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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Document Type: Research Article
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, P.R. China,Xinyang Agricultural College, Xinyang, P.R. China
National Reference Laboratory of Veterinary Drug Residues, Huazhong Agricultural University, P.R. China
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, P.R. China
June 1, 2009
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