Development of Fluorescence Polarization Immunoassay With scFv to Detect Fumonisin Bs in Maize and Simultaneous Study of Their Molecular Recognition Mechanism

Abstract

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 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-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 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 FB_s and scFvs, which provided a reference for the better understanding of the immunoassay mechanism.