Protocol for isolating human extracellular vesicles from nephrotic urine and enriching autoimmunoglobulin-triggered extracellular vesicles

This protocol describes the isolation of total urinary extracellular vesicles (EVs) from nephrotic patient urines and enrichment of AutoImmunoglobulin-Triggered Extracellular Vesicles (AIT-EVs). Procedures for urine collection, sample processing, buffer preparation, antibody coupling to DynaBeads, urine ultrafiltration, and total EV isolation are provided. The protocol further details EV concentration measurement and selective AIT-EV capture and elution using anti-human IgG4 antibodies. This approach enables reproducible enrichment of AIT-EVs and can be adapted to isolate other EV subtypes by substituting the capture antibody.