A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma
Entity
UAM. Departamento de Biología MolecularPublisher
BMCDate
2022-02-08Citation
10.1186/s12951-022-01256-5
Journal of Nanobiotechnology 20.1 (2022): 72
ISSN
1477-3155DOI
10.1186/s12951-022-01256-5Project
Gobierno de España. RTI2018-093569-B-I00; Gobierno de España. RED2018102411-T; Comunidad de Madrid. 2017/BMD-3733-2/IMMUNOTHERCANEditor's Version
https://doi.org/10.1186/s12951-022-01256-5Subjects
Cancer; Colloids; ELISA; Extracellular vesicles; Flocculation; Flow cytometry; Liquid biopsy; Biología y Biomedicina / BiologíaRights
© The Author(s) 2022
Esta obra está bajo una licencia de Creative Commons Reconocimiento-CompartirIgual 4.0 Internacional.
Abstract
Background: Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors. Results: Here we describe a method that, using just a few microliters of patient’s plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. Conclusions: This optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones
Files in this item
Google Scholar:Campos Silva, Carmen
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Cáceres-Martell, Yaiza
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Sánchez Herrero, Estela
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Sandúa, Amaia
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Beneitez-Martínez, Alexandra
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González, Álvaro
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Provencio Pulla, Mariano
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Romero, Atocha
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Jara-Acevedo, Ricardo
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Yáñez Mo, María
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Valés Gómez, Mar
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