Izon's size exclusion chromatography (SEC) qEV isolation platform. qEV columns offered a particularly good balance between EV yield and purity for plasma EV isolation. Others had reported SEC as having the highest efficiency for removing unbound fluorescent labels. They incorporated qEV Isolation into the workflow to reduce F-NTA's background noise and improve specificity. They found qEV Isolation to be highly effective in removing unbound fluorescent labels. This was critical for reliable F-NTA measurements and enabled them to quantitatively resolve EV subpopulations based on surface marker expression. The group validated the method's ability to resolve EV subpopulations, and the effectiveness of qEV in removing unbound fluorescent labels. Using EVs from different cell lines, they confirmed: The effective removal of unbound anti-ApoB labels (AF488 streptavidin) from liposomes. Snapshots from F-NTA visualisation showed a marked reduction in background noise after qEV isolation. The versatility of qEV Isolation in removing different labels, as assessed through the quantification of labelled HT29 EVs and very low-density lipoprotein (VLDL) from human plasma. Unremoved labels accounted for 4.22% and 8.66% of total fluorescent particles for EVs and VLDL, respectively. While complete removal of free labels is rarely achievable in fluorescence-based EV workflows, these levels were considered negligible – particularly given that downstream F-NTA analysis applies intensity thresholds to distinguish true particle signals from low-level background.
