摘要
酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)具有高通量、操作简单、检测结果明显等优点,是一种较为理想的检测食品中真菌污染物的免疫分析技术。然而,传统的ELISA方法具有较大的局限性,比如使用时抗体易受温度影响,容易变性,贮存期短,易受到检测环境中其他物质干扰而造成假阳性或假阴性等。该实验使用实验室前期获得的抗黄曲霉毒素M_(1)(aflatoxin M_(1),AFM_(1))纳米抗体(nanobody-M4,Nb-M4)与C4结合蛋白(recombinant C4 binding protein alpha,C4BPa)C端片段融合形成自组装七聚体融合蛋白(Nb-M4-C4 bpα),并基于该七聚体开发应用于乳制品中AFM_(1)的间接竞争酶联免疫吸附法(indirect competitive enzyme-linked immunosorbent assay,icELISA)。在最佳实验参数下,AFM_(1)的IC_(50)为0.038 ng/mL,检测限为0.009 ng/mL,较单体Nb-M4的提高了8.63倍和5.56倍。与AFM_(1)类似物和其他常见真菌毒素的交叉反应可忽略不计,且在加标乳样中获得了良好的回收率和可重复性。此外,将所开发的方法应用于实际样品中AFM_(1)的分析,结果与HPLC的结果具有很好的相关性(R^(2)=0.995)。该研究表明,自组装的七聚化纳米抗体是改善亲和力和信号放大的有效策略,今后可用于灵敏、选择性和快速检测食品中的霉菌毒素和其他小分子污染物。
Enzyme-linked immunosorbent assay(ELISA)has the advantages of high throughput,simple operation and obvious results,which makes it a more ideal immunoassay technique for detecting fungal contaminants in food.However,traditional ELISA methods have major limitations,such as the use of antibodies susceptible to temperature,easy denaturation,short storage period,susceptible to false positives or false negatives due to the interference of other substances in the detection environment,etc.In this experiment,we used the anti-AFM_(1)nanobody(Nb-M4)obtained in the laboratory in the previous stage to fuse with the C-terminal fragment of C4-binding protein(C4 bpα)to form a self-assembled heptameric fusion protein(Nb-M4-C4 bpα),and based on this heptamer,we developed an indirect competitive enzyme-linked immunosorbent assay(icELISA)applied to AFM_(1)in dairy products.Under the optimal experimental parameters,the IC_(50)of AFM_(1)was 0.038 ng/mL and the LOD was 0.009 ng/mL,which were 8.63-fold and 5.56-fold higher than those of monomeric Nb-M4.Negligible cross-reactivity with AFM_(1)analogs and other common mycotoxins and good recoveries and reproducibility in spiked milk samples were obtained.In addition,the developed method was applied to the analysis of AFM_(1)in real samples,and the results correlated well with those of HPLC(R^(2)=0.995).This work shows that self-assembled heptamerized nanobodies are an effective strategy for improved affinity and signal amplification,which could be used in the future for sensitive,selective and rapid detection of mycotoxins and other small molecule contaminants in food.
作者
刘颖达
刘海媛
苏娜
李庆辉
刘佳
TUYATSETSEG Jambel
伊丽
吉日木图
LIU Yingda;LIU Haiyuan;SU Na;LI Qinghui;LIU Jia;TUYATSETSEG Jambel;YI Li;JI Rimutu(Key Laboratory of Dairy Biotechnology and Engineering,Ministry of Education,Inner Mongolia Agriculture University,Hohhot 010018,China;China-Mongolia Joint laboratory for Biomacromolecule Research,Ulaanbaatar 016199,Mongolia;Inner Mongolia China-Kazakhstan Camel Research Institute,Alxa 737300,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2024年第18期291-299,共9页
Food and Fermentation Industries
基金
内蒙古自然科学基金(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)
内蒙古农业大学食品科学与工程学院科技计划项目(SPKJ201913)。
关键词
竞争性酶联免疫吸附实验
纳米抗体
多聚化
灵敏度增强方法
competitive enzyme-linked immunosorbent assay
nanobody
polymerization
sensitivity enhancement method
