| Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 90 === An useful、simple and convenient ELISA with a sensitivity of less than 10 μg/kg was used for the detection of cloxacillin in milk samples. The labeled antigen was produced by combining cloxacillin with horseradish peroxidase（HRP）, while the anti-cloxacillin antibody was obtained from Biogenesis®. Checkerboard method was used to determine the optimal concentrations of antibody and enzyme conjugate. The working antibodies were diluted into 1600x, and the same with enzyme conjugates. Standard curves were obtained by the competitive immunoassay in PBS and in milk, respectively, and both the detectable ranges were from 5 to 1000 ng/ml. The detection limit in PBS and in milk was 3.16 ng/ml and 5.68 ng/ml, respectively. The cross-reactivity of oxacillin and dicloxacillin in this method was 22.6% and 55.3%, respectively, while it was less than 1% with other β-lactam antibiotics. Intra- and inter-assay variations in PBS and in milk were less than 10 %. Intramammary infusion of cloxacillin and ampicillin was given triply into the infected quarter in a cow with mastitis at 12 hours interval. The maximal concentration of cloxacillin in milk and in serum continued until 2 hours after treatment with 366,944 ng/ml and 55.74 ng/ml, respectively. Cloxacillin residue in milk and in serum was detectable for 10 days and 12 hours, respectively. The result of detecting all the different market milk showed that the cloxacillin residue was lower than 10 ng/ml. The ELISA kit used in this study was successfully applied to analyze cloxacillin in milk, and was not interfered by other antibiotics. Therefore, it needs a further study to produce the antibody of cloxacillin to complete this ELISA kit.
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