6-Plex microsphere immunoassay with imaging planar array detection for mycotoxins in barley.
6-Plex microsphere immunoassay with imaging planar array detection for mycotoxins in barley.
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DOI：
10.1039/c4an00368c
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发表时间：
2014-07
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影响因子：
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通讯作者：
J. Peters;A. Cardall;W. Haasnoot;M. Nielen
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作者：
J. Peters;A. Cardall;W. Haasnoot;M. Nielen
霉菌毒素是真菌作为次生代谢产物产生的。它们经常对食品和饲料商品造成多重污染，对人类和动物造成健康风险。对这些商品进行快速且易于应用的多重筛选可能有助于检测多重污染。为此，我们开发了一种半定量六重免疫测定法，使用顺磁颜色编码微球悬浮阵列结合成像平面阵列检测来检测霉菌毒素黄曲霉毒素 B1、赭曲霉毒素 A、玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、T2 毒素、HT-2 毒素和伏马菌素 B1。将霉菌毒素特异性单克隆抗体与不同组的微球偶联，并将与荧光蛋白 R-藻红蛋白缀合的霉菌毒素作为报告分子。样品中的游离霉菌毒素与混合报告分子之间对混合微球上抗体结合位点的竞争产生了多重直接抑制免疫测定。选择的试剂是为了测定之间没有或很少有交叉相互作用，并且与代谢物发生交叉反应，并确定了可能的掩蔽形式。使用空白和加标大麦样品进行了实验室内验证。此外，6-plex 用于筛选可用的大麦和大麦麦芽参考材料。验证显示所有样本的日间和日内精度非常高，最大相对标准偏差值为 10%。根据欧盟立法，筛选测定可以轻松快速地多重检测大麦中的目标霉菌毒素。根据欧盟最高水平，截止因子为 50%，我们能够在 2 μg kg(-1) 下筛选黄曲霉毒素 B1、2.5 μg kg(-1) 赭曲霉毒素 A、625 μg kg(-1) 脱氧雪腐镰刀菌烯醇、50 μg kg(-1) 玉米赤霉烯酮、1000 μg kg(-1) 伏马菌素 B1 和T-2毒素为25 μg kg(-1)。得益于可移动平面阵列系统，开发的 6-plex 具有未来现场测试的潜力。未来在仪器分析之前将该方法用作预筛选工具非常有吸引力，因为可以避免对低于最高水平的样品进行昂贵的 LC-MS/MS 分析。
Mycotoxins are produced by fungi as secondary metabolites. They often multi-contaminate food and feed commodities posing a health risk to humans and animals. A fast and easy to apply multiplex screening of these commodities could be useful to detect multi-contamination. For this, we developed a semi-quantitative 6-plex immunoassay using a suspension array of paramagnetic colour-coded microspheres combined with imaging planar array detection for the mycotoxins aflatoxin B1, ochratoxin A, zearalenone, deoxynivalenol, T2-toxin, HT-2 toxin and fumonisin B1. Mycotoxin specific monoclonal antibodies were coupled to different sets of microspheres and mycotoxins conjugated to the fluorescent protein R-phycoerythrin served as reporter molecules. Competition between free mycotoxins in the sample and mixed reporter molecules for antibody binding sites on mixed microspheres created a multiplex direct inhibition immunoassay. The reagents were selected for no or low cross-interactions between the assays and cross-reactions with metabolites and possible masked forms were determined. A within-laboratory validation was carried out using blank and spiked barley samples. Furthermore, the 6-plex was used to screen available barley, and malted barley, reference materials. The validation showed very high inter and intra-day precision for all samples with a maximum relative standard deviation value of 10%. The screening assay allows easy and rapid multiplex detection of the target mycotoxins in barley according to EU legislation. With a cut off factor of 50%, based on the EU maximum levels, we were able to screen at 2 μg kg(-1) for aflatoxin B1, 2.5 μg kg(-1) for ochratoxin A, 625 μg kg(-1) for deoxynivalenol, 50 μg kg(-1) for zearalenone, 1000 μg kg(-1) for fumonisin B1 and 25 μg kg(-1) for T-2 toxin. Thanks to the transportable planar array system, the developed 6-plex has potential for future on-site testing. Future implementation of this method as a pre-screening tool, prior to instrumental analysis, is highly attractive since costly LC-MS/MS analysis of samples below the maximum levels can be avoided.