Rapid Isolation and Characterization of Exosomes through a Single-Step, Label-Free Protein Biomarker Analysis

Exosomes, small extracellular vesicles with compositions reflecting their cell of origin, serve as sensitive and specific biomarkers for disease detection. We herein report a protocol for rapid isolation and characterization of exosomes by a single-step and label-free protein biomarker analysis. Using a simple centrifugation-filtration-concentration (CFC) method, exosomes are isolated and enriched 50-fold from conditioned cell culture media. For protein biomarker analysis, unconjugated antibodies are added directly to the isolated exosome solution. The specific interaction between the antibodies and exosomes leads to aggregation of exosomes, and subsequently, an average particle size increase of the assay solution. This average particle size increase can be detected using dynamic light scattering and correlated to the presence or absence of protein biomarkers on the exosomes. In this study, exosomes from three cell types, human embryonic kidney (HEK293) cells, genetically modified HEK-GFP cells, and GBM/NSC CD133⁺ cells were isolated. The exosomes released from HEK293, HEK-GFP, and GBM/NSC CD133⁺ cells exhibited monodispersed size distributions with an average particle size centered around 70, 66, and 249 nm, respectively. Positive antibody binding to exosome surface proteins resulted in a peak shift, increasing particle size by 25, 32, and 148 nm, respectively, for the HEK293, HEK-GFP, and GBM/NSC CD133⁺ exosomes, while the size increase upon addition of a negative antibody remained minimum. This protocol provides a convenient platform for the design and development of rapid diagnostic tests targeting disease specific exosomes.