Abstract
In order to develop a sensitive immunoassay for Aflatoxin B1 (AFB1) monitoring, a hybridoma secreting anti-AFB1 monoclonal antibody with high binding affinity was screened. A new type of CdTe/CdS/ZnS quantum dot was synthesized and conjugated with an artificial antigen for use as a fluorescent probe in a simple one-step fluorescence immunoassay (FLISA). The developed FLISA allowed a sensitive determination of AFB1 in cereal samples in a wide linear range, from 0.08 to 1.97 ng mL−1, with a detection limit of 0.01 ng mL−1 in cereal samples. The corresponding immunoglobulin genes of the Fab fragment were cloned and sequenced, and expression of Fab was successfully verified in HEK293 cells, with a KD value of 1.09 × 10−7 mol L−1 for AFB1. To investigate the interactions between the antibody and AFB1, molecular docking, molecular dynamic simulation, and quantum-chemical computation were performed on AFB1 and a homology model of the Fab fragment. Our results showed that residues Ser32, Trp93, and Trp98 played the most important roles in the binding through hydrogen bonds formation, Pi-Pi stacked/Pi-alkyl interactions, and van der Waals interactions. In addition, the electrostatic potential study of AFB1 demonstrated that electrostatic interactions also played an important role in the recognition process. Results from theoretical studies provide guidance for hapten design and antibody improvement through genetic engineering.
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