一种基于高灵敏和高特异性单克隆抗体的快速同时检测食品和饲料中黄曲霉毒素 B1 和玉米赤霉烯酮的新型侧向流动免疫层析分析方法。
A Novel Lateral Flow Immunochromatographic Assay for Rapid and Simultaneous Detection of Aflatoxin B1 and Zearalenone in Food and Feed Samples Based on Highly Sensitive and Specific Monoclonal Antibodies.
机构信息
Henan Institute of Science and Technology, College of Animal Science and Veterinary Medicine, Xinxiang 453003, China.
Faculty of Veterinary Medicine, Sumy National Agrarian University, 40021 Sumy, Ukraine.
出版信息
Toxins (Basel). 2022 Sep 2;14(9):615. doi: 10.3390/toxins14090615.
Simultaneous aflatoxin (AFB1) and zearalenone (ZEN) contamination in agro-products have become widespread globally and have a toxic superposition effect. In the present study, we describe a highly sensitive and specific dual lateral flow immunochromatographic assay (dual test strip) for rapid and simultaneous detection of AFB1 and ZEN in food and feed samples based on respective monoclonal antibodies (mAbs). Two immunogens AFB1-BSA (an AFB1 and bovine serum albumin (BSA) conjugate) and ZEN-BSA (a ZEN and BSA conjugate) were synthesized in oximation active ester (OAE) and amino glutaraldehyde (AGA). The molecular binding ratio of AFB1:BSA was 8.64:1, and that of ZEN:BSA was 17.2:1, identified by high-resolution mass spectrometry (HRMS) and an ultraviolet spectrometer (UV). The hybridoma cell lines 2A11, 2F6, and 3G2 for AFB1 and 2B6, 4D9 for ZEN were filtered by an indirect non-competitive enzyme-linked immunosorbent assay (inELISA) and an indirect competitive enzyme-linked immunosorbent assay (icELISA), respectively. As AFB1 mAb 2A11 and ZEN mAb 2B6 had the lowest 50% inhibitive concentration (IC50) and cross-reactivity (CR), they were selected for subsequent experiments. By systematically optimizing the preparation condition of gold nanoparticles (AuNPs), AuNPs-labeled mAbs, and detection condition, the visual limit of detection (LOD) of the dual test strip was 1.0 μg/L for AFB1 and 5.0 μg/L for ZEN, whereas that of the test strip reader was 0.23 μg/L for AFB1 and 1.53 μg/L for ZEN. The high reproducibility and stability of the dual test were verified using mycotoxin-spiked samples. The dual test strips were highly specific and sensitive for AFB1 and ZEN, which were validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Thus, the proposed AFB1 and ZEN dual test strip is suitable for rapid and simultaneous detection of AFB1 and ZEN contamination in food and feed samples.
同时，农产品中黄曲霉毒素（AFB1）和玉米赤霉烯酮（ZEN）的污染已在全球范围内广泛存在，并具有毒性叠加效应。本研究基于各自的单克隆抗体（mAb），描述了一种用于快速同时检测食品和饲料样品中 AFB1 和 ZEN 的高灵敏度和特异性双重侧向流动免疫层析测定法（双重测试条）。两种免疫原 AFB1-BSA（AFB1 和牛血清白蛋白（BSA）的缀合物）和 ZEN-BSA（ZEN 和 BSA 的缀合物）是通过肟活性酯（OAE）和氨戊二醛（AGA）合成的。通过高分辨率质谱（HRMS）和紫外分光光度计（UV）鉴定，AFB1：BSA 的分子结合比为 8.64：1，ZEN：BSA 的分子结合比为 17.2：1。通过间接非竞争酶联免疫吸附测定（inELISA）和间接竞争酶联免疫吸附测定（icELISA）分别筛选出针对 AFB1 的杂交瘤细胞系 2A11、2F6 和 3G2 以及针对 ZEN 的 2B6、4D9。由于 AFB1 mAb 2A11 和 ZEN mAb 2B6 的 50%抑制浓度（IC50）和交叉反应性（CR）最低，因此选择它们进行后续实验。通过系统优化金纳米粒子（AuNPs）、AuNPs 标记的 mAb 和检测条件的制备条件，双重测试条的目视检测限（LOD）分别为 AFB1 的 1.0 μg/L 和 ZEN 的 5.0 μg/L，而测试条读取器的 LOD 分别为 AFB1 的 0.23 μg/L 和 ZEN 的 1.53 μg/L。使用真菌毒素污染的样品验证了双重测试的高重复性和稳定性。双重测试条对 AFB1 和 ZEN 具有高度特异性和灵敏度，这已通过液相色谱-串联质谱（LC-MS/MS）进行了验证。因此，所提出的 AFB1 和 ZEN 双重测试条适用于快速同时检测食品和饲料样品中 AFB1 和 ZEN 的污染。
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