Work

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

Year: 2026

Type: article

Abstract: 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.

Source: Journal of Extracellular Vesicles

DOI: 10.1002/jev2.70392