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2016 ; 17
(10
): ä Nephropedia Template TP
gab.com Text
Twit Text FOAVip
Twit Text #
English Wikipedia
Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA
Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies
#MMPMID27681727
Nameta M
; Saijo Y
; Ohmoto Y
; Katsuragi K
; Yamamoto K
; Yamamoto T
; Ishibashi K
; Sasaki S
Int J Mol Sci
2016[Sep]; 17
(10
): ä PMID27681727
show ga
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.