Production and Characterization of Monoclonal Antibodies against Norfloxacin

To monitor the abuse of Norfloxacin (NFLX), developing a monoclonal antibody (mAb) based enzyme linked immunosorbent assay (ELISA) kit is our future goal. For this purpose, Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mouse. Noncompetitive and competitive indirect ELISA was employed to screen positive cell clones. Caprylic acid ammonium sulphate (CAAP) method was used to purify NFLX mAb and the Batty saturation method was used to determine the affinity constant (Ka). Finally, six hybridoma cell lines named N1-A2, N2-B3, N2-B5, N3-C6, N4-D5, and N4-D8 were screen out, their corresponding mAbs were of the IgG1 isotype with k light chain, and the Kas of all mAbs were between 3.2×109 and 2.6×1010 L/mol. Based on the square matrix titration, two representative icELISA curves were established. The dynamic range for N3-C6 in assay buffer was from 0.003 to 38 ng/mL, with LOD and IC50 value of 0.002 ng/mL and 0.16 ng/mL, respectively. As to N2-B5, it was from 0.004 to 26 ng/mL, with LOD and IC50 value of 0.002 and 0.22 ng/mL, respectively. Except for a high cross-reactivity (CR) to Pefloxacin (33.6%) and Lomefloxacin (21.8%), this mAb originated from N3-C6 exhibited negligible CR values to other chemicals. Therefore, the established hybridomas put a potential for the development of a rapid test kit and provide an alternative method for the detection of NFLX residues in food producing animals.