Anti-CD63-Oligonucleotide Functionalized Magnetic Beads for the Rapid Isolation of Small Extracellular Vesicles and Detection of EpCAM and HER2 Membrane Receptors using DARPin Probes
The development of a sensitive and reliable method for the isolation and detection of pathologically relevant small extracellular vesicles (sEVs) remains a challenge. Herein we present a method for characterization of sEVs that is based on the isolation of sEVs by anti-CD63 aptamer conjugated magnetic beads and detecting the presence of the membrane proteins on their surface using anti-EpCAM and anti-HER2 designed ankyrin repeat proteins (DARPins). We perform a comparative study for identification of EpCAM and HER2 surface proteins in sEVs derived from MCF7, SKOV3, MDA-MB-231 and CHO cell lines and blood plasma of a healthy donor. Both DRAPins show high specificity to EpCAM and HER2 receptors with a limit of detection about 104 vesicles for MCF7 and Plasma sEVs and 105 vesicles for SKOV3 sEVs. Combination of magnetic capture with highly reactive DARPin probes made it possibly to reduce total assay time to 1 h. This method provides a basis for isolating and quantifying various sEV subpopulations that contain disease specific surface-markers.
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