Developing EV isolation methods is also particularly challenging due to the inability of most methods, such as the commonly used Nanoparticle Tracking Analysis (NTA) to differentiate between EVs and similarly sized lipoproteins ( Johnsen et al., 2019). This is particularly the case for separating EVs and lipoproteins, as these two classes of particles have overlapping size ranges ( Simonsen, 2017). Since EVs are much less abundant than free proteins and lipoproteins in plasma, enriching them without the co-isolation of these impurities remains highly challenging ( Sódar et al., 2016 Smolarz et al., 2019). EVs contain RNA and protein cargo from their cell of origin and are a promising class of biomarkers in biofluids such as plasma ( Shah et al., 2018). Editor's evaluationĮxtracellular vesicles (EVs) are membrane vesicles released by all cells. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. 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 developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma.
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