An immunoassay for urinary extracellular vesicles

American Journal of Physiology. Renal Physiology
Mahdi SalihEwout J Hoorn

Abstract

Although nanosized urinary extracellular vesicles (uEVs) are increasingly used for biomarker discovery, their isolation currently relies on time-consuming techniques hindering high-throughput application. To navigate this problem, we designed an immunoassay to isolate, quantify, and normalize uEV proteins. The uEV immunoassay consists of a biotinylated CD9 antibody to isolate uEVs, an antibody against the protein of interest, and two conjugated antibodies to quantify the protein of interest and CD9. As a proof of principle, the immunoassay was developed to analyze the water channel aquaporin-2 (AQP2) and the sodium-chloride cotransporter (NCC). CD9 was used as a capture antibody because immunoprecipitation showed that anti-CD9 antibody, but not anti-CD63 antibody, isolated AQP2 and NCC. CD9 correlated strongly with urine creatinine, allowing CD9 to be used for normalization of spot urines. The uEV immunoassay detected AQP2 and NCC with high sensitivity, low coefficients of variance, and stability in dilution series. After water loading in healthy subjects, the uEV immunoassay detected decreases in AQP2 and NCC equally well as the traditional method using ultracentrifugation and immunoblot. The uEV immunoassay also reliably dete...Continue Reading

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Citations

Sep 20, 2018·American Journal of Physiology. Renal Physiology·Robert A Fenton
Sep 29, 2018·Channels·Hila WardakJenny Van Der Wijst
Nov 24, 2018·Proteomics·Bas W M van BalkomSai Kiang Lim
Oct 26, 2018·American Journal of Physiology. Renal Physiology·Lennart TonneijckDaniël H van Raalte
Aug 15, 2019·American Journal of Physiology. Renal Physiology·Charles J Blijdorp, Ewout J Hoorn
Jun 29, 2018·Frontiers in Oncology·Matteo GiuliettiRodolfo Montironi
Aug 8, 2019·American Journal of Physiology. Renal Physiology·Erik H KoritzinskyPeter S T Yuen
Oct 17, 2020·Pharmaceutical Research·Menchus Quan, Shihuan Kuang
Mar 31, 2021·Journal of the American Society of Nephrology : JASN·Charles J BlijdorpEwout J Hoorn
May 27, 2021·Journal of Extracellular Vesicles·Uta ErdbrüggerElena S Martens-Uzunova
Jul 1, 2021·Expert Review of Proteomics·Qi Wu, Robert A Fenton

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