Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins

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. Researchers at the Wyss Institute for Biologically Inspired Engineering have 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), the researchers were able to measure the separation of EVs from both lipoproteins and free proteins. They used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. They also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. The researchers 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. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.

Development and validation of automated device for running
size exclusion chromatography (SEC) columns in parallel

(A) CAD image of semi-automated SEC stand designed to hold eight columns at once with sliding collection tube holder that allows liquid to drip either into 2 ml collection tubes, or to waste. (B) Photograph of stand connected to a Tecan Cavro syringe pump controlled by a Raspberry Pi. (C) Simoa comparison of CD9, CD63, CD81, ApoB-100, and albumin when SEC was performed on 16 samples of 1 ml plasma using either manual SEC (8 samples) or SEC on the automated device (8 samples). Each point is the average of two Simoa measurements (technical replicates).

Ter-Ovanesyan D, Gilboa T, Budnik B, Nikitina A, Whiteman S, Lazarovits R, Trieu W, Kalish D, Church GM, Walt DR. (2023) Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins. Elife 12:e86394. [article]

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