Abstract: 【Objective】The goals of this study were to obtain immunogen and coating antigen, generate its mice polyclonal antiserum and develop FLE-Kit by its competitive indirect enzyme-linked immunosorbent assay (ciELISA).【Method】Artificial antigen BSA-FLE and OVA-FLE were synthesized using DCC and the mixed anhydride reaction methods by linking carrier proteins BSA and OVA to FLE. The antigens BSA-FLE and OVA-FLE were identified by ultraviolet scanning and SDS-PAGE, then five female white rats were subcutaneously immunized with the immunogen Fle-BSA at multiple sites in the back. The initial immunization was subcutaneously injected with 189 μL of conjugate in 311 μL of PBS (0.01 mol•L-1, pH 7.4) and 0.5 mL of Freund’s complete adjuvant. The rest of five booster immunizations were conducted by injecting 189 μL of FLE-BSA in 311 μL of PBS (0.01 mol•L-1, pH 7.4) and 0.5 mL of iFA at 20-day intervals. After obtained mice polyclonal antiserum, the ciELISA method and rapid detection kit of FLE were developed. The kit was compared with HPLC method to ensure its quality.【Result】The ultraviolet scanning and SDS-PAGE showed that FLE artificial antigen was synthesized successfully. Five BALB/c mice indirect ELISA titer against FLE were all above 2.5×104 and the IC50 of No.4 mice was the lowest (162.18 ng•mL-1). After optimized the ELISA method, the icELISA revealed that the optimal concentration of mice serum was 1:6400, the optimal concentration of coated antigen was 5 µg•mL-1, and the optimal concentration of sheep anti-rabbit IgG was 1:1000. The regression equation was Y =-0.3627x + 1.3517（R2 = 0.9956), the lowest detection limit of the kit was 16.22 ng•mL-1, the assay measured drug residue in pork liver spiked with FLE with coefficient of variation between 9.34%-10.7%, and the average recovery rates between 67.5%-87.9%, respectively. The assay measured drug residue in pork meats spiked with FLE with coefficient of variation between 8.3%-10.4%, the average recovery rates were between 74%-88.2%. Good agreement of the results obtained by ELISA and high performance liquid chromatography (HPLC) further confirmed the reliability and accuracy of the icELISA for rapid detection of SPFX in pork meats. The coefficients of variation of intra-assay and inter-assay were 5.07% and 7.44% during five standard concentrations. The antiserum of FLE had no cross reactivity with other competitors.【Conclusion】The FLE polyclonal antiserum has been generated. The icELISA method and rapid detection kit were developed for the detection of FLE residues with the characters of sensitivity, accuracy, convenience and rapid in this study and they laid a foundation for establishing immunoassay of FLE residues.

Key words: fleroxacin , artificial antigen , polyclonal antiserum , ELISA , kit