纳米抗体(nanobody,Nb)具有稳定性高、特异性强、易于表达等优势,已经成为小分子免疫分析的重要工具。为了监测乳制品中黄曲霉毒素M1(aflatoxin M1,AFM1)的污染情况,以先前从双峰驼AFM1免疫文库中淘选得到的抗AFM1型纳米抗体M6为研究对象,评估其理化性质并建立间接竞争酶联免疫吸附测定法(indirect competitive enzyme-linked immunosorbent assay,icELISA)。优化反应体系中封闭方法、甲醇溶液、pH值和离子强度等参数后,建立竞争抑制标准曲线,并进行方法学验证特异性和准确性。结果表明,Nb-M6具有良好的亲和力和热稳定性;Nb-icELISA的检测限(limit of detection,LOD)为0.111 ng/mL,半数抑制浓度(half maximal inhibitory concentration,IC50)为1.498 ng/mL,乳制品加标回收率为89.8%~104.1%,与高效液相色谱法的检测结果趋于一致。该方法操作简单、便捷,可应用于乳制品中AFM1的初步批量筛查。
Nanobody has become an important tool for immunoassays because of its stability, specificity, and ease of expression.To monitor the aflatoxin M1 in dairy products, the anti-AFM1 nanobody, named M6, selected from the Bactrian camel AFM1 immune library, evaluated its properties, and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA).After optimizing the parameters, including the blocking method, methanol concentrations, pH values, and ionic strength in the reaction system, a standard competitive inhibition curve was established, and methodological specificity and accuracy was verified.Results showed that Nb-M6 had a good affinity and thermal stability, the limit of detection (LOD) of Nb-icELISA was 0.111 ng/mL, and the half maximal inhibitory concentration (IC50) was 1.498 ng/mL.The recoveries of dairy products spiked were 89.8%-104.1%, which were in agreement with the high-performance liquid chromatography (HPLC).The Nb-icELISA is simple, convenient and can be applied to batch screening of AFM1 in dairy products.
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