Production and characterization of monoclonal antibodies against small hapten-ciprofloxacin

Abstract

High affinity and specificity monoclonal antibody against small hapten-ciprofloxaicn (CPFX) was produced and was used for rapid CPFX immunoassay in food stuffs of animal origin. Firstly, two kinds of antigens for CPFX were made using carrier proteins, bovine serum albumin (BSA) and ovalbumin (OVA), by a modified carbodiimide method and then the hapten-protein conjugates were characterized by ultraviolet spectrophotometry to detect hapten density before being used for the immunization and detection purposes. The production of monoclonal antibodies (Mabs) was sought following the generation of appropriate CPFX-BSA conjugate. Spleen cells from Balb/c mice immunized with CPFXBSA conjugate were fused with SP2/0 myeloma cells, and hybridomas secreting antibodies against CPFX were selected and cloned. One Mab against CPFX was produced and the average affinity of the Mab was 2.88×10⁹ L/mol. The number of the hybridoma chromosome was 90 to 105. Except for enrofloxacin, the Mab had not any cross-reactivity with other fluoroquinolones. In the optimized ELISA, the Mab gave a 50% inhibition of 7.77 ng/ml with a detection limit of 1.56 ng/ml.

Key words: Ciprofloxacin, hapten, antibody, immunoassay.