Haasnoot Willem, du Pré Jolanda G
RIKILT-Institute of Food Safety, Wageningen UR, AE Wageningen, The Netherlands.
J Agric Food Chem. 2007 May 16;55(10):3771-7. doi: 10.1021/jf063281o. Epub 2007 Apr 25.
An automated fluorescent microsphere-based flow cytometric triplex immunoassay, using the Luminex 100 flow analyzer with MultiAnalyte Profiling (xMAP) technology, was developed for the simultaneous detection of proteins from three vegetable sources as potential fraudulent adulterants in milk powder. In the final triplex inhibition immunoassay, soluble wheat proteins (SWP) and proteins from soy and pea were coupled to three different microsphere sets. A mixture of these microsphere sets was transferred to a microtiter plate well together with the sample and a mixture of three affinity-purified polyclonal antibodies raised against the proteins and labeled with a fluorophore (Alexa 532). After incubation for 1.5 h at room temperature in the dark, the fluorescence intensities on the microspheres were directly measured (no wash procedure) in the Luminex during 10 s per well (100 microspheres per set). The sensitivities of the three assays for plant protein extracts were determined as 0.5-0.6 microg/mL at 50% inhibition. For the detection of the vegetable proteins in milk powder, the samples were dissolved in buffer (0.1 g in 10 mL) and further diluted (20 times) to create a 50% inhibition at approximately 0.5% of the vegetable proteins in the total protein content of milk powder. With the help of calibration standards, prepared under conditions comparable to those for sample materials, the triplex immunoassay proved to be quantitative above 0.1%, although concentrations in high-heated milk powders were underestimated. Due to the xMAP technology, in which 100 different microsphere sets can be distinguished, this triplex immunoassay can easily be extended to detect other possible adulterants.
开发了一种基于自动化荧光微球的流式细胞术三联免疫分析法，使用配备多分析物谱分析（xMAP）技术的Luminex 100流式分析仪，用于同时检测来自三种植物源的蛋白质，这些蛋白质可能是奶粉中的欺诈性掺假物。在最终的三联抑制免疫分析中，将可溶性小麦蛋白（SWP）以及大豆和豌豆中的蛋白质与三种不同的微球组偶联。将这些微球组的混合物与样品以及针对这些蛋白质制备的并标记有荧光团（Alexa 532）的三种亲和纯化多克隆抗体的混合物一起转移到微量滴定板孔中。在黑暗中于室温孵育1.5小时后，直接在Luminex中测量微球上的荧光强度（无需洗涤步骤），每孔测量10秒（每组100个微球）。三种植物蛋白提取物检测方法在50%抑制率时的灵敏度测定为0.5 - 0.6微克/毫升。对于奶粉中植物蛋白的检测，将样品溶解在缓冲液中（0.1克溶于10毫升）并进一步稀释（20倍），以使奶粉总蛋白含量中约0.5%的植物蛋白产生50%的抑制率。借助在与样品材料相当的条件下制备的校准标准品，三联免疫分析法在高于0.1%时被证明是定量的，尽管高温处理奶粉中的浓度被低估。由于xMAP技术能够区分100种不同的微球组，这种三联免疫分析法可以轻松扩展以检测其他可能的掺假物。
