Heterologous Expression, Characterization, and Fish Matrix Performance of a Shark-Derived Single-Domain Antibody Fusion Protein against Enrofloxacin

Abstract

The abuse of enrofloxacin (ENR) in aquaculture and the lack of monitoring of other metabolites may lead to unknown harmful effects on human health. In this study, a shark-derived single-domain antibody fusion protein 2E6-SUMO, which was specific to ENR, was heterologously expressed. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using 2E6-SUMO as the recognition element was established to detect ENR in fish samples. The results showed that the 2E6-SUMO fusion protein has good thermal performance compared to traditional IgG antibodies. The half-maximal inhibitory concentration (IC50) of 2E6-SUMO to ENR was determined as 42.08 ng/mL, with a detection limit of 3.84 ng/mL and a linear range (IC20 − 80) of 9.94–376.17 ng/mL, respectively. In our experiments, the ENR-negative fish matrix did not seriously interfere with the recognition of the 2E6-SUMO to ENR, with the matrix index between 3.31% and 6.85%. The recovery experiment indicated that the recovery rates of spiked samples ranged between 83.33% and 123.06%. Our study broadens the application scenario of shark-derived single-domain antibodies, providing new immunoassay method for detecting ENR.