Fig. 2: Orthogonal validation of RPPA results on serum-derived EVs.

A Representative scheme of our homemade ELISA test, i.e. ELEXO, including EV-coated microtiter plate, primary antibody, HRP-conjugated secondary antibody and colorimetric reaction with TMB substrate. B H1975 and H1299 EVs analyzed by ELEXO assay. IgG isotype and Phosphate-Buffered Saline (PBS) were used as internal controls. Data are reported as mean and SD (n = 3) of arbitrary units of O.D. (Optical Density) at the specified wavelength (nm) (*p = 0.02). C, D Representative images of SEM (C) and TEM (D) analysis of EVs isolated from the sera of prostate cancer patients. Images are representative of pivotal and training cohort PCas. E Box plot representative of serum EV size distribution evaluated by SEM analysis. F, G ELEXO measurement of EpCAM (F) and PD-L1 (G) antigens in EVs isolated from colon and lung cancer patients, respectively. CD81 has been used as endogenous EV control. Data are reported as mean and SD (n = 3) of arbitrary units of O.D. (Optical Density) at the specified wavelength (nm). Internal reference controls were reported as IgG, CD-81, EpCAM, or PD-L1 antibody in PBS condition. H Absolute RPPA quantification of EGFR_pY1068 synthetic peptide in dilution curve along with isolated EVs from sera of colon and lung cancer patients. The expression levels in EVs fall below the lowest EGFR_pY1068 peptide dilution point and their scale is magnified in the left panel of the plot. The reference curve in the lower panel was obtained as in Fig. 1D to predict absolute EGFR_pY1068 RPPA levels in EV samples.