Antigenic composition of single nano-sized extracellular blood vesicles

Abstract

Extracellular vesicles (EVs) are important in normal physiology and are altered in various pathologies. EVs produced by different cells are antigenically different. Since the majority of EVs are too small for routine flow cytometry, EV composition is studied predominantly in bulk, thus not addressing their antigenic heterogeneity. Here, we describe a nanoparticle-based technique for analyzing antigens on single nano-sized EVs. The technique consists of immuno-capturing of EVs with 15-nm magnetic nanoparticles, staining captured EVs with antibodies against their antigens, and separating them from unbound EVs and free antibodies in a magnetic field, followed by flow analysis. This technique allows us to characterize EVs populations according to their antigenic distribution, including minor EV fractions. We demonstrated that the individual blood EVs carry different sets of antigens, none being ubiquitous, and quantified their distribution. The physiological significance of antigenically different EVs and their correlation with different pathologies can now be directly addressed.

Flow analysis of individual blood extracellular vesicles in acute coronary syndrome

“…Earlier we demonstrated that the efficiency of our technique for capture of viruses and EVs of interest is about 99% [25,30]. In the current work we confirmed this result: we captured EVs via MNP-coupled antibodies against CD41a, stained them with antibodies against CD31 and CD63 and isolated on magnetic columns.…”

“…Here, we attempted to trace the origin of EVs by determining the antigenic composition of individual EVs isolated from blood of healthy volunteers and from blood of patients with acute forms of coronary artery disease, in particular myocardial infarction. This task was accomplished with the new technology “flow virometry”, which was originally developed for the analysis of individual virions [30] and later adapted to analyze individual small EVs [25]. …”

“…Recently, we overcame these obstacles and developed a nanotechnology-based system that permits the analysis of the antigenic composition of individual EVs [25]. Here, we applied this method to identify the antigenic composition of individual EVs in the blood of healthy volunteers and patients with acute forms of coronary artery disease.…”

Extracellular vesicles and viruses: Are they close relatives?

“…For example, recent developments in flow cytometrybased techniques have opened up the possibility to quantify and characterize particles 50-200 nm in size. Such developments include not only hardware adaptations in high-end flow cytometers to improve signal-to-noise ratios, but also optimized staining protocols for general labeling of EVs and the use of magnetic nanoparticles to screen the surface antigenic composition of EVs (47,48).…”

Therapeutic and diagnostic applications of extracellular vesicles

“…However, for diagnostic purposes such an approach will require screening of large amounts of vesicles as 'diseased' EVs are rare among the total isolated EV population. One promising approach relies on modifying flow cytometry equipment/protocols to detect single, nanosized EVs [244][245][246]. However, to date FACS is not able to detect the lower size range of EVs and requires antibodies (and hence also knowledge of a particular disease marker) to phenotype EVs.…”