Characterization, specific recognition, and the performance in fish matrix of a shark-derived single-domain antibody against enrofloxacin

Abstract

The third generation of genetic engineering antibodies, single-domain antibodies, have been widely reported as potential biomaterials in recognizing small molecular hazards. In this study, a shark-derived single-domain antibody was used as the recognition element for the first time to detect enrofloxacin (ENR), one of the most representative hazards in aquaculture. An ENR-specific clone named 2E6 was isolated by phage display technology. Experimental results proved that 2E6 ssdAb showed high affinity to ENR-PEI complete antigen, with the highest OD450 value of 1.348 in binding ELISA. Through icELISA, it was determined that the IC50 of 2E6 ssdAb to ENR was 19.230 ng/mL, while the IC10 was 0.975 ng/mL, with rare recognition to other fluoroquinolones, which showed high sensitivity and specificity to ENR. The 2E6 ssdAb also performed excellently in fish matrix immunoassay. Results showed that the ENR-negative fish matrix did not seriously interfere with the recognition of 2E6 ssdAb to ENR-OVA, with the matrix index between 4.85% and 11.75%, while the results of icELISA in ENR-spiked fish matrix showed that 2E6 ssdAb could recognize the target ENR in different ENR-spiked concentrations of the fish matrix (10–1000 ng/mL), with the recovery between 89.30% and 126.38% and the RSD between 1.95% and 9.83%. This study broadens the application scenario of shark-derived single-domain antibodies as small molecule recognition biomaterials, providing a new recognition element on ENR detection for immunoassay.