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

Abstract

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 10 4 vesicles for MCF7 and Plasma sEVs and 10 5 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.

Progress in Isolation and Molecular Profiling of Small Extracellular Vesicles via Bead-Assisted Platforms

“…The sEVs in PBS and in spiked serum samples were successfully quantified with the limit of detection of 1 × 10 2 vesicles per µL. A comparative study for the identification of EpCAM and HER2 surface proteins in the sEVs derived from MCF7, SKOV3, MDA-MB-231 and CHO cell lines and blood plasma of a healthy donor was carried out using anti-EpCAM and anti-HER2 designed ankyrin repeat proteins (DARPins) [66]. Both DRAPins showed high specificity to EpCAM and HER2 receptors, whereas the highly reactive nature DARPins, along with magnetic capture, allowed reducing total assay time.…”

Studying the small extracellular vesicle capture efficiency of magnetic beads coated with tannic acid

“…19 Among the numerous types of beads, magnetically responsive beads are particularly attractive because they can be collected by a magnet, which in most cases is advantageous compared to centrifugation, because it can reduce the isolation time and minimise impurities. 20,21 Tauro and co-workers compared immunomagnetic capture of sEVs (IAC-Exos) with ultracentrifugation (UC-Exos) and OptiPrepTM density-based separation (DG-Exos) methods and found that IAC-Exos is the most effective method to isolate sEVs enabling identification of cancer-related proteins and signalling molecules in sEVs. 22 Brett et al concluded that commercial isolation kits are not able to remove plasma proteins from sEV samples, while an immunoaffinity-based approach was highly effective for trapping highly purified sEVs.…”

Filtration-based technologies for isolation, purification and analysis of extracellular vesicles

“…[11][12][13] Sucrose or iodixanol can also be used to form a density gradient allowing to separate and purify EVs based on their buoyant density. 13,14 Other developed EV isolation methods include size-exclusion chromatography, 15,16 field-flow fractionation (FFF), 3,17 immuno-affinity capture, 18,19 polymer-based precipitation, [20][21][22] and microfluidics. 23 Combinations of such methods were also previously presented with the main focus being mostly to achieve an increase in purity and yield.…”