Peters Jeroen, Cardall Alice, Haasnoot Willem, Nielen Michel W F
RIKILT Wageningen UR (Institute of Food Safety), Akkermaalsbos 2, 6700 AE Wageningen, The Netherlands.
Analyst. 2014 Aug 21;139(16):3968-76. doi: 10.1039/c4an00368c.
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.
霉菌毒素是真菌产生的次生代谢产物。它们常常对食品和饲料造成多种污染，对人类和动物健康构成风险。对这些商品进行快速且易于应用的多重筛选，有助于检测多重污染情况。为此，我们开发了一种半定量的6重免疫分析方法，该方法使用顺磁颜色编码微球的悬浮阵列，并结合成像平面阵列检测技术，用于检测黄曲霉毒素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等霉菌毒素。将霉菌毒素特异性单克隆抗体偶联到不同组的微球上，将与荧光蛋白R - 藻红蛋白偶联的霉菌毒素用作报告分子。样品中的游离霉菌毒素与混合报告分子竞争混合微球上的抗体结合位点，从而形成多重直接抑制免疫分析。所选试剂在各分析之间无交叉相互作用或交叉相互作用较低，并测定了与代谢物的交叉反应以及可能的隐蔽形式。使用空白和加标的大麦样品进行了实验室内部验证。此外，该6重免疫分析方法还用于筛选现有的大麦、麦芽大麦参考材料。验证结果表明，所有样品的日内和日间精密度都非常高，最大相对标准偏差值为10%。该筛选分析方法能够根据欧盟法规轻松快速地对大麦中的目标霉菌毒素进行多重检测。基于欧盟最大限量，以50%的截断因子为依据，我们能够对黄曲霉毒素B1以2 μg kg⁻¹、赭曲霉毒素A以2.5 μg kg⁻¹、脱氧雪腐镰刀菌烯醇以625 μg kg⁻¹、玉米赤霉烯酮以50 μg kg⁻¹、伏马菌素B1以1000 μg kg⁻¹以及T - 2毒素以25 μg kg⁻¹进行筛选。由于该可运输的平面阵列系统，所开发的6重免疫分析方法具有未来现场检测的潜力。在仪器分析之前，将该方法作为预筛选工具的未来应用极具吸引力，因为可以避免对低于最大限量的样品进行成本高昂的液相色谱 - 串联质谱分析。
