Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples
Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples
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DOI：
10.1002/pros.23393
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发表时间：
2017-09-15
期刊：
影响因子：
2.8
通讯作者：
Leong, Hon S.
中科院分区：
文献类型：
--
作者：
Brett, Sabine I.;Lucien, Fabrice;Leong, Hon S.
背景分离细胞外囊泡（EV）如外来体或微粒的能力是一种重要的方法，目前尚未标准化。虽然市售试剂盒提供从生物流体中纯化EV，但此类纯化的EV样品还将含有非EV实体，例如可溶性蛋白质和核酸，其可能混淆后续实验。理想情况下，只有EV将被隔离，没有可溶性蛋白将存在于最终EV preparation.MethodsWe比较市售EV隔离试剂盒与免疫亲和纯化技术，并评估我们最终EV制剂使用原子力显微镜（AFM）和纳米级流式细胞术（NFC）。AFM是唯一能够检测区分可溶性蛋白质与EV的方法，这对下游蛋白质组学方法很重要。NFC是唯一的技术能够定量的比例目标EV的非目标EV在最终EV preparation.ResultsTo确定富集的前列腺衍生EV相对于非目标MP，抗PSMA（前列腺特异性膜抗原）抗体中使用NFC。基于抗体的免疫亲和纯化产生了最高质量的前列腺衍生EV制剂，因为样品中缺乏蛋白质和RNA。所有试剂盒产生的纯度差EV制剂，未能耗尽血浆protein.ConclusionsWhile吸引人，由于其易用性，EV纯化试剂盒不提供从生物流体，如血浆中分离EV的重大改进。免疫亲和方法更有效和经济，并且还将消除下游方法所必需的大部分血浆蛋白。
BackgroundThe ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation.MethodsWe compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation.ResultsTo determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein.ConclusionsWhile attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches.