Fig. 2

Binding characteristics of phage-displayed nanobodies isolated by direct panning on EV-enriched samples. a Phage-ELISA of clones selected by panning was performed to confirm their specific binding to EV-enriched fractions recovered from SKBR3 and HEK293 cells. Assay microplates were coated with EV-enriched samples (5 μg/well of protein) and bound phages were detected with HRP-labelled anti-M13 antibodies. An irrelevant phage-displayed nanobody was used as a negative control. b Characterization of phages binding to EVs by double-staining flow-cytometry. The vesicle fractions (positive for anti-CD9 APC labelled antibodies) were further incubated with the nanobody-displaying phages previously isolated. Bound phages were visualized after staining by the addition of anti-M13 monoclonal antibodies and goat anti mouse PE antibodies. Gates were set according to the values of autofluorescence of naked beads (not coated with EVs) and an irrelevant phage-displayed nanobody was used as a negative control