Abstract
Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly-sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan*, Norman* et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography (SEC) with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main contaminants of EV isolation in plasma and apply this approach to develop novel methods for isolating highly-pure EVs from human plasma. These methods will enable applications where high purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.
Competing Interest Statement
DRW is a founder and equity holder in Quanterix. His interests were reviewed and are managed by BWH and Partners HealthCare in accordance with their conflict of interest policies. GMC Disclosures: https://arep.med.harvard.edu/gmc/tech.html. The authors have filed IP on methods for EV isolation and analysis.
