Performance difference of enrofloxacin monoclonal antibody and nanobody elicited from the same immunogen and the underlying molecular mechanism

Enrofloxacin (ENR) is a widely used fluoroquinolone antibiotic in animal husbandry, aquaculture, and humans. Here, a monoclonal antibody (mAb 2D3) and a nanobody (Nb22) against ENR were generated with the same immunogen. Nb22 had the nanobody common property of greater stability in harsh conditions, but its assay sensitivity was approximately 30-fold lower than that of mAb 2D3. Nb22 showed better selectivity, which the cross-reactivity to each of ENR analogs was less than or equal to that of mAb 2D3. The VH and VL gene sequences were amplified from the hybridoma cell line 2D3. Molecular docking revealed that mAb 2D3 had stronger hydrogen bonds and formed a flat and wide binding pocket to accommodate other analogs of ENR. The average recoveries of ENR from milk, milk powder, egg and fish determined by mAb 2D3 and Nb22 based ic-ELISAs ranged from 77.7% to 119% and 88.2% to 116%, respectively. This study confirmed that the direct application of nanobody in immunoassay is no better than the conventional monoclonal antibody. Improving the sensitivity of nanobody is an essential prerequisite for taking advantage of its stability and specificity.