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Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test

Extracellular vesicles (EVs) have become a topic of interest within the field of diagnostic biomarkers; however, recent developments in the study of EVs have increased the need for simpler but still comprehensive methods for characterization. Here, we describe how to simultaneously measure several s...

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Autores principales: Clegg, Lee-Ann Marie, Sloth, Jenni Kathrine, Bæk, Rikke, Jørgensen, Malene Møller
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9642848/
https://www.ncbi.nlm.nih.gov/pubmed/36387270
http://dx.doi.org/10.3389/fmolb.2022.917487
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author Clegg, Lee-Ann Marie
Sloth, Jenni Kathrine
Bæk, Rikke
Jørgensen, Malene Møller
author_facet Clegg, Lee-Ann Marie
Sloth, Jenni Kathrine
Bæk, Rikke
Jørgensen, Malene Møller
author_sort Clegg, Lee-Ann Marie
collection PubMed
description Extracellular vesicles (EVs) have become a topic of interest within the field of diagnostic biomarkers; however, recent developments in the study of EVs have increased the need for simpler but still comprehensive methods for characterization. Here, we describe how to simultaneously measure several surface or surface-associated proteins on EVs using a multiparametric microarray-based analysis termed Extracellular Vesicle Array (EV Array), which is developed to catch and phenotypically characterize small EVs. Previously, this analysis has been limited to measuring only one fluorescent signal per analysis. The analysis relies on antibodies printed onto a solid surface, for catching the EVs carrying the specific surface or surface-associated proteins, and on the subsequent fluorescent detection. For the optimization of detection, two antibodies with attached Cy3 or Cy5 were added to various combinations of the EV surface or surface-associated proteins: CD9, CD63, CD81, flotillin-1, and HSP90. In this study, the EV surface or surface-associated proteins were analyzed in human plasma from six healthy subjects. Changes observed in signal intensities from Cy3 and Cy5 related specifically to these combinations and allowed for a comparison of the two different fluorescent signals. When comparing the results, it was observed that it is possible to measure the EV surface or surface-associated proteins at both 532 nm (Cy3) and 635 nm (Cy5) simultaneously without a significant change in signals from the detection molecules. This allows us to measure multiple EV marker proteins in a single analysis, thereby more quickly finding complex biomarker patterns in a sample.
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spelling pubmed-96428482022-11-15 Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test Clegg, Lee-Ann Marie Sloth, Jenni Kathrine Bæk, Rikke Jørgensen, Malene Møller Front Mol Biosci Molecular Biosciences Extracellular vesicles (EVs) have become a topic of interest within the field of diagnostic biomarkers; however, recent developments in the study of EVs have increased the need for simpler but still comprehensive methods for characterization. Here, we describe how to simultaneously measure several surface or surface-associated proteins on EVs using a multiparametric microarray-based analysis termed Extracellular Vesicle Array (EV Array), which is developed to catch and phenotypically characterize small EVs. Previously, this analysis has been limited to measuring only one fluorescent signal per analysis. The analysis relies on antibodies printed onto a solid surface, for catching the EVs carrying the specific surface or surface-associated proteins, and on the subsequent fluorescent detection. For the optimization of detection, two antibodies with attached Cy3 or Cy5 were added to various combinations of the EV surface or surface-associated proteins: CD9, CD63, CD81, flotillin-1, and HSP90. In this study, the EV surface or surface-associated proteins were analyzed in human plasma from six healthy subjects. Changes observed in signal intensities from Cy3 and Cy5 related specifically to these combinations and allowed for a comparison of the two different fluorescent signals. When comparing the results, it was observed that it is possible to measure the EV surface or surface-associated proteins at both 532 nm (Cy3) and 635 nm (Cy5) simultaneously without a significant change in signals from the detection molecules. This allows us to measure multiple EV marker proteins in a single analysis, thereby more quickly finding complex biomarker patterns in a sample. Frontiers Media S.A. 2022-10-25 /pmc/articles/PMC9642848/ /pubmed/36387270 http://dx.doi.org/10.3389/fmolb.2022.917487 Text en Copyright © 2022 Clegg, Sloth, Bæk and Jørgensen. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Clegg, Lee-Ann Marie
Sloth, Jenni Kathrine
Bæk, Rikke
Jørgensen, Malene Møller
Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test
title Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test
title_full Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test
title_fullStr Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test
title_full_unstemmed Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test
title_short Photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test
title_sort photometric method for dual targeting of surface and surface-associated proteins on extracellular vesicles in the multiparametric test
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9642848/
https://www.ncbi.nlm.nih.gov/pubmed/36387270
http://dx.doi.org/10.3389/fmolb.2022.917487
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