Rapid, accurate isolation and quantitation of extracellular vesicles

Isolation of EVs is challenging due to their small size, interaction with proteins, and other molecules, and susceptibility to deformation. Furthermore, many purification techniques are not suitable for raw biofluids such as plasma, meaning that additional, laborious preparative steps are required. Izon’s qEV Isolation columns are based on size-exclusion chromatography to enable rapid and gentle purification of EVs with almost complete (>97%) removal of contaminating proteins, including free proteins and viral-protein aggregates (Figure 1). The biophysical integrity of the vesicles is unchanged, and EVs are eluted into buffers suitable for downstream analyses with user-specified volumes. Combining qEV Isolation columns with Izon’s Automated Fraction Collector (AFC) creates a streamlined workflow, which achieves reliable and reproducible isolation in under 15 minutes with minimal inter- and intra-operator variability.

Despite their importance in almost all cell functions and potential therapeutic applications, accurate characterisation of EVs is technically challenging. The concentration of EVs in biofluids has been suggested to be indicative of health status and have potential diagnostic applications; therefore, direct and accurate quantification of EVs is essential. However, the field is limited by a lack of standardised, robust techniques as well as a historical reliance on ensemble and indirect techniques. Ensemble techniques do not provide sufficient resolution for diagnostic purposes and overlook the presence of potentially important subpopulations, while indirect techniques—such as detection of EV proteins—are influenced by heterogeneity within the sample, leading to inaccurate quantification.

The Exoid offers the only method of direct quantification of EVs that does not require laborious or complex protocols. Prior knowledge or input of particle properties is not necessary, removing any guesswork and ensuring that even previously uncharacterised EV samples are quantified with high accuracy and precision (Figure 2). As the only method of determining true particle concentration and size distribution, TRPS offers fast, accurate, and reproducible analysis. The semi-automated Exoid system enables high-throughput measurements to be carried out with ease and minimises intra-/inter-operator variability.

While size is the most commonly analysed EV parameter, there is growing evidence of the importance of other properties such as surface charge. Multi-parameter measurement techniques are becoming critical in research as well as the development of EV-based therapeutics. Regulatory bodies are recognising that reliable, reproducible, and comparable multi-parameter characterisation data are essential for such particles. The Exoid offers the only standardisable method of characterising EV size, concentration, and zeta potential. As every particle in the sample is characterised and individually calibrated to NIST-traceable particles, the accuracy of single-particle data are assured.  
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Reliable and accurate data enable inter- and intra-laboratory comparisons, essential for progress in fundamental research and therapeutic development. Data obtained from Exoid analysis will be acceptable to regulatory agencies, who are likely to mandate the use of validated, reliable methods of EV characterisation in the near future.

The heterogeneity of EV samples presents a challenge to research. Discriminating between particles with very similar diameters within the nanorange can be difficult, if not impossible. However, the presence and characteristics of subpopulations is becoming recognised as an important aspect of EV analysis, and individual analysis of these subpopulations is essential. The sophisticated software of the Exoid measures the size and zeta potential of every particle individually, ensuring that subpopulations are identified with high resolution (Figure 3). Up to four subpopulations can be identified within a sample with ease and unmatched resolution (Figure 4). Furthermore, changes in zeta potential or particle size in response to the binding of an antibody or detector molecule such as an aptamer can be evaluated using TRPS, which may enable the identification and quantification of specific surface markers (Figure 4). Where other techniques would require incredibly complex protocols and data analysis to evaluate these factors, the semi-automated system enables pre-programmed, multi-step experiments to be run. This enables, for the very first time, high-throughput analysis and evaluation of multiple parameters of numerous subpopulations with minimal user input.