Studies on Preparation and Characteristics of Monoclonal AntibodiesAgainst Enrofloxacin and Cross-Reactivity of Related Fluoroquinolones

An ester activation method was employed to couple enrofloxacin(ENFX) to the carrierproteins BSA and OVA. The conjugates ENFX-BSA and ENFX-OVA were identified with an UVspectrophotometer and amino acid automation analysis instrument, and resulted in conjugateswith 48 ENFX molecules per carrier molecule(BSA). Splenocytes from mice immunized withENFX-BSA were fused with SP2/0 myeloma cells and hybridomas secreting antibodies againstenrofloxacin were selected and cloned. Two stable monoclonal antibodies, 2C5, 5D5 of thesubclass IgG2a, were isolated. Using antibody 5D5, an indirect competitive inhibitionenzyme-linked immunosorbent assay (Ci-ELISA) was developed for the quantitative detectionof enrofloxacin and its metabolites. The IC50 of the standard curve was 21.67 ng mL^(-1) andthe limit of detection for enrofloxacin was 0.13 ng mL^(-1). This method was sensitive and hada linear range from 0.13 to I0 000ngmL^(-1) (r=-0.9782). Monoclonal antibody 5D5 exhibitedhigh relative affinity to enrofloxacin, and the cross-reactivities with ciprofloxacin,marbofloxacin, sarafloxacin and danorfloxacin were 110.8, 27.40, 71.05 and 37.41%,respectively. Three non-fluoroquinolones of cefadroxil, chloramphenicol, sulfadimethoxinewere tested and there was no cross-reaction between them.