In‐Solution Characterization of Extracellular Vesicles: A New Approach to Evaluating Antibody Binding and Surface Interactions

Extracellular vesicles (EVs) are emerging as diagnostic and therapeutic agents, yet their nanoscale size limits quantitative single-vesicle analysis of surface epitopes and molecular interactions. Flow-Induced Dispersion Analysis (FIDA) is a microfluidic technique enabling real-time measurement of hydrodynamic size and interaction kinetics using fluorescent ligands without requiring surface immobilization. Here, FIDA was applied to characterize EV-antibody interactions using anti-CD63 antibodies across three EV sources: EVs from genetically engineered HEK cells expressing CD63-eGFP or CD63-NeonGreen-Fc domain (CD63-NG-Fc), and EVs from clonally expanded immortalized mesenchymal stromal cells (ciMSCs). FIDA-measured EV diameter sizes ranged from 40 to 90 nm, compared to 70-200 nm as determined by Nanoparticle Tracking Analysis (NTA), likely reflecting methodological differences or NTA's sensitivity to non-EV particles. CD63-binding EC₅₀values were 1.9 × 10⁸ particles/mL for CD63-eGFP EVs and 8.4 × 10⁸ to 2.4 × 10⁹ particles/mL for ciMSC EVs, indicating higher epitope abundance in engineered vesicles. Within a broader antibody titration, maximal antibody loading to the Fc-receptor domain on EVs was observed at 0.5-5 nM, reaching 30%-40% for IgG1 and 15%-20% for Cetuximab. These results establish FIDA as a high-resolution, label-efficient tool for quantifying EV-antibody interactions and epitope accessibility, supporting its integration into EV standardization and quality control workflows.