Framework for rapid comparison of extracellular vesicle isolation methods

  1. Dmitry Ter-Ovanesyan
  2. Maia Norman
  3. Roey Lazarovits
  4. Wendy Trieu
  5. Ju-Hyun Lee
  6. George Church
  7. David R Walt  Is a corresponding author
  1. Wyss Institute for Biologically Inspired Engineering, United States

Abstract

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Dmitry Ter-Ovanesyan

    Wyss Institute for Biologically Inspired Engineering, Boston, United States
    Competing interests
    Dmitry Ter-Ovanesyan, The authors have filed intellectual property related to methods for isolating extracellular vesicles..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1134-0073
  2. Maia Norman

    Wyss Institute for Biologically Inspired Engineering, Boston, United States
    Competing interests
    Maia Norman, The authors have filed intellectual property related to methods for isolating extracellular vesicles..
  3. Roey Lazarovits

    Wyss Institute for Biologically Inspired Engineering, Boston, United States
    Competing interests
    No competing interests declared.
  4. Wendy Trieu

    Wyss Institute for Biologically Inspired Engineering, Boston, United States
    Competing interests
    No competing interests declared.
  5. Ju-Hyun Lee

    Wyss Institute for Biologically Inspired Engineering, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6728-2071
  6. George Church

    Wyss Institute for Biologically Inspired Engineering, Boston, United States
    Competing interests
    George Church, GMC commercial interests: http://arep.med.harvard.edu/gmc/tech.html..
  7. David R Walt

    Wyss Institute for Biologically Inspired Engineering, Boston, United States
    For correspondence
    dwalt@bwh.harvard.edu
    Competing interests
    David R Walt, DRW has a financial interest in Quanterix Corporation, a company that develops an ultra-sensitive digital immunoassay platform. He is an inventor of the Simoa technology, a founder of the company and also serves on its Board of Directors. Dr. Walt's interests were reviewed and are managed by BWH. The authors have filed aprovisional patent (WO2021163416A1) on methods for EV isolationmeasuring andpurifying EVs..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5524-7348

Funding

Chan Zuckerberg Initiative (NDCN Collaborative Science Award)

  • Dmitry Ter-Ovanesyan
  • Maia Norman
  • Roey Lazarovits
  • Wendy Trieu
  • Ju-Hyun Lee
  • George Church
  • David R Walt

Open Philanthropy Project

  • Dmitry Ter-Ovanesyan
  • Maia Norman
  • Roey Lazarovits
  • Wendy Trieu
  • Ju-Hyun Lee
  • David R Walt

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Ter-Ovanesyan et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,889
    views
  • 1,021
    downloads
  • 91
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Dmitry Ter-Ovanesyan
  2. Maia Norman
  3. Roey Lazarovits
  4. Wendy Trieu
  5. Ju-Hyun Lee
  6. George Church
  7. David R Walt
(2021)
Framework for rapid comparison of extracellular vesicle isolation methods
eLife 10:e70725.
https://doi.org/10.7554/eLife.70725

Share this article

https://doi.org/10.7554/eLife.70725

Further reading

    1. Biochemistry and Chemical Biology
    Adrian CD Fuchs
    Research Article

    The protein ligase Connectase can be used to fuse proteins to small molecules, solid carriers, or other proteins. Compared to other protein ligases, it offers greater substrate specificity, higher catalytic efficiency, and catalyzes no side reactions. However, its reaction is reversible, resulting in only 50% fusion product from two equally abundant educts. Here, we present a simple method to reliably obtain 100% fusion product in 1:1 conjugation reactions. This method is efficient for protein-protein or protein-peptide fusions at the N- or C-termini. It enables the generation of defined and completely labeled antibody conjugates with one fusion partner on each chain. The reaction requires short incubation times with small amounts of enzyme and is effective even at low substrate concentrations and at low temperatures. With these characteristics, it presents a valuable new tool for bioengineering.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Joar Esteban Pinto Torres, Mathieu Claes ... Yann G-J Sterckx
    Research Article

    African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.