Affinity-based isolation of extracellular vesicles and the effects on downstream molecular analysis.

Extracellular vesicles (EVs) are transport vesicles with diameters ranging from 30 to 1000 nm, secreted by cells in both physiological and pathological conditions. By using the EV shuttling system, biomolecular cargo such as proteins and genetic materials travels between cells resulting in intercellular communication and epigenetic regulation. Because the presence of EVs and cargo molecules in body fluids can predict the state of the parental cells, EV isolation techniques from complex biofluids have been developed. Further exploration of EVs through downstream molecular analysis depends heavily on those isolation technologies. Methodologies based either on physical separation or on affinity binding have been used to isolate EVs. Affinity-based methods for EV isolation are known to produce highly specific and efficient isolation results. However, so far, there is a lack of literature summarizing these methods and their effects on downstream EV molecular analysis. In the present work, we reviewed recent efforts on developing affinity-based methods for the isolation of EVs, with an emphasis on comparing their effects on downstream analysis of EV molecular cargo. Antibody-based isolation techniques produce highly pure EVs, but the harsh eluents damage the EV structure, and some antibodies stay bound to the EVs after elution. Aptamer-based methods use relatively mild elution conditions and release EVs in their native form, but their isolation efficiencies need to be improved. The membrane affinity-based method and other affinity-based methods based on the properties of the EV lipid bilayer also isolate intact EVs, but they can also result in contaminants. From the perspective of affinity-based methods, we investigated the influence of the isolation methods of choice on downstream EV molecular analysis.