Broadly speaking there are two main applications within extracellular vesicle research, namely; diagnostics and therapeutics. From a diagnostic perspective, sample purification is a means to an end. Purification is only performed because of the limitations associated with the instrumentation used to measure extracellular vesicles. The ideal tool would by-pass sample purification and be able to fully characterize vesicles direct from biological samples, with high specificity, in low volumes with adequate detection sensitivities to make clinical diagnoses. The ExoView platform is such a tool, it provides phenotyping, sizing and concentration measurements direct from biological fluids therefore bypassing arduous purification protocols which bias the data generated.
For those working in therapeutics, it is desirable to produce highly pure samples of vesicles. In this case, purification is required. However, because most analytical techniques do not have the ability to measure vesicles in both un-purified and purified samples, it can be difficult to assess the efficiency of the purification techniques used. The ExoView platform provides exactly this capability and provides a label free characterization tool to measure final product as well as a tool that can be used to monitor the efficiency of the purification techniques used in the manufacturing process.
The ExoView platform works with as little as 50µL of sample and captures EVs directly from cell culture medium or plasma onto a fuctionalized array as is shown below. Each spot on the array can be functionalized with different antibodies, peptides or aptomers. Once bound the concentration and size or antigen positive EVs can be measured in parallel providing the ideal platform for streamlined exosome research and biomarker discovery.
Common Issues with Purification
As Coumens et al (2017) describe, the methods used in sample collection and isolation can influence significantly the downstream analysis of vesicles in terms of their function, content and physical form/concentration. The study goes on to explain that no technique that will isolate extracellular vesicles only and that soluble proteins, protein aggregates, lipoproteins and cell organelles and viruses are candidates for co-purification. Techniques which are unable to discriminate those things which co-purify and report them alongside EVs (techniques such as NTA and TRPS) are therefore subject to errors in data interpretation. The paper goes on to describe the main limitations associated with all commonly used techniques to purify (summarized below), making the point that the choice of purification is the single most important determinant of the results of subsequent analysis.
|CONTAMINANTS||Lipoproteins, protein aggregates, viruses||Lipoproteins (HDLs)||Lipoproteins, protein, protein aggregates, viruses||Same size particles||Soluble proteins||Protein|
|MAJOR ARTEFACTS||EV-particle aggregates||EV-particle aggregates||Protein complex, EV - particle aggregates|
|EV RECOVERY %||2 to 80||10||40 to 90||10 to 80||90|
|ASSAY TIME (h)||3 to 9||16 to 90||0.3||0.5||4 to 20||0.3 to 12|
DC = Differential Centrifugation
DGC = Density Gradient Centrifugation
SEC = Size Exclusion Chromatography
UF = Ultrafiltration
IC = Immuno Capture
P = Precipitation
So why purify? Why not measure the parameters that matter, with confidence, direct from complex biological media like blood serum and urine? ExoView is the world’s first immuno-based detection method for single vesicle characterization. Save costs, save time and remove the variability associated with sample purification and find out more about the ExoView platform today!