Single-Cell-Derived Rabbit mAb-Based Immunoassay for Simultaneous Detection of Florfenicol and Florfenicol Amine

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Previously, we speculated that rabbits could produce more diverse and higher-affinity antibodies for small molecules due to their specific, unique antibody structure and antibody evolution mechanism. Here, using a single antibody-secreting cell (ASC) precise localization and sorting strategy, we discovered five rabbit monoclonal antibodies (RmAbs) with distinct recognition profiles to florfenicol (FF) and FF amine (FFA). Among them, RmAb-A4# exhibited superior performance, featuring high-affinity dual recognition of FF and FFA, with IC50 values of 0.07 ng/mL and 0.16 ng/mL in ELISA, demonstrating approximately 40-fold and 20-fold greater affinity compared to the best mouse monoclonal antibody (MmAb) 4F8 we previously reported, surpassing the reported antibodies derived from the mouse by 2–463-fold. As revealed by molecular docking and dynamic simulations, this enhancement is attributed to a more open binding pocket of RmAb-A4# and more hydrogen bonding interactions with FF and FFA. More importantly, RmAb-A4# displayed remarkable halotolerance (18–5000 mM NaCl) and enhanced pH stability (pH 4–9) in comparison to MmAb-4F8. Based on RmAb-A4#, a lateral flow immunoassay enabling rapid detection of FF and FFA was developed without pretreatment for milk and with a 3-fold dilution for eggs. The semiquantitative limit of detection reached 0.22 μg/L (FF) and 0.17 μg/L (FFA) in milk and 0.74 μg/kg (FF) and 0.78 μg/kg (FFA) in eggs. This work adopted a practical single ASC sorting strategy, discovered RmAb to small molecule, and validated its applicability for on-site sensitive immunoassay detection.