogeneous method in solid phase that is limited by incubation and washing steps (Sheng et al, 2012). Much effort needs to be focused on some one-step homogeneous assay without multiple washing steps
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
