Immunocapture and Flow Cytometry of Extracellular Vesicles for Antigenic Characterization

Begin with a tube carrying cell-derived membrane-bound particles termed extracellular vesicles, or EVs, that exhibit unique antigenic compositions.

Introduce magnetic nanoparticles, MNPs, containing capture antibodies conjugated with fluorescent-labeled Fab fragments.

Incubate to facilitate the interaction between the MNPs with specific antigens of EVs, forming MNP-EV complexes.

Add immunoglobulin-G to block Fc binding sites, then introduce fluorescently labeled detection antibodies to stain other antigens on the captured particles.

Load this mixture onto a pre-washed magnetic column placed in a magnetic separator.

The magnetic field retains MNP-captured particles within the column. Exchange the buffer to remove unbound antibodies.

Retrieve the column from the magnetic separator, elute MNP-captured particles with a buffer, and fix them to preserve their structural integrity.

Analyze the immunocaptured particles by flow cytometry; distinct fluorescence signals from stained antigens correlate with unique antigenic characteristics of EVs.