Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.
Cancer Research Institute Ghent, Ghent, Belgium.
Nat Protoc. 2021 Feb;16(2):603-633. doi: 10.1038/s41596-020-00446-5. Epub 2021 Jan 15.
The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires ~4-5 d.
细胞外囊泡（EV）的诊断和治疗用途正在受到深入研究，可能会给社会带来益处。参考物质是开发、改进和评估受监管的 EV 应用性能以及进行定量和客观数据解释的宝贵资源。我们已经将重组 EV（rEV）设计为一种生物参考物质。rEV 具有与样本 EV 相似的生化和物理特性，并在整个分析过程中充当内部定量和定性控制。在进行 EV 分析之前，将 rEV 掺入体液中可以映射 EV 应用的技术变异性，并促进实验室内部和实验室之间的研究。本方案是对我们之前发表的方案（Tulkens 等人，2020 年）的扩展，描述了 rEV 的生产、分离和质量保证，以及它们在体液中的稀释和添加，以及基于互补荧光、核酸和蛋白质测量的检测步骤。我们展示了 rEV 在方法开发、数据归一化和评估分析前变量方面的用途。该方案可供具有标准实验室和基本 EV 分离/表征经验的研究人员采用，大约需要 4-5 天。
