RESUMO
Contamination in food and the environment with fluoroquinolones (FQs) has become a serious threat to the global ecological balance and public health safety. Ofloxacin (OFL) is one of the most widely utilized sterilization agents in FQs. In the process of monitoring OFL, broad-spectrum monoclonal antibodies (mAb) cannot meet the demand for monospecific detection. Here, a computational chemistry-assisted hapten screening strategy was proposed in this study. Differences in the properties of antigenic epitopes were precisely extracted through a comprehensive comparative study of 16 common FQs molecules and a monospecific and ultrasensitive mAb-3B4 for OFL was successfully prepared. The screened fleroxacin (FLE) hapten was applied in a heterologous competition strategy resulting in a 20-fold improvement in the half inhibitory concentration (IC50) of mAb-3B4 to 0.0375 µg L-1 and cross-reacted only with marbofloxacin (MAR) in regulated FQs. In addition, a single-chain variable fragment (scFv) for OFL was constructed for the first time with an IC50 of 0.378 µg L-1. Molecular recognition mechanism studies validated the reliability of this strategy and revealed the key amino acid sites responsible for OFL specificity and sensitivity. Finally, ic-ELISA and GICA were established for OFL in real samples. This work provides new ideas for the preparation of monospecific mAb and improves the monitoring system of FQs.