The expressions of MCSP and ErbB3 on the surface of EVs were measured using commercial ELISA kits: MCSP (Sigma-Aldrich, RAB1594) and ErbB3 (Sigma-Aldrich, RAB0174). The assays were conducted according to the manufacturer’s instructions. Briefly, 100 μl of standards, controls, and unknown samples was introduced to selected wells and incubated at 4°C overnight. Afterward, the reaction wells were washed four times with wash buffer. One hundred microliters of biotinylated detection antibody was then added to all wells and incubated for 1 hour at RT with gentle shaking. The wells were subsequently washed four times with wash buffer. The horseradish peroxidase–streptavidin reagent was added to all wells and incubated for 45 min at RT. The reaction wells were then washed four times with wash buffer, followed by 30-min incubation with 100 μl of 3,3′,5,5′-tetramethylbenzidine reagent at RT in the dark with gentle shaking. Last, all wells were supplemented with 50 μl of stop solution and analyzed for absorbance at 450 nm.

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