喹乙醇时间分辨荧光免疫分析方法的建立
首发时间:2016-05-17
摘要:本研究是在制备抗喹乙醇单克隆抗体的基础上,建立了一种以Eu3+为探针的直接竞争时间分辨荧光免疫分析(TRFIA)方法。采用金属螯合剂环化二乙烯三胺五醋酸酐(cyDTPA)将Eu3+与OLAmAb偶联,制备铕标抗体。开展TRFIA最佳反应条件的优化实验表明,其包被原最佳浓度为1.0 μg/mL,Eu3+标抗体(Eu3+-OLAmAb)最佳稀释度为1000倍稀释;最佳包被缓冲液为0.05 mol/L,pH为9.6的 CBS;Eu3+-OLA-mAb最佳稀释液为0.01 mol/L,pH为7.0的PBS;OLA标准溶液配制时的最佳有机溶剂为含5%甲醇的PBS。条件优化后进行标准曲线拟合,得到一条S型曲线,其IC10和IC50值分别为0.62 ng/mL 和12.64 ng/mL。对饲料进行添加回收率实验,其回收率处于84.80~114.21%之间,变异系数小于15%。经HPLC验证,TRFIA分析灵敏度高,稳定性良好,应用价值非常高。
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Development of an TRFIA method for Olaquindox based on monoclonal antibody
Abstract:In this paper,a direct competition TRFIA method based on monoclonal antibody to olaquindox and optimized its reaction system were established. The optimal concentration of coating antigen was 1.0 μg/mL, the optimal dilution of Eu3+-OLAmAb was 1000#, the optimum coating buffer was 0.05 M pH9.6 CBS, the best ionic strength of Eu3+-OLAmAb dilutions was 0.01 M and its optimum pH was 7.0, and the preferred organic solvent was "5% methnol-PBS". Finally we got a sigmoid curve, the detection limit for OLA was 0.62 ng/mL and its IC50 was 12.64 ng/mL. The recoveries from feedstuff, pond were all among 84.80~114.21% when OLA were spiked. The coefficient variation were below 15%. The analytical sensitivity of TRFIA was higher than HPLC, so it has a great application value with its good stability.
Keywords: Olaquindox monoclonal antibody TRFIA
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No.4687107973747146****
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