An Immunoassay for Urinary Extracellular Vesicles.

An Immunoassay for Urinary Extracellular Vesicles. Am J Physiol Renal Physiol. 2016 Jan 28;:ajprenal.00463.2015 Authors: Salih M, Fenton RA, Knipscheer J, Janssen JW, Vredenbregt-van den Berg MS, Jenster G, Zietse R, Hoorn EJ 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-immunoa...
Source: Am J Physiol Renal P... - Category: Urology & Nephrology Authors: Tags: Am J Physiol Renal Physiol Source Type: research