A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma

Journal of Nanobiotechnology

Yaiza Cáceres-Martell¹  Carmen Campos-Silva¹  Mar Valés‐Gómez¹  María Yáñez-Mó²  Ricardo Jara-Acevedo³  Alexandra Beneitez-Martínez³  Estela Sánchez-Herrero⁴  Atocha Romero⁵  Mariano Provencio⁵  Amaia Sandúa⁶  Álvaro González⁶ 

[1] Department of Immunology and Oncology, Spanish National Centre for Biotechnology, CNB-CSIC, Madrid, Spain;Department of Molecular Biology, UAM - Centro de Biología Molecular Severo Ochoa, Madrid, Spain;Instituto de Investigación del Hospital Universitario La Princesa, Madrid, Spain;Immunostep, S.L., Salamanca, Spain;Laboratorio de Biopsia Líquida, Instituto de Investigación Sanitaria Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain;Atrys Health, Barcelona, Spain;Laboratorio de Biopsia Líquida, Instituto de Investigación Sanitaria Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain;Medical Oncology Department, Hospital Universitario Puerta de Hierro, Majadahonda, Madrid, Spain;Service of Biochemistry, Clínica Universidad de Navarra, Pamplona, Spain;

关键词: Extracellular vesicles;    Cancer;    Colloids;    Flocculation;    ELISA;    Flow cytometry;    Liquid biopsy;   

DOI  :  10.1186/s12951-022-01256-5

来源: Springer

【 摘 要 】

BackgroundExtracellular 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.ResultsHere 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.ConclusionsThis 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.Graphical Abstract

【 授权许可】

CC BY