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CC BY 4.0 · TH Open 2018; 02(03): e250-e260
DOI: 10.1055/s-0038-1667204
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Sensitive and Specific Detection of Platelet-Derived and Tissue Factor–Positive Extracellular Vesicles in Plasma Using Solid-Phase Proximity Ligation Assay

Åsa Thulin
1   Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
,
Junhong Yan
2   Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden
3   Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, The Netherlands
,
Mikael Åberg
1   Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
,
Christina Christersson
4   Department of Medical Sciences, Cardiology, Uppsala University, Uppsala, Sweden
,
Masood Kamali-Moghaddam
2   Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden
,
Agneta Siegbahn
1   Department of Medical Sciences, Clinical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
› Author Affiliations Funding The study was supported by the Swedish Research Council (K2013–65X-11568–18–5 and K2011–67X-13096–13–4), the Swedish Heart-Lung foundation, the Swedish Foundation for Strategic Research, and the Science for Life Laboratory, Uppsala University.
Further Information

Publication History

14 February 2018

13 June 2018

Publication Date:
27 July 2018 (online)

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Abstract

Extracellular vesicles (EVs) derived from blood cells are promising biomarkers for various diseases. However, they are difficult to measure accurately in plasma due to their small size. Here, we demonstrate that platelet-derived EVs in plasma can be measured using solid-phase proximity ligation assay with high sensitivity and specificity using very small sample volume of biological materials. The results correlate well with high-sensitivity flow cytometry with the difference that the smallest EVs are detected. Briefly, the EVs are first captured on a solid phase, using lactadherin binding, and detection requires recognition with two antibodies followed by qPCR. The assay, using cholera toxin subunit-B or lactadherin as capture agents, also allowed detection of the more rare population of tissue factor (TF)-positive EVs at a concentration similar to sensitive TF activity assays. Thus, this assay can detect different types of EVs with high specificity and sensitivity, and has the potential to be an attractive alternative to flow cytometric analysis of preclinical and clinical samples. Improved techniques for measuring EVs in plasma will hopefully contribute to the understanding of their role in several diseases.

Keywords

microvesicles - myocardial infarction - cardiovascular diseases - vascular homeostasis

Authors' Contributions

A.S. conceived the research; M.K-M. and J.Y. participated in the initial discussions and provided the technical protocols for SP-PLA with antibodies as catchers and antibody conjugations with oligonucleotides; J.Y. performed preliminary experiments. A.S. and Å.T. designed the SP-PLA using cholera toxin subunit B and lactadherin to capture EVs; and A.S., Å.T., M.Å., and C.C. designed the research. Å.T. performed the experiments. C.C. provided plasma samples from patients with myocardial infarction. Å.T. and A.S. analyzed the results and Å.T. wrote the first draft of the manuscript. All authors read and commented on the manuscript and approved for submission.


 

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