An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles

Campos-Silva, Carmen; Cáceres‐Martell, Yaiza; López‐Cobo, Sheila; Rodriguez, María Josefa; Jara, Ricardo; Yáñez-Mó, Marı́a; Valés‐Gómez, Mar

doi:10.1007/978-1-0716-1205-7_24

Cited by 10 publications

(9 citation statements)

References 39 publications

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“…We have recently described a high sensitivity method for immunocapture and detection of EVs by flow cytometry, based on the use of antibody-coated beads followed by detection with a labelled antibody [ 22 , 29 ]. During the optimisation of that method, we calculated the theoretical number of EVs that would bind the antibody-coated microspheres and analysed the saturation curve in experiments with increasing amounts of EVs.…”

Section: Resultsmentioning

confidence: 99%

“…Cells were grown until 70% confluence and then changed into medium prepared with 1% EV-free FBS (prepared by ultracentrifugation at 100,000× g for 20 h), for EV accumulation during 3–4 days. Cell supernatants were centrifuged for 10 min at 200× g and small EVs enriched by sequential centrifugation as previously described [ 29 , 70 ]. After ultracentrifugation at 100,000× g for 2 h at 4 °C, EVs were resuspended in 0.22 µm filtered HEPES-buffered saline (HBS: 10 mM HEPES pH 7.2, 150 mM NaCl) (2.67 µl/ml of starting cell culture supernatant) and stored at − 20 °C, for short term use, or at − 80 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Beads were acquired by flow cytometry using Gallios, Cytomics FC 500 (Beckman Coulter) or CytoFLEX (Beckman Coulter) and data were analysed using Kaluza (Beckman Coulter) or FlowJo (Tree Star, Inc) software. Single beads were gated in Forward Scatter in the region corresponding to 6 μm [established using calibration beads (FlowCheck ProTM fluorospheres, Beckman Coulter, Brea, CA, USA)], excluding bead doublets and selecting APC-positive events [ 29 ]. PE MFI was analysed within the 6 μm-APC-positive events.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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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. Graphical Abstract

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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“…Cells were grown until 70% confluence and then changed into medium prepared with 1% EV-free FBS (prepared by ultracentrifugation at 100,000 × g for 20 hours), for EV accumulation during 3-4 days. Cell supernatants were centrifuged for 10 min at 200 × g and small EVs enriched by sequential centrifugation as previously described 32, 33 . After ultracentrifugation at 100,000 × g for 2 hours at 4°C, EVs were resuspended in 0.22 µm filtered HEPES-buffered saline (HBS: 10mM HEPES pH 7.2, 150mM NaCl) (2.67 µl/ml of starting cell culture supernatant) and stored at −20°C, for short term use, or at −80°C.…”

Section: Methodsmentioning

confidence: 99%

“…Beads were acquired by flow cytometry using Gallios, Cytomics FC 500 (Beckman Coulter) or CytoFLEX (Beckman Coulter) and data were analysed using Kaluza (Beckman Coulter) or FlowJo (Tree Star, Inc) software. Single beads were gated in Forward Scatter in the region corresponding to 6 μm [established using calibration beads (FlowCheck ProTM fluorospheres, Beckman Coulter, Brea, CA, USA)], excluding bead doublets and selecting APC-positive events 33 . PE MFI was analysed within the 6 μm-APC-positive events.…”

Section: Methodsmentioning

confidence: 99%

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

Campos-Silva

Cáceres‐Martell

Sánchez‐Herrero

et al. 2021

Preprint

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Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. Here we describe a method that, using just a few microliters of patient 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. This 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.

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Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography

Benayas,

Morales,

Egea

et al. 2023

J of Extracellular Bio

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Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non‐EV contaminants present in biofluid samples in a one‐step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation.

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An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles

Cited by 10 publications

References 39 publications

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

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

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

Optimization of extracellular vesicle isolation and their separation from lipoproteins by size exclusion chromatography

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