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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. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for Ap...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10229111/ https://www.ncbi.nlm.nih.gov/pubmed/37252755 http://dx.doi.org/10.7554/eLife.86394 |
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author | Ter-Ovanesyan, Dmitry Gilboa, Tal Budnik, Bogdan Nikitina, Adele Whiteman, Sara Lazarovits, Roey Trieu, Wendy Kalish, David Church, George M Walt, David R |
author_facet | Ter-Ovanesyan, Dmitry Gilboa, Tal Budnik, Bogdan Nikitina, Adele Whiteman, Sara Lazarovits, Roey Trieu, Wendy Kalish, David Church, George M Walt, David R |
author_sort | Ter-Ovanesyan, Dmitry |
collection | PubMed |
description | 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 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 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. |
format | Online Article Text |
id | pubmed-10229111 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-102291112023-05-31 Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins Ter-Ovanesyan, Dmitry Gilboa, Tal Budnik, Bogdan Nikitina, Adele Whiteman, Sara Lazarovits, Roey Trieu, Wendy Kalish, David Church, George M Walt, David R eLife Biochemistry and Chemical Biology 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 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 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. eLife Sciences Publications, Ltd 2023-05-30 /pmc/articles/PMC10229111/ /pubmed/37252755 http://dx.doi.org/10.7554/eLife.86394 Text en © 2023, Ter-Ovanesyan, Gilboa et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Biochemistry and Chemical Biology Ter-Ovanesyan, Dmitry Gilboa, Tal Budnik, Bogdan Nikitina, Adele Whiteman, Sara Lazarovits, Roey Trieu, Wendy Kalish, David Church, George M Walt, David R Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins |
title | Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins |
title_full | Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins |
title_fullStr | Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins |
title_full_unstemmed | Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins |
title_short | Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins |
title_sort | improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins |
topic | Biochemistry and Chemical Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10229111/ https://www.ncbi.nlm.nih.gov/pubmed/37252755 http://dx.doi.org/10.7554/eLife.86394 |
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