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

Kakavoulia, Maria‐Anthi; Spies, Peter; Kutzner, Tanja J.; Tertel, Tobias; Franzmeier, Sophie; Andaloussi, Samir El; Giebel, Bernd; Kaltner, Herbert; Meinel, Dominik; Görgens, André; Ludwig, Anna‐Kristin (2026): In‐Solution Characterization of Extracellular Vesicles: A New Approach to Evaluating Antibody Binding and Surface Interactions. Journal of Extracellular Vesicles, 15 (4): e70269. ISSN 2001-3078

Abstract

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 EC50 values 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.

• Dokumententyp: Artikel (LMU)
• Organisationseinheit (Fakultäten): 18 Chemie und Pharmazie > Department Biochemie
• DFG-Fachsystematik der Wissenschaftsbereiche: Naturwissenschaften
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 491502892