Abstract: Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method was employed to synthesize the artificial antigen of enrofloxacin (ENR), and New Zealand rabbits were used to produce anti-ENR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (ELISA) standard curve was established. This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg (R²=0.9567), with the half maximal inhibitory concentration (IC₅₀) and limit of detection (LOD) values of 9.4 μg/kg and 0.2 μg/kg, respectively. Of all the competitive analogues, the produced pAb exhibited a high cross-reactivity to ciprofloxacin (CIP) (87%), the main metabolite of ENR in tissues. After optimization, the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork. The overall recoveries and coefficients of variation (CVs) were in the ranges of 86%–109% and 6.8%–13.1%, respectively. It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.

[1]Ahmad, B., Parveen, S., Khan, R.H., 2006. Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site. Biomacromolecules, 7(4):1350-1356.

[2]Bucknall, S., Silverlight, J., Coldham, N., Thorne, L., Jackman, R., 2003. Antibodies to the quinolones and fluoroquinolones for the development of generic and specific immunoassays for detection of these residues in animal products. Food Addit. Contam., 20(3):221-228.

[3]Christodoulou, E.A., Samanidou, V.F., Papadoyannis, I.N., 2008. Development of an HPLC multi-residue method for the determination of ten quinolones in bovine liver and porcine kidney according to the European Union Decision 2002/657/EC. J. Sep. Sci., 31(1):119-127.

[4]Delepine, B., Hurtaud-Pessel, D., Sanders, P., 1998. Simultaneous determination of six quinolones in pig muscle by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Analyst, 123(12):2743-2747.

[5]Dufresne, G., Fouquet, A., Forsyth, D., Tittlemier, S.A., 2007. Multiresidue determination of quinolone and fluoroquinolone antibiotics in fish and shrimp by liquid chromatography/tandem mass spectrometry. J. AOAC Int., 90(2):604-612.

[6]Hassouan, M.K., Ballesteros, O., Zafra, A., Vílchez, J.L., Navalón, A., 2007. Multiresidue method for simultaneous determination of quinolone antibacterials in pig kidney samples by liquid chromatography with fluorescence detection. J. Chromatogr. B, 859(2):282-288.

[7]Hermo, M.P., Nemutlu, E., Kir, S., Barrón, D., Barbosa, J., 2008. Improved determination of quinolones in milk at their MRL levels using LC-UV, LC-FD, LC-MS and LC-MS/MS and validation in line with regulation 2002/657/EC. Anal. Chim. Acta, 613(1):98-107.

[8]Huang, B., Yin, Y., Lu, L., Ding, H., Wang, L., Yu, T., Zhu, J.J., Zheng, X.D., Zhang, Y.Z., 2010. Preparation of high-affinity rabbit monoclonal antibodies for ciprofloxacin and development of an indirect competitive ELISA for residues in milk. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 11(10):812-818.

[9]Huet, A.C., Charlier, C., Tittlemier, S.A., Singh, G., Benrejeb, S., Delahaut, P., 2006. Simultaneous determination of (fluoro)quinolone antibiotics in kidney, marine products, eggs, and muscle by enzyme-linked immunosorbent assay (ELISA). J. Agric. Food Chem., 54(8):2822-2827.

[10]Liu, Y., Zhang, C.Z., Yu, X.Y., Zhang, Z.Y., Zhang, X., Liu, R.R., Liu, X.J., Gong, Z.M., 2007. Development and evaluation of immunoassay for zeranol in bovine urine. J. Zhejiang Univ.-Sci. B, 8(12):900-905.

[11]Lu, S., Zhang, Y., Liu, J., Zhao, C., Liu, W., Xi, R., 2006. Preparation of anti-pefloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of pefloxacin residue in chicken liver. J. Agric. Food Chem., 54(19):6995-7000.

[12]San Martín, B., Cornejo, J., Iragüen, D., Hidalgo, H., Anadón, A., 2007. Depletion study of enrofloxacin and its metabolite ciprofloxacin in edible tissues and feathers of white leghorn hens by liquid chromatography coupled with tandem mass spectrometry. J. Food Prot., 70(8):1952-1957.

[13]Tong, C., Zhuo, X., Liu, W., Wu, J., 2010. Synchronous fluorescence measurement of enrofloxacin in the pharmaceutical formulation and its residue in milks based on the yttrium (III)-perturbed luminescence. Talanta, 82(5):1858-1863.

[14]Volmer, D.A., Mansoori, B., Locke, S.J., 1997. Study of 4-quinolone antibiotics in biological samples by short-column liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Anal. Chem., 69(20):4143-4155.

[15]Wang, L., Zhang, Y., Gao, X., Duan, Z., Wang, S., 2010. Determination of chloramphenicol residues in milk by enzyme-linked immunosorbent assay: improvement by biotin-streptavidin-amplified system. J. Agric. Food Chem., 58(6):3265-3270.

[16]Wang, Z., Zhu, Y., Ding, S., He, F., Beier, R.C., Li, J., Jiang, H., Feng, C., Wan, Y., Zhang, S., et al., 2007. Development of a monoclonal antibody-based broad-specificity ELISA for fluoroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds. Anal. Chem., 79(12):4471-4483.

[17]Wu, J.X., Zhang, S.E., Zhou, X.P., 2010. Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 11(1):52-60.

[18]Yan, H., Wang, H., Qin, X., Liu, B., Du, J., 2011. Ultrasound-assisted dispersive liquid-liquid microextraction for determination of fluoroquinolones in pharmaceutical wastewater. J. Pharm. Biomed. Anal., 54(1):53-57.

[19]Yang, G., Lin, B., Zeng, Z., Chen, Z., Huang, X., 2005. Multiresidue determination of eleven quinolones in milk by liquid chromatography with fluorescence detection. J. AOAC Int., 88(6):1688-1694.

[20]Yorke, J.C., Froc, P., 2000. Quantification of nine quinolones in chicken tissues by HPLC with fluorescence detection. J. Chromatogr. A, 882(1-2):63-77.

[21]Zhao, S., Jiang, H., Li, X., Mi, T., Li, C., Shen, J., 2007. Simultaneous determination of trace levels of 10 quinolones in swine, chicken, and shrimp muscle tissues using HPLC with programmable fluorescence detection. J. Agric. Food Chem., 55(10):3829-3834.

[22]Zhu, Y., Li, L., Wang, Z., Chen, Y., Zhao, Z., Zhu, L., Wu, X., Wan, Y., He, F., Shen, J., 2008. Development of an immunochromatography strip for the rapid detection of 12 fluoroquinolones in chicken muscle and liver. J. Agric. Food Chem., 56(14):5469-5474.