Indirect capillary electrophoresis immunoassay of membrane protein in extracellular vesicles

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.