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Improved Isolation of Extracellular Vesicles by Removal of Both Free Proteins and Lipoproteins

View ORCID ProfileDmitry Ter-Ovanesyan, View ORCID ProfileTal Gilboa, Bogdan Budnik, Adele Nikitina, Sara Whiteman, Roey Lazarovits, Wendy Trieu, David Kalish, View ORCID ProfileGeorge M Church, View ORCID ProfileDavid R Walt
doi: https://doi.org/10.1101/2023.01.20.524891
Dmitry Ter-Ovanesyan
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
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Tal Gilboa
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
2Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA
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Bogdan Budnik
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
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Adele Nikitina
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
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Sara Whiteman
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
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Roey Lazarovits
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
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Wendy Trieu
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
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David Kalish
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
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George M Church
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
3Harvard Medical School, Boston, MA, USA
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  • ORCID record for George M Church
David R Walt
1Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA
2Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA
3Harvard Medical School, Boston, MA, USA
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  • For correspondence: dwalt@bwh.harvard.edu
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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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted January 21, 2023.
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Improved Isolation of Extracellular Vesicles by Removal of Both Free Proteins and Lipoproteins
Dmitry Ter-Ovanesyan, Tal Gilboa, Bogdan Budnik, Adele Nikitina, Sara Whiteman, Roey Lazarovits, Wendy Trieu, David Kalish, George M Church, David R Walt
bioRxiv 2023.01.20.524891; doi: https://doi.org/10.1101/2023.01.20.524891
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Improved Isolation of Extracellular Vesicles by Removal of Both Free Proteins and Lipoproteins
Dmitry Ter-Ovanesyan, Tal Gilboa, Bogdan Budnik, Adele Nikitina, Sara Whiteman, Roey Lazarovits, Wendy Trieu, David Kalish, George M Church, David R Walt
bioRxiv 2023.01.20.524891; doi: https://doi.org/10.1101/2023.01.20.524891

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