Development and Validation of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Screening of Tylosin and Tilmicosin in Muscle, Liver, Milk, Honey and Eggs

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• 作者：Dapeng Peng ; Shengqiang Ye ; Yulian Wang ; Dongmei Chen ; Yanfei Tao ; Lingli Huang ; Zhenli Liu ; Menghong Dai ; Xiaoqing Wang ; Zonghui Yuan
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2012
• 出版时间：January 11, 2012
• 年：2012
• 卷：60
• 期：1
• 页码：44-51
• 全文大小：251K
• 年卷期：v.60,no.1(January 11, 2012)
• ISSN：1520-5118

文摘

Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 渭g L^鈥?, with an IC₅₀ value of 6.1 渭g L^鈥? and 12.1 渭g L^鈥?, respectively. The limits of detection of the ic-ELISA ranged from 5.1 渭g kg^鈥? to 13.8 渭g kg^鈥? in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0鈥?00.0 渭g kg^鈥?. Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.

Keywords:

tylosin; tilmicosin; monoclonal antibody; indirect competitive enzyme-linked immunosorbent assay; muscle; liver; honey; egg; milk