Monoclonal antibody based enzyme-linked immunosorbent assay for the specific detection of ciprofloxacin and enrofloxacin residues in fishery products

Ciprofloxacin (CPFX) residues in fishery products were dangerous for human health and still remain a big challenge in China. However, there are a few available assays to detect specifically CPFX residues in fishery products. This study aimed at generating CPFX-specific monoclonal antibodies (mAbs) and developing a competitive indirect enzyme-linked immunosorbent assay (ci-ELISA) for the sensitive and specific detection of CPFX residues in fishery products. Firstly, the CPFX was conjugated with BSA and the conjugates were immunized to Balb/c mice. After characterizing anti-CPFX sera, we generated hybridoma cell lines and found that one hybridoma clone, 1C9, produced anti-CPFX mAb. Characterization of IC9 mAb revealed that it belonged to IgG2a, with an affinity of 3.75 × 10 10 mol/l for CPFX. Furthermore, we developed ic-ELISA using 150 ng/ml of CPFX-OVA for coating antigen and 0.22 μg of mAb. We found that this assay detected CPFX at 45.25 ng/ml and had 84.60% of cross-activity to enrofloxacin (ENR), but not to ofloxacin, levofloxacin, difloxacin, sarafloxacin, furacilin, chloramphenicol and nalachite green. Using this assay, we detected the CPFX in fishery products prepared with recovery rates from 80.57% to 94.61% and relative standard deviation (RSD) varying from 1.78% to 8.02%, similar to that by HPLC analysis. These data, together the protocol for the preparation of fishery products, indicate that the ic-ELISA is able to detect CPFX and ENR residues in fishery products sensitively and specifically within 2 h. Therefore, this assay can be used for monitoring CPFX and ENR in commercial fishery products, improving the safety of commercial meats in China.