Generation of broad-spectrum recombinant antibody and construction of colorimetric immunoassay for tropane alkaloids: Recognition mechanism and application

Abstract

Tropane alkaloids (TAs) have emerged as plant toxins, related to poisoning events. The development of stable antibodies is crucial to ensure the effectiveness of immunological methods in quickly and accurately monitoring these alkaloids. In this study, based on hybridoma, the variable region gene of monoclonal antibody (mAb) was amplified, and the recombinant antibody (rAb) gene sequence (VH-Linker-VL) was successfully constructed and expressed in HEK293F. The obtained rAb has kept the same performance as mAb, and the IC50 of 29 TAs ranged from 0.12 to 2642.78 ng/mL. In the recognition mechanism, the docking and dynamics model identified hydrophobic interaction as the most critical force. Substituent will impact recognition by influencing the spatial structure and hydrophobic properties. Then, a colorimetric immunoassay based on rAb was established, five types of water and thirty-nine nectars of honey were tested. The results demonstrated the absence of TAs in environmental water, whereas atropine was detected in more than 13.47% of honey samples at concentrations exceeding 1 μg/kg. The results show a good correlation with UHPLC-MS/MS, suggesting that the immunoassay has excellent screening ability. The data on TAs in honey and water could serve as a foundation for developing relevant policies.