Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma

Background Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer...

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Autores: López-Cobo, Sheila, Campos-Silva, Carmen, Moyano, Amanda, Oliveira-Rodríguez, Myriam, Paschen, Annette, Yáñez-Mó, María, Blanco-López, María Carmen, Valés-Gómez, Mar
Tipo de recurso: artículo
Fecha de publicación:2018
País:España
Institución:Universidad Loyola Andalucía
Repositorio:Brújula
OAI Identifier:oai:repositorio.uloyola.es:20.500.12412/5646
Acceso en línea:https://hdl.handle.net/20.500.12412/5646
Access Level:acceso abierto
Palabra clave:Lateral flow
Immune capture
Exosomes
Tumour antigens
Steric hindrance
Aggregation
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repository_id_str
spelling Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanomaLópez-Cobo, SheilaCampos-Silva, CarmenMoyano, AmandaOliveira-Rodríguez, MyriamPaschen, AnnetteYáñez-Mó, MaríaBlanco-López, María CarmenValés-Gómez, MarLateral flowImmune captureExosomesTumour antigensSteric hindranceAggregationBackground Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. Methods Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. Results Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. Conclusions These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques.2018info:eu-repo/semantics/articlehttps://hdl.handle.net/20.500.12412/5646reponame:Brújulainstname:Universidad Loyola AndalucíaInglésThis work has been supported by Grants from Madrid Regional Government [IMMUNOTHERCAN‑CM (S2010/BMD‑2326)] and the Spanish Ministry of Economy [SAF2015‑69169‑R (MINEICO/FEDER) and MAT2017‑84959‑C2‑1‑R.; the Network of Excellence for Research in Exosomes, Rediex (MINEICO/FEDER); the Consejería de Economía y Empleo del Principado de Asturias (Plan de Ciencia, Tecnología e Innovación 2013‑2017), under the Grant GRUPIN14‑022. SL‑C was a recipient of a FPU fellowship (MECD), and a travel fellowship from the Spanish Group of Extracellular Vesicles (GEIVEX); CC‑S was a recipient of a master’s fellowship from “Postgrado de la Fundación Ramón Areces‑UAM"http://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:repositorio.uloyola.es:20.500.12412/56462026-06-24T12:48:37Z
dc.title.none.fl_str_mv Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
title Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
spellingShingle Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
López-Cobo, Sheila
Lateral flow
Immune capture
Exosomes
Tumour antigens
Steric hindrance
Aggregation
title_short Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
title_full Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
title_fullStr Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
title_full_unstemmed Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
title_sort Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
dc.creator.none.fl_str_mv López-Cobo, Sheila
Campos-Silva, Carmen
Moyano, Amanda
Oliveira-Rodríguez, Myriam
Paschen, Annette
Yáñez-Mó, María
Blanco-López, María Carmen
Valés-Gómez, Mar
author López-Cobo, Sheila
author_facet López-Cobo, Sheila
Campos-Silva, Carmen
Moyano, Amanda
Oliveira-Rodríguez, Myriam
Paschen, Annette
Yáñez-Mó, María
Blanco-López, María Carmen
Valés-Gómez, Mar
author_role author
author2 Campos-Silva, Carmen
Moyano, Amanda
Oliveira-Rodríguez, Myriam
Paschen, Annette
Yáñez-Mó, María
Blanco-López, María Carmen
Valés-Gómez, Mar
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Lateral flow
Immune capture
Exosomes
Tumour antigens
Steric hindrance
Aggregation
topic Lateral flow
Immune capture
Exosomes
Tumour antigens
Steric hindrance
Aggregation
description Background Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. Methods Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. Results Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. Conclusions These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques.
publishDate 2018
dc.date.none.fl_str_mv 2018
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/20.500.12412/5646
url https://hdl.handle.net/20.500.12412/5646
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv This work has been supported by Grants from Madrid Regional Government [IMMUNOTHERCAN‑CM (S2010/BMD‑2326)] and the Spanish Ministry of Economy [SAF2015‑69169‑R (MINEICO/FEDER) and MAT2017‑84959‑C2‑1‑R.; the Network of Excellence for Research in Exosomes, Rediex (MINEICO/FEDER); the Consejería de Economía y Empleo del Principado de Asturias (Plan de Ciencia, Tecnología e Innovación 2013‑2017), under the Grant GRUPIN14‑022. SL‑C was a recipient of a FPU fellowship (MECD), and a travel fellowship from the Spanish Group of Extracellular Vesicles (GEIVEX); CC‑S was a recipient of a master’s fellowship from “Postgrado de la Fundación Ramón Areces‑UAM"
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.source.none.fl_str_mv reponame:Brújula
instname:Universidad Loyola Andalucía
instname_str Universidad Loyola Andalucía
reponame_str Brújula
collection Brújula
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,81155

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