Development of Fluorescence Polarization Immunoassay With scFv to Detect Fumonisin B(s) in Maize and Simultaneous Study of Their Molecular Recognition Mechanism

In this study, a fluorescence polarization immunoassay (FPIA) was developed based on the single-chain variable fragments (scFvs) for fumonisin B(s) (FB(s)). The scFvs were prepared from FB(s)-specific monoclonal antibody secreting hybridomas (4F5 and 4B9). The established FPIA could determine the sum of fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) within a short time. The IC(50) of FPIA for the detection of FB(1) and FB(2) 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 FB(1) and FB(2) 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-FB(s) 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 FB(1)/FB(2) could be inserted into this binding cavity stably through strong hydrogen bonds and other interactions. While in 4F5 scFv, only the FB(1) stably inserted in the binding pocket formed by four CDRs through strong hydrogen bonds, and FB(2) 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 FB(2)-4B9 scFv complex is higher than the FB(2)-4F5 scFv complex. This study established a FPIA method with scFv for the detection of FB(1) and FB(1) in maize, and systematically predicted recognition mechanism of FB(s) and scFvs, which provided a reference for the better understanding of the immunoassay mechanism.