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Simultaneous detection of tylosin and tilmicosin in honey using a novel immunoassay and immunochromatographic strip based on an innovative hapten

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Tylosin (TYL) and tilmicosin (TIM) are macrolide antibiotics, and maximum residue limits (MRLs) have been set to control their illegal usage in food. We developed a sensitive indirect competitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip (ICS) test to simultaneously detect TYL and TIM based on an innovative hapten (TYL-carboxymethoxylamine hemihydrochloride). The monoclonal antibody 2B3 was obtained with the isotype IgG1, and TYL and TIM had half maximal inhibitory concentrations of 0.57 and 2.10 ng/ml, respectively. The cross-reactivity values were 100%, 27.57%, 97.43%, and 62.42% for TYL, TIM, desmycosin, and acetylisovaleryl tylosin, respectively. By optimizing, the standard dilution buffer was based on 0.8% NaCl, pH 7.4 and 5% acetonitrile. The recoveries ranged from 89.37% to 112.00% in the honey samples, which suggests that the assay would be reliable in the analysis of real honey samples. In addition, an ICS was developed for qualitative, semi-quantitative, and spot detection analysis, with cut-off values of 5 and 15 ng/ml for TYL and TIM in phosphate-buffered saline, and 10 and 20 ng/ml for TYL and TIM in honey, respectively. The results show that these assays can be used to analyze real samples with the strictest MRL being 50 ng/ml for both TYL and TIM.
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Keywords: ELISA; immunochromatographic strip; monoclonal antibody; tilmicosin; tylosin

Document Type: Research Article

Affiliations: State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, People's Republic of China

Publication date: May 3, 2016

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