Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma
Abstract 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 hum...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-05-01
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| Series: | Journal of Nanobiotechnology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12951-018-0372-z |
| _version_ | 1817989374479958016 |
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| author | Sheila López-Cobo Carmen Campos-Silva Amanda Moyano Myriam Oliveira-Rodríguez Annette Paschen María Yáñez-Mó María Carmen Blanco-López Mar Valés-Gómez |
| author_facet | Sheila López-Cobo Carmen Campos-Silva Amanda Moyano Myriam Oliveira-Rodríguez Annette Paschen María Yáñez-Mó María Carmen Blanco-López Mar Valés-Gómez |
| author_sort | Sheila López-Cobo |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-14T00:46:09Z |
| format | Article |
| id | doaj.art-6ddd4a4f12704d90bf1f49fb3ac900a4 |
| institution | Directory Open Access Journal |
| issn | 1477-3155 |
| language | English |
| last_indexed | 2024-04-14T00:46:09Z |
| publishDate | 2018-05-01 |
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| series | Journal of Nanobiotechnology |
| spelling | doaj.art-6ddd4a4f12704d90bf1f49fb3ac900a42022-12-22T02:22:01ZengBMCJournal of Nanobiotechnology1477-31552018-05-0116111210.1186/s12951-018-0372-zImmunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanomaSheila López-Cobo0Carmen Campos-Silva1Amanda Moyano2Myriam Oliveira-Rodríguez3Annette Paschen4María Yáñez-Mó5María Carmen Blanco-López6Mar Valés-Gómez7Department of Immunology and Oncology, National Centre for Biotechnology, CNB-CSICDepartment of Immunology and Oncology, National Centre for Biotechnology, CNB-CSICDepartment of Physical and Analytical Chemistry, University of OviedoDepartment of Physical and Analytical Chemistry, University of OviedoDepartment of Dermatology, University Hospital Essen, University Duisburg-Essen, and German Cancer Consortium (DKTK) Partner Site Essen/DüsseldorfDepartment of Molecular Biology, UAMDepartment of Physical and Analytical Chemistry, University of OviedoDepartment of Immunology and Oncology, National Centre for Biotechnology, CNB-CSICAbstract 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.http://link.springer.com/article/10.1186/s12951-018-0372-zLateral flowImmune captureExosomesTumour antigensSteric hindranceAggregation |
| spellingShingle | Sheila López-Cobo Carmen Campos-Silva Amanda Moyano Myriam Oliveira-Rodríguez Annette Paschen María Yáñez-Mó María Carmen Blanco-López Mar Valés-Gómez Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma Journal of Nanobiotechnology Lateral flow Immune capture Exosomes Tumour antigens Steric hindrance Aggregation |
| title | 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_short | 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 |
| topic | Lateral flow Immune capture Exosomes Tumour antigens Steric hindrance Aggregation |
| url | http://link.springer.com/article/10.1186/s12951-018-0372-z |
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