基于纳米抗体建立icELISA检测乳制品中黄曲霉毒素M1  

Nanobody-based icELISA for aflatoxin M1 in dairy products

作　　者：刘海媛 刘颖达 Tuyatsetseg Jambal 吉日木图[1,4] 伊丽 LIU Haiyuan;LIU Yingda;Tuyatsetseg Jambal;JI Rimutu;YI Li(Key Laboratory of Dairy Biotechnology and Bioengineering,Ministry of Education,Inner Mongolia Agriculture University,Hohhot 010018,China;China-Mongolia Joint Laboratory for Biomacromolecule Research,Ulaanbaatar 999097,Mongolia;College of Industrial Technology,Mongolian University of Science and Technology,Ulaanbaatar 999097,Mongolia;Inner Mongolia China-Kazakhstan Camel Research Institute,Alxa 737300,China)

机构地区：[1]内蒙古农业大学,乳品生物技术与工程教育部重点实验室,内蒙古呼和浩特010018 [2]中-蒙生物高分子应用研究联合实验室,蒙古国乌兰巴托999097 [3]蒙古国立科技大学轻工业学院,蒙古国乌兰巴托999097 [4]内蒙古中哈骆驼研究院,内蒙古阿拉善737300

出　　处：《食品与发酵工业》2024年第12期46-52,62,共8页Food and Fermentation Industries

基　　金：内蒙古农业大学食品科学与工程学院科技计划项目(SPKJ201913);内蒙古自治区自然科学基金(2020BS03011);国家重点研发计划项目(2020YFE0203300);内蒙古自治区科技成果转化专项资金项目(2021CG0021)。

摘　　要：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,IC_(50))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(IC_(50))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.

关 键 词：纳米抗体 间接竞争酶联免疫吸附测定法 黄曲霉毒素M1 乳制品 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]