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 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.
Electron Microscope Core Facility, Niigata University, Niigata 951-851, Japan. nametan@med.niigata-u.ac.jp.
Masaaki
M
Nameta
Department of Research and Development, Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokusima 771-0182, Japan. saijoy@otsuka.jp.
Yoko
Y
Saijo
Department of Medical Innovations, New Drug Research Division, Otsuka Pharmaceutical Co., Ltd., Tokusima 771-0192, Japan. ohmoto.yasukazu@otsuka.jp.
Yasukazu
Y
Ohmoto
Department of Research and Development, Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokusima 771-0182, Japan. katsuragik@otsuka.jp.
Kiyonori
K
Katsuragi
Biofluid Biomarker Center, Institute of Social Innovation and Promotion, Niigata University, Niigata 950-2181, Japan. keikoy-bbc@ccr.niigata-u.ac.jp.
Keiko
K
Yamamoto
Biofluid Biomarker Center, Institute of Social Innovation and Promotion, Niigata University, Niigata 950-2181, Japan. tadashiy-bbc@ccr.niigata-u.ac.jp.
Tadashi
T
Yamamoto
Department of Pathophysiology, Meiji Pharmaceutical University, Tokyo 204-8588, Japan. kishiba@my-pharm.ac.jp.
Kenichi
K
Ishibashi
Department of Pathophysiology, Meiji Pharmaceutical University, Tokyo 204-8588, Japan. ssasaki.kid@tmd.ac.jp.|Department of Nephrology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan. ssasaki.kid@tmd.ac.jp.
Sei
S
Sasaki
ELISA; aquaporin-2 (AQP2); biomarker; exosome; extracellular vesicle; kidney; water-balance
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