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, researchers from the Erasmus Medical Center 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 detected lower and higher AQP2 or NCC levels in uEVs from patients with pathological water or salt reabsorption, respectively.
uEV-immunoassay set-up showing the neutravidin coated plate and the four antibodies, including the biotinylated anti-CD9 (capture) antibody, anti-AQP2 or anti-NCC antibody (protein of interest) targeting an intracellular domain, and the two luminescent antibodies to quantify the number of uEVs (Europium-conjugated anti-CD9 antibody) and the protein of interest (horseradish-peroxide-conjugated antibody).