Standardized Microplate-Based Assays for Quantifying Antioxidant Activity in Extracellular Vesicles and Nanovesicles Across Biological Kingdoms

The antioxidant activity of extracellular vesicles (EVs) and nanovesicles are increasingly linked to their physiological and therapeutic functions, yet its accurate quantification remains limited by low EV yield, co-isolated contaminants and the lack of standardized methodologies. Here, we establish optimized microplate-based protocols for ABTS and ORAC assays specifically adapted to highly purified EVs and nanovesicles obtained by ultracentrifugation followed by size-exclusion chromatography (UC + SEC). By minimizing sample input, implementing protein-based normalization (TEAC/g EV protein), and introducing assay-specific controls-including a self-blank correction for ORAC-we achieve sensitive and reproducible measurements across plant-, human plasma- and bacterial-derived EVs and nanovesicles. Our results demonstrate that conventional isolation methods substantially overestimate antioxidant activity due to non-vesicular contaminants, underscoring the need for rigorous purification. Using a detergent-based disruption strategy compatible with ABTS, we further distinguish surface-associated from cargo-derived antioxidant activity, revealing a two-tier functional organization of EVs. Notably, we report for the first time measurable antioxidant activity in bacterial extracellular vesicles (BEVs). Application of these methods to a human dietary intervention shows that EV antioxidant capacity is dynamically modulated in vivo. Collectively, this work provides a standardized and broadly applicable framework for the reliable assessment of EV and nanovesicles antioxidant properties across biological kingdoms.