Abstract
Extracellular vesicles (EVs) hold great promise in the fields of diagnostics and therapeutics. However, the heterogeneity of these membrane-enclosed messengers and the complexity of the biological samples in which they occur pose significant research challenges. The aim of this study was to improve the reliability of size-exclusion chromatography (SEC) and immunolabelling as common approaches in the EV field, and to provide a comprehensive characterisation tool for diverse EV preparations. Profiling of SEC-separated sample components was conducted through light absorbance, fluorescence, and light scattering, providing insights into particle content, size, protein content, and specific markers. Key considerations for assay robustness, including SEC mobile phase composition and immunostaining parameters, were addressed. Respecting the importance of controlled immunolabelling and preanalytical factors, the method efficiently reveals changes in the sample profile with respect to particles and small impurities. The detailed analytical capabilities and method adaptability offer a practical way to enhance the efficiency of EV research and applications.