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Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies

Aquaporin-2 (AQP2) is present in urine extracellular vesicles (EVs) and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at −25 °C is required for enzyme-linked immunosorbent assay (ELISA) measurement. We speculated t...

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Detalles Bibliográficos
Autores principales: Nameta, Masaaki, Saijo, Yoko, Ohmoto, Yasukazu, Katsuragi, Kiyonori, Yamamoto, Keiko, Yamamoto, Tadashi, Ishibashi, Kenichi, Sasaki, Sei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085667/
https://www.ncbi.nlm.nih.gov/pubmed/27681727
http://dx.doi.org/10.3390/ijms17101634
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author Nameta, Masaaki
Saijo, Yoko
Ohmoto, Yasukazu
Katsuragi, Kiyonori
Yamamoto, Keiko
Yamamoto, Tadashi
Ishibashi, Kenichi
Sasaki, Sei
author_facet Nameta, Masaaki
Saijo, Yoko
Ohmoto, Yasukazu
Katsuragi, Kiyonori
Yamamoto, Keiko
Yamamoto, Tadashi
Ishibashi, Kenichi
Sasaki, Sei
author_sort Nameta, Masaaki
collection PubMed
description Aquaporin-2 (AQP2) is present in urine extracellular vesicles (EVs) and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at −25 °C is required for enzyme-linked immunosorbent assay (ELISA) measurement. We speculated that disruptions of EVs membranes are necessary to allow for the direct contact of antibodies with their epitopes. Human urine EVs were prepared using an ultracentrifugation method. Urine EV samples were stored at different temperatures for a week. Electron microscopy showed abundant EVs with diameters of 20–100 nm, consistent with those of exosomes, in normal urine, whereas samples from alkali/detergent pre-treated urine showed fewer EVs with large swollen shapes and frequent membrane disruptions. The abundance and structures of EVs were maintained during storage at −80 °C, but were severely damaged at −25 °C. Binding and competitive inhibition assays showed that epitopes of monoclonal antibody and polyclonal antibody were the hydrophilic Loop D and C-terminus of AQP2, respectively, both of which are present on the inner surface of EVs. Thus, urine storage at −25 °C or pre-treatment with alkali/detergent disrupt EVs membranes and allow AQP2 antibodies to bind to their epitopes located inside EVs.
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spelling pubmed-50856672016-11-01 Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies Nameta, Masaaki Saijo, Yoko Ohmoto, Yasukazu Katsuragi, Kiyonori Yamamoto, Keiko Yamamoto, Tadashi Ishibashi, Kenichi Sasaki, Sei Int J Mol Sci Communication Aquaporin-2 (AQP2) is present in urine extracellular vesicles (EVs) and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at −25 °C is required for enzyme-linked immunosorbent assay (ELISA) measurement. We speculated that disruptions of EVs membranes are necessary to allow for the direct contact of antibodies with their epitopes. Human urine EVs were prepared using an ultracentrifugation method. Urine EV samples were stored at different temperatures for a week. Electron microscopy showed abundant EVs with diameters of 20–100 nm, consistent with those of exosomes, in normal urine, whereas samples from alkali/detergent pre-treated urine showed fewer EVs with large swollen shapes and frequent membrane disruptions. The abundance and structures of EVs were maintained during storage at −80 °C, but were severely damaged at −25 °C. Binding and competitive inhibition assays showed that epitopes of monoclonal antibody and polyclonal antibody were the hydrophilic Loop D and C-terminus of AQP2, respectively, both of which are present on the inner surface of EVs. Thus, urine storage at −25 °C or pre-treatment with alkali/detergent disrupt EVs membranes and allow AQP2 antibodies to bind to their epitopes located inside EVs. MDPI 2016-09-26 /pmc/articles/PMC5085667/ /pubmed/27681727 http://dx.doi.org/10.3390/ijms17101634 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Nameta, Masaaki
Saijo, Yoko
Ohmoto, Yasukazu
Katsuragi, Kiyonori
Yamamoto, Keiko
Yamamoto, Tadashi
Ishibashi, Kenichi
Sasaki, Sei
Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies
title Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies
title_full Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies
title_fullStr Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies
title_full_unstemmed Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies
title_short Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies
title_sort disruption of membranes of extracellular vesicles is necessary for elisa determination of urine aqp2: proof of disruption and epitopes of aqp2 antibodies
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085667/
https://www.ncbi.nlm.nih.gov/pubmed/27681727
http://dx.doi.org/10.3390/ijms17101634
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