Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
Department of Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
J Chromatogr A. 2020 Oct 11;1629:461513. doi: 10.1016/j.chroma.2020.461513. Epub 2020 Aug 29.
Extracellular vesicles (EVs) exist in biological fluids such as blood, urine, and cerebrospinal fluid, and these have shown promise for use as biomarkers of cancers. Conventional methods for determination of EVs include direct detection via enzyme-linked immunosorbent assay and detection of their membrane proteins via western blotting. These techniques, however, have individual shortcomings in terms of the need for large sample consumption, processes that are time-consuming, and a lack of the capacity for quantification. In this study, we developed a method to determine the EV membrane protein, CD63, by coupling capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF). In this process, the EVs were isolated from a culture medium and were subsequently reacted with a fluorescently labeled anti-CD63 antibody to form a CD63 complex localized on the surface of EVs. After removing the EVs containing the CD63 immune complex by centrifugation, the supernatant containing the free fluorescent antibody was injected into a capillary to serve as a sample. A decrease in the peak area of the free fluorescent antibody became apparent when the amount of EVs was increased while that of the fluorescent antibody remained constant. The peak areas were decreased proportionally against the increased amounts of EVs. The concentration of the CD63 could then be estimated based on the slope of the linear relationship. This study is the first to quantify CD63 immobilized on EVs via CEIA-LIF, which is a novel method with the potential to determine membrane proteins localized on the surface of EVs.
细胞外囊泡（EVs）存在于血液、尿液和脑脊液等生物体液中，它们已被证明可作为癌症生物标志物的用途。传统的 EVs 测定方法包括通过酶联免疫吸附测定法进行直接检测和通过 Western blot 检测其膜蛋白。然而，这些技术在需要大量样本消耗、耗时的过程以及缺乏定量能力方面都存在各自的缺点。在本研究中，我们开发了一种通过毛细管电泳免疫分析与激光诱导荧光（CEIA-LIF）相结合的方法来测定 EV 膜蛋白 CD63。在这个过程中，EVs 从培养基中分离出来，然后与荧光标记的抗 CD63 抗体反应，形成位于 EV 表面的 CD63 复合物。通过离心去除含有 CD63 免疫复合物的 EV 后，将含有游离荧光抗体的上清液注入毛细管作为样品。当 EV 数量增加而荧光抗体数量保持不变时，游离荧光抗体的峰面积明显减小。峰面积与 EV 数量成比例减少。然后可以根据线性关系的斜率来估计 CD63 的浓度。本研究首次通过 CEIA-LIF 定量测定了固定在 EV 上的 CD63，这是一种具有潜在能力来测定位于 EV 表面的膜蛋白的新方法。
