Fig. 5

Sandwich assay for EV capture with VHH-GFP constructs tested by flow-cytometry and ELISA. Latex beads were coated with purified nanobodies, EVs were captured from cell culture and detected by flow-cytometry with anti-CD9-PE labelled antibodies. Bars indicate median fluorescence intensity of bound anti-CD9–PE antibodies to H1-GFP, H6-GFP, and irrelevant (EV-neg) VHH-GFP coated beads (a). Direct capture of EVs from plasma (b) was assessed by flow-cytometry using VHH-GFP coated beads. Anti-CD9-PE was used for detection and bead autofluorescence was measured in the absence of plasma (controls). c ELISA microplates were either directly coated with EV-enriched fractions or EVs were bound by means of previously immobilized purified anti-CD9 nanobodies. Such EV-prepared microplates were used to capture anti-EV nanobodies displayed on phages which were detected by adding anti-M13, HRP-labelled antibodies. The error bars indicate standard deviations for triplicate measurements