Department of Biochemistry, Clínica Universidad de Navarra, Pamplona, Spain.
Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany.
Clin Chem Lab Med. 2019 Sep 25;57(10):1539-1545. doi: 10.1515/cclm-2018-1297.
Background Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis. Methods Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay. Results The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits. Conclusions Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.
背景 外泌体是由细胞释放的纳米囊泡，可以在血液中检测到。外泌体包含多种分子，如细胞因子，具有作为疾病生物标志物的潜在用途。本研究的目的是比较六种适用于临床实验室的商业试剂盒在分离外泌体的效率和纯度方面的差异，并比较它们对外泌体后续细胞因子分析的影响。
方法 使用六种商业试剂盒（exoEasy、ExoQuick、Exo-spin、ME 试剂盒、ExoQuick Plus 和 Exo-Flow）从 10 名志愿者的血清中提取外泌体。使用纳米颗粒跟踪分析定量测定外泌体浓度和粒径分布。通过 Western blot 测定外泌体标记物 CD63、CD9 和 TSG101。使用散射比浊法测定 ApoB 和白蛋白。使用 Luminex 测定 S100A9、CXCL5 和 CXCL12。
结果 不同试剂盒提取的颗粒浓度差异可达 100 倍。不同试剂盒之间的颗粒浓度提取无相关性，除了 ExoQuick 和 Exo-Flow 之间。ExoQuick Plus 和 exoEasy 获得的外泌体纯度最高，而 ME 和 ExoQuick 获得的纯度最低。所有分析的外泌体提取物中均存在白蛋白，除了用 Exo-Flow 和 ME 提取的外泌体外，所有提取物中均存在 ApoB。细胞因子的检测取决于所使用的纯化试剂盒，不同试剂盒获得的样本之间细胞因子浓度没有相关性。
结论 样品和使用的商业试剂盒类型都会影响外泌体分离的效率和纯度。此外，外泌体的纯化方法对外泌体中细胞因子的检测和定量有很大影响。
