High sensitivity detection of extracellular vesicles immunecaptured from urine by conventional flow cytometry
High sensitivity detection of extracellular vesicles immunecaptured from urine by conventional flow cytometry
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DOI：
10.1038/s41598-019-38516-8
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发表时间：
2019-02-14
影响因子：
4.6
通讯作者：
Vales-Gomez, Mar
中科院分区：
文献类型：
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作者：
Campos-Silva, Carmen;Suarez, Henar;Vales-Gomez, Mar
细胞外囊泡（EV）提供了分析生理过程的宝贵工具，因为它们在生物流体中运输从个体的不同组织分泌的生物分子。EV生物标志物检测需要能够识别单个分子的高灵敏度技术。然而，缺乏广泛的，负担得起的高通量EV分析方法意味着生物标志物的研究尚未在大型患者队列中进行。为了开发用于生物样品中EV分析的工具，我们在此评估了关键参数，以优化基于EV免疫捕获然后进行流式细胞术的测定。我们描述了一种使用一般EV标志物（如四跨膜蛋白CD 9、CD 63和CD 81）进行EV检测的简单方法，该方法允许高度灵敏地检测尿液EV，而无需事先富集。在概念验证实验中，在来自细胞系的EV中和直接在尿液样品中鉴定了癌细胞中富集的上皮标志物EpCAM。然而，尽管EpCAM的蛋白质印迹检测需要从5-10 ml尿中分离的EV，但仅500 μ l尿就足以通过流式细胞术可视化EpCAM表达。这种方法有可能允许任何使用常规流式细胞术的实验室使用最低限度处理的生物样品来鉴定EV上的表面标志物，甚至是非丰富的蛋白质。
Extracellular vesicles (EVs) provide an invaluable tool to analyse physiological processes because they transport, in biological fluids, biomolecules secreted from diverse tissues of an individual. EV biomarker detection requires highly sensitive techniques able to identify individual molecules. However, the lack of widespread, affordable methodologies for high-throughput EV analyses means that studies on biomarkers have not been done in large patient cohorts. To develop tools for EV analysis in biological samples, we evaluated here the critical parameters to optimise an assay based on immunocapture of EVs followed by flow cytometry. We describe a straightforward method for EV detection using general EV markers like the tetraspanins CD9, CD63 and CD81, that allowed highly sensitive detection of urinary EVs without prior enrichment. In proof-of-concept experiments, an epithelial marker enriched in carcinoma cells, EpCAM, was identified in EVs from cell lines and directly in urine samples. However, whereas EVs isolated from 5-10 ml of urine were required for western blot detection of EpCAM, only 500 mu l of urine were sufficient to visualise EpCAM expression by flow cytometry. This method has the potential to allow any laboratory with access to conventional flow cytometry to identify surface markers on EVs, even non-abundant proteins, using minimally processed biological samples.