Logozzi Mariantonia, Di Raimo Rossella, Mizzoni Davide, Fais Stefano
Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
Methods Enzymol. 2020;645:155-180. doi: 10.1016/bs.mie.2020.06.011. Epub 2020 Jul 9.
The immunocapture-based ELISA for extracellular vesicles (EVs)/exosomes, originally described in 2009 by Logozzi and colleagues, allows to capture, detect, characterize and quantify extracellular vesicles in both human body fluids and cell culture supernatants. It is based on the use of two antibodies directed one against a typical exosomal housekeeping protein and the second against either another exosomal housekeeping protein or a potential disease marker: the first antibody is used for the capture of exosomes, the second for the quantification and characterization of the captured vesicles. In fact, with this method it is possible both to characterize and count exosomes and to detect the presence of disease, including tumor, biomarkers. This needs of course to preliminary obtain an EVs purification from the clinical sample; the most agreed method to get to an EVs purification is the repeated rounds of ultracentrifugation, that, while far to be perfect, is the methodological approach allowing to not exclude EVs subpopulation from the separation procedure and to analyze a full range of EVs from both qualitative and quantitative point of view. The immunocapture-based approach has proven to be highly useful in screening, diagnosis and prognosis of tumors, in plasma samples. One amazing information provided by this method is that cancer patients have always significantly higher levels of EVs, in particular of exosomes, independently from the histological nature of the tumor. One microenvironmental factor that is fully involved in the increased exosome release by tumors is the extracellular acidity. However, few pre-clinical data suggest that plasmatic levels of exosomes may correlate with the tumor mass. Some recent clinical reports suggest also that circulating exosomes represent the real delivery system for some known tumor markers that are presently on trial (e.g., PSA). Here we review the pros and cons of the immunocapture-based technique in quantitative and qualitative evaluation of EVs in both health and disease.
基于免疫捕获的细胞外囊泡（EVs）/外泌体酶联免疫吸附测定（ELISA）最初由洛戈齐及其同事于2009年描述，可用于捕获、检测、表征和定量人体体液和细胞培养上清液中的细胞外囊泡。它基于使用两种抗体，一种针对典型的外泌体管家蛋白，另一种针对另一种外泌体管家蛋白或潜在的疾病标志物：第一种抗体用于捕获外泌体，第二种用于对捕获的囊泡进行定量和表征。事实上，通过这种方法既可以表征和计数外泌体，又可以检测疾病（包括肿瘤）生物标志物的存在。当然，这需要首先从临床样本中获得EVs的纯化；实现EVs纯化的最公认方法是重复进行超速离心，虽然远非完美，但这种方法学途径可以在分离过程中不排除EVs亚群，并从定性和定量角度分析全范围的EVs。基于免疫捕获的方法已被证明在血浆样本中肿瘤的筛查、诊断和预后方面非常有用。该方法提供的一个惊人信息是，癌症患者的EVs水平，尤其是外泌体水平，始终显著高于正常人，与肿瘤的组织学性质无关。肿瘤细胞外酸度是完全参与肿瘤外泌体释放增加的一个微环境因素。然而，很少有临床前数据表明血浆中外泌体水平可能与肿瘤大小相关。最近的一些临床报告还表明，循环外泌体是一些目前正在试验的已知肿瘤标志物（如前列腺特异性抗原）的真正递送系统。在这里，我们回顾了基于免疫捕获技术在健康和疾病状态下对EVs进行定量和定性评估的优缺点。
