Abstract

Low specificity and sensitivity often restrict the fluoroquinolones (FQs) detection when traditional IgG antibodies are used as recognition elements in immunoassay. Herein, shark-derived single-domain antibodies (ssdAbs) were used as new recognition elements to enhance the sensitivity and specificity for norfloxacin detection in immunoassay. Firstly, NOR-specific clones were isolated by phage display technology. Finally, five clones were successfully isolated from a NOR-immunized library. Secondly, a soluble expression system was constructed to produce ssdAbs. The 1N9 ssdAb exhibits the best affinity to the NOR complete antigen, with a limit of detection of 0.392 ng/mL and the cross-reaction rates of less than 8.240%, indicating its high sensitivity and specificity as a new recognition element in NOR detection. More importantly, the ELISA result proved that in NOR-negative fish matrix, no serious interference was observed, the 1N9 ssdAb could still recognize complete antigen NOR-OVA effectively. While in icELISA, 1N9 ssdAb could recognize NOR in different NOR-spiked concentrations between 10 to 1000 ng/mL, with the recovery rates between 93.66 - 116.49% with high reproducibility. Our study provides a new and effective recognition element with great potential for NOR detection.