A novel multiplexed immunoassay for surface‐exposed proteins in plasma extracellular vesicles
Abstract Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal su...
Saved in:
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2024-11-01
|
| Series: | Journal of Extracellular Vesicles |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jev2.70007 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1846149716529643520 |
|---|---|
| author | Emma Tordoff Jillian Allen Katya Elgart Ahmed Elsherbini Vrinda Kalia Haotian Wu Erden Eren Dimitrios Kapogiannis Olesia Gololobova Kenneth Witwer Olga Volpert Erez Eitan |
| author_facet | Emma Tordoff Jillian Allen Katya Elgart Ahmed Elsherbini Vrinda Kalia Haotian Wu Erden Eren Dimitrios Kapogiannis Olesia Gololobova Kenneth Witwer Olga Volpert Erez Eitan |
| author_sort | Emma Tordoff |
| collection | DOAJ |
| description | Abstract Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal surface protein composition of heterogeneous EV populations in culture supernatants and in biofluids. Tetraspanins CD9, CD63, and CD81 are common and abundant EV markers. However, their relative enrichment (profile) on EVs of specific cellular origins is not fully elucidated. We introduce LuminEV, a novel version of the Luminex assay for the multiplexed analysis of EV surface proteins. Optimized LuminEV reagents enable direct, specific, and sensitive measurements of EV markers in biofluids and in culture supernatants, bypassing EV isolation step. LuminEV assay for CD9, CD63, and CD81 was validated by comparing simplex and multiplex measurements, establishing linearity, spike‐in recovery, inter‐ and intra‐assay precision, and reproducibility between operators. LuminEV measurements of CD9, CD63, and CD81 in conditioned media from 15 cell lines revealed strong variations between cell types and showed high sensitivity, which enabled EV detection without prior concentration. Using tetraspanin levels as a readout, we noted suppression and induction of EV release from the cultured cells by GW6869 and monensin. Measurement of EV CD9, CD63, and CD81 in blood plasma from 70 disease‐free donors showed respective abundance of 72, 16, and 12%. CD63 displayed weak, albeit significant, negative correlation with age and was slightly lower in female samples. The assay was then used to detect cell type‐specific EV surface markers, including CD235a (erythrocytes), GAP43 (neurons), and CD68 (macrophages), and to detect differences in tetraspanin profiles between healthy and diseased donors. In summary, LuminEV offers robust and sensitive approach for multiplexed assessment of EV surface proteins, to facilitate the research into EV biology, biomarker, and therapeutic applications. |
| format | Article |
| id | doaj-art-fbe12b47bc20462b80379b836781f1c1 |
| institution | Kabale University |
| issn | 2001-3078 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj-art-fbe12b47bc20462b80379b836781f1c12024-11-29T11:34:28ZengWileyJournal of Extracellular Vesicles2001-30782024-11-011311n/an/a10.1002/jev2.70007A novel multiplexed immunoassay for surface‐exposed proteins in plasma extracellular vesiclesEmma Tordoff0Jillian Allen1Katya Elgart2Ahmed Elsherbini3Vrinda Kalia4Haotian Wu5Erden Eren6Dimitrios Kapogiannis7Olesia Gololobova8Kenneth Witwer9Olga Volpert10Erez Eitan11NeuroDex, Inc. Natick Massachusetts USANeuroDex, Inc. Natick Massachusetts USANeuroDex, Inc. Natick Massachusetts USANeuroDex, Inc. Natick Massachusetts USADepartment of Environmental Health Sciences, Mailman School of Public Health Columbia University New York New York USADepartment of Environmental Health Sciences, Mailman School of Public Health Columbia University New York New York USALaboratory of Neurosciences, Intramural Research Program National Institute on Aging/National Institutes of Health (NIA/NIH) Baltimore Maryland USALaboratory of Neurosciences, Intramural Research Program National Institute on Aging/National Institutes of Health (NIA/NIH) Baltimore Maryland USADepartment of Molecular and Comparative Pathobiology Johns Hopkins University Baltimore Maryland USADepartment of Molecular and Comparative Pathobiology Johns Hopkins University Baltimore Maryland USANeuroDex, Inc. Natick Massachusetts USANeuroDex, Inc. Natick Massachusetts USAAbstract Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal surface protein composition of heterogeneous EV populations in culture supernatants and in biofluids. Tetraspanins CD9, CD63, and CD81 are common and abundant EV markers. However, their relative enrichment (profile) on EVs of specific cellular origins is not fully elucidated. We introduce LuminEV, a novel version of the Luminex assay for the multiplexed analysis of EV surface proteins. Optimized LuminEV reagents enable direct, specific, and sensitive measurements of EV markers in biofluids and in culture supernatants, bypassing EV isolation step. LuminEV assay for CD9, CD63, and CD81 was validated by comparing simplex and multiplex measurements, establishing linearity, spike‐in recovery, inter‐ and intra‐assay precision, and reproducibility between operators. LuminEV measurements of CD9, CD63, and CD81 in conditioned media from 15 cell lines revealed strong variations between cell types and showed high sensitivity, which enabled EV detection without prior concentration. Using tetraspanin levels as a readout, we noted suppression and induction of EV release from the cultured cells by GW6869 and monensin. Measurement of EV CD9, CD63, and CD81 in blood plasma from 70 disease‐free donors showed respective abundance of 72, 16, and 12%. CD63 displayed weak, albeit significant, negative correlation with age and was slightly lower in female samples. The assay was then used to detect cell type‐specific EV surface markers, including CD235a (erythrocytes), GAP43 (neurons), and CD68 (macrophages), and to detect differences in tetraspanin profiles between healthy and diseased donors. In summary, LuminEV offers robust and sensitive approach for multiplexed assessment of EV surface proteins, to facilitate the research into EV biology, biomarker, and therapeutic applications.https://doi.org/10.1002/jev2.70007assay validationextracellular vesiclessurface marker analysistetraspanin composition |
| spellingShingle | Emma Tordoff Jillian Allen Katya Elgart Ahmed Elsherbini Vrinda Kalia Haotian Wu Erden Eren Dimitrios Kapogiannis Olesia Gololobova Kenneth Witwer Olga Volpert Erez Eitan A novel multiplexed immunoassay for surface‐exposed proteins in plasma extracellular vesicles Journal of Extracellular Vesicles assay validation extracellular vesicles surface marker analysis tetraspanin composition |
| title | A novel multiplexed immunoassay for surface‐exposed proteins in plasma extracellular vesicles |
| title_full | A novel multiplexed immunoassay for surface‐exposed proteins in plasma extracellular vesicles |
| title_fullStr | A novel multiplexed immunoassay for surface‐exposed proteins in plasma extracellular vesicles |
| title_full_unstemmed | A novel multiplexed immunoassay for surface‐exposed proteins in plasma extracellular vesicles |
| title_short | A novel multiplexed immunoassay for surface‐exposed proteins in plasma extracellular vesicles |
| title_sort | novel multiplexed immunoassay for surface exposed proteins in plasma extracellular vesicles |
| topic | assay validation extracellular vesicles surface marker analysis tetraspanin composition |
| url | https://doi.org/10.1002/jev2.70007 |
| work_keys_str_mv | AT emmatordoff anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT jillianallen anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT katyaelgart anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT ahmedelsherbini anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT vrindakalia anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT haotianwu anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT erdeneren anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT dimitrioskapogiannis anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT olesiagololobova anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT kennethwitwer anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT olgavolpert anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT erezeitan anovelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT emmatordoff novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT jillianallen novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT katyaelgart novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT ahmedelsherbini novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT vrindakalia novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT haotianwu novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT erdeneren novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT dimitrioskapogiannis novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT olesiagololobova novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT kennethwitwer novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT olgavolpert novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles AT erezeitan novelmultiplexedimmunoassayforsurfaceexposedproteinsinplasmaextracellularvesicles |