A novel two-step EV isolation from plasma using size-exclusion chromatography and antibody-mediated removal of lipoproteins

Background: Owing to extracellular vesicles (EVs) ubiquitous distribution within tissues and bio-fluids, EV isolation is an essential part of all EV research. Unfortunately, EV isolation remains a challenging task, especially when isolating EVs from complex bio-fluids such as plasma. The biggest challenge is the co-isolation of non-EV proteins and lipoproteins, both of which are abundantly present in plasma. In an attempt to understand these challenges, our group has previously examined several commonly used EV isolation methods for plasma, where we demonstrated that EV isolates obtained by size-exclusion chromatography (SEC) contained minimal levels of non-EV proteins, however, high levels of lipoproteins. Recently, our research group has also showed that lipoproteins can be removed from plasma by antibody-mediated removal. Based on these findings, the aim of this study was to evaluate a novel two-step EV isolation by SEC and subsequent lipoprotein removal, for an ultra-pure EV isolate. Methods: EV isolation will be performed in five replicates from a single plasma pool collected from healthy donors. Briefly explained, EVs are first isolated from platelet poor plasma using commercially available qEV Original SEC columns. The resulting EV fractions are incubated with magnetic beads conjugated with antibodies against apolipoprotein B-48 and B-100 (ApoB). After incubation, the magnetic beads and lipoproteins are removed, leaving a final EV isolate. For comparison, the procedure is performed both with and without lipoprotein removal. The isolated EVs will be characterized using transmission electron microscopy with CD9 immunoblotting, nanoparticle tracking analysis and Western blotting against CD9 and ApoB. Results: This two-step EV isolation should mitigate the current limitation of SEC when used on plasma, where we previously found that EV isolates produced by SEC have a significantly higher lipoprotein- and lower non-EV protein content compared to conventional ultracentrifugation (unpublished). Potentially, this novel method could result in the generation of an ultra-pure EV isolation with minimal co-isolation of non-EV components. Summary/Conclusion: If successful, this EV isolate would allow for greatly improved plasma EV characterization, a process that has previously been difficult due to varying degrees of non-EV contamination.