Production and Identification of Monoclonal Antibody Against Flumequine and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay

Abstract

The hapten of Flumequine (FLU) with four carbon atoms spacer arm (FLUABA) was synthesized and coupled to bovine serum albumin (BSA) for immunogen by the activated ester method. BALB/C mice were immunized with the artificial immunogen and the splenocytes of immunized mice were fused with SP2/0 cells to obtain the monoclonal antibody (McAb). A hybridoma cell line (named DB6-E7) secreting anti-FLU McAbs was obtained by limited dilution method and screened by heterologous enzyme-linked immunosorbent assay (ELISA). The results showed that the subtype of the obtained McAb was IgG1, and the affinity was about 8.19 × 108 M−1. The haptens of FLU, FLUABA, and FLUACA with different space arms were linked to ovalbumin (OVA) for heterologous or homologous coating antigens. The results of indirect ELISA and indirect competitive ELISA indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. Using FLU-OVA as coating antigen, the ELISA showed an IC50 value of 26.33 μg L−1, a limit of detection (LOD) of 4.0 μg L−1, and the workable range of 8–114 μg L−1 (IC20–IC80). Cross-reactivity studies showed that the McAbs were quiet specific for FLU, no cross-reactivity (< 0.1%) was detected between the obtained McAbs and the several major quinolones compounds or other structural analogs. The developed ELISA can satisfy the detection criteria of FLU in animal food-products.