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Development of an indirect competitive ELISA for the detection of doxycycline residue in animal edible tissues

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A synthetic hapten doxycycline (DOX) with a spacer-arm (para-aminobenzoic acid (PABA)) was attached to bovine serum albumin (BSA) or ovalbumin (OVA) by the diazonium coupling reaction and mixed anhydride methods. Then, DOX-PABA-OVA conjugate was used as a coating antigen in enzyme-linked immunosorbent assay (ELISA), while DOX-PABA-BSA was used as an immunogen to produce polyclonal antibodies. A reliable and sensitive indirect competitive enzyme-linked immunosorbent assay was developed and applied to the quantitative determination of DOX residue in muscle and liver samples. After the optimisation of the main parameters, 50% inhibition was 8.74 µg/l and the limit of detection was 1.96 µg/l. A weak cross-reactivity (CR) was also observed with other structurally related compounds, such as oxytetracycline (10.71%), tetracycline (4.10%) and chlortetracycline (1.89%). The CRs with other antibiotics were all below 0.1%. With the ELISA method, the recoveries were demonstrated to be from 80.19 to 89.41% in liver samples and 83.98-94.75% in muscle samples. The mean of the coefficients of variation with the intra-assay test were 5.75 and 7.53% in liver and muscle samples, respectively. For the inter-assay test, the average of the coefficients of variation was 5.92% in liver and 7.21% in muscle samples. The ELISA method is accurate and reliable for the detection of DOX residue in edible foods of animal origins.
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Keywords: ELISA; doxycycline; hapten synthesis; polyclonal antibody

Document Type: Research Article

Affiliations: 1: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, P.R. China,Xinyang Agricultural College, Xinyang, P.R. China 2: National Reference Laboratory of Veterinary Drug Residues, Huazhong Agricultural University, P.R. China 3: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, P.R. China

Publication date: June 1, 2009

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