The above research provided us new information to better understand the FBs-antibody acknowledgement mechanism, which will lead future design and antibody improvement. Conclusion In this study, scfvs were successfully constructed based on 4F5 and 4B9 hybridoma cells. that six complementarity-determining regions (CDRs) were involved in 4B9 scFv acknowledgement, forming a thin binding cavity, and FB1/FB2 could be inserted into this binding Harringtonin cavity stably through Harringtonin strong hydrogen bonds and other interactions. While in 4F5 scFv, only the FB1 stably inserted in the binding pocket created 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 important acknowledgement 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 acknowledgement mechanism of FBs and scFvs, which provided a reference for the better understanding of the immunoassay mechanism. pathogens, are mycotoxins present in maize and other grains during storage and pose a serious threat to humans and domestic animals worldwide. FBs consist of different chemotypes, and fumonisin B1 (FB1) and fumonisin B2 (FB2) are believed to be the most prevalent and harmful in naturally contaminated cereals (Weidenb?rner, 2001). Numerous guidance or regulations for FBs have been enforced to protect public health. The Food and Drug Administration stated the maximum levels of total FB1, FB2, and fumonisin B3 (FB3) were 2C4?mg/kg in human food, and the European Union has established a maximum residue limit in human maize of 1 1?mg/kg FB1 + FB2 (Weidenb?rner, 2001). Many methods have been developed for FBs detection, including the golden method of mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) for the simultaneous multiple mycotoxin analysis (Songsermsakul and Razzazi-Fazeli, 2008). 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 usually a heterogeneous method in solid phase that is limited by incubation and washing actions (Sheng et al., 2012). Thus, much effort needs to be focused on some one-step homogeneous assay without multiple washing actions. Fluorescence polarization immunoassay (FPIA) is usually a homogeneous assay and has been applied to detect numerous mycotoxins, including aflatoxins (AFs), FBs, deoxynivalenol (DON), T-2 toxin, ochratoxin A (OTA), and zearalenone (ZEA) (Zhang et al., 2017). Li Rabbit polyclonal to AIRE et al. revealed a limit of detection (LOD) of 157.4?g/kg for FB1 and 290.6?g/kg for FB2 in the optimized FPIA with the pair of FB1-FITC and 4B9 traditional monoclonal antibody (mAb) (Li et al., 2015). However, these studies are based on mAbs whose production usually takes Harringtonin several months. With the development of recombinant antibody expression technology, an increasing number of studies have been developed based on the recombinant antibodies due to their simple preparation process and without sacrifice of animals. Single-chain variable fragments (scFvs), as one of the most standard recombinant antibodies, contain the antibody variable regions of the light chain (VH) and variable regions of heavy chain (VL) and are usually linked by a small, flexible peptide chain. ScFv can be generated by many techniques including ribosome display technology (Mahalakshmi et al., 2019), phage display technology (Hu et al., 2016), and also directly cloned from your obtained hybridoma.