Wang Feng, Yang Yuan-Yuan, Wan De-Bin, Li Jia-Dong, Liang Yi-Fan, Li Zhen-Feng, Shen Yu-Dong, Xu Zhen-Lin, Yang Jin-Yi, Wang Hong, Gettemans Jan, Hammock Bruce D, Sun Yuan-Ming
Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, P. R. China.
Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California, Davis, California 95616, United States.
Food Control. 2022 Jun;136. doi: 10.1016/j.foodcont.2022.108835. Epub 2022 Jan 20.
The accurate analysis of chemical isomers plays an important role in the study of their different toxic effects and targeted detection of pollutant isomers in foods. The mycotoxins tenuazonic acid (TeA) and iso-tenuazonic acid (ITeA) are two isomer mycotoxins with the lack of single analysis methods due to the similar structures. Antibody-based immunoassays exhibit high sensitivity and superior application in isomer-specific determination. Previously, various kinds of antibodies for TeA have been prepared in our group. Herein, highly specific nanobodies (Nbs) against ITeA mycotoxin were selected from immune nanobody phage display library, and one of Nbs, namely Nb(B3G3) exhibited excellent affinity, thermal stability as well as organic solvent tolerance. By molecular simulation and docking technology, it was found that stronger interaction between Nb(B3G3) and ITeA lead to higher affinity than that for its isomer TeA. Furthermore, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was established with a limit of detection (LOD) of 0.09 ng/mL for ITeA mycotoxin. The recovery rate of ITeA in spiked samples was analyzed with 84.8%-89.5% for rice, 78.3%-96.3% for flour, and 79.5%-90.7% for bread. A conventional LC-MS/MS method was used to evaluate the accuracy of this proposed icELISA, which showed a satisfactory consistent correlation. Since the convenient strategy for nanobody generation by phage display technology, this study provide new biorecognition elements and sensitive immunoassay for analysis of ITeA in foods.
化学异构体的准确分析在研究其不同毒性效应以及食品中污染物异构体的靶向检测方面发挥着重要作用。霉菌毒素细交链孢菌酮酸（TeA）和异细交链孢菌酮酸（ITeA）是两种异构体霉菌毒素，由于结构相似，缺乏单一的分析方法。基于抗体的免疫分析在异构体特异性测定中表现出高灵敏度和卓越的应用性能。此前，我们团队已制备了多种针对TeA的抗体。在此，从免疫纳米抗体噬菌体展示文库中筛选出了针对ITeA霉菌毒素的高特异性纳米抗体（Nbs），其中一种纳米抗体Nb(B3G3)表现出优异的亲和力、热稳定性以及有机溶剂耐受性。通过分子模拟和对接技术发现，Nb(B3G3)与ITeA之间更强的相互作用导致其对ITeA的亲和力高于对其异构体TeA的亲和力。此外，建立了一种灵敏的间接竞争酶联免疫吸附测定法（icELISA），对ITeA霉菌毒素的检测限（LOD）为0.09 ng/mL。加标样品中ITeA在大米中的回收率为84.8% - 89.5%，在面粉中的回收率为78.3% - 96.3%，在面包中的回收率为79.5% - 90.7%。采用传统的液相色谱 - 串联质谱法（LC - MS/MS）评估所提出的icELISA的准确性，结果显示具有令人满意的一致相关性。由于通过噬菌体展示技术产生纳米抗体的便捷策略，本研究为食品中ITeA的分析提供了新的生物识别元件和灵敏的免疫测定方法。
