Background/Objective
Accurate quantification of antibody binding efficiency and EV-epitope accessibility remains a major challenge in EV-based therapeutic development. Conventional assays using surface immobilization may distort EV structure and mask epitopes. Here we introduce Flow-Induced Dispersion Analysis (FIDA), a solution-based alternative to bead-based or immobilization assays that quantifies antibody binding to EVs in real time and under laminar flow by monitoring hydrodynamic radius changes of a fluorescent indicator.
Methods
EVs from two donors of clonal immortalized mesenchymal stromal cells (ciMSC-EVs) and EVs from HEK293 cells expressing CD63-NeonGreen with Fc-binding domains (Fc-EVs) were analyzed. ciMSC-EVs were prepared by ultrafiltration and ultracentrifugation; Fc-EVs by tangential flow filtration and ultrafiltration. FIDA was used to determine hydrodynamic radii, binding kinetics, and accessible antibody binding sites per vesicle. Particle concentration was measured by NTA and IFCM, and antibody binding was assessed with fluorophore-conjugated and therapeutic antibodies.
Results
Hydrodynamic radii of 17–42 nm (mean ± SD: 17.0 ± 0.7 to 41.8 ± 1.0 nm) were detected, consistent with small EVs (34–84 nm). For ciMSC-EVs, FIDA detected accessible CD63 binding sites per vesicle ranging from 70 ± 9 to 90 ± 11 (NTA-based particle concentration). Binding sites in Fc-EVs varied with particle quantification method: IFCM yielded counts 5–15 times higher (5,400–6,600 ± 460 per EV) than NTA (500–1,400 ± 130 per EV), underscoring substantial discrepancies between current enumeration with or without labeling techniques. While FIDA proved robust for diverse EV preparations, sensitivity was reduced in highly protein-rich environments.
Conclusion
FIDA enables high-resolution, native-condition analysis of EV–antibody interactions and provides a quantitative platform for antibody loading and quality control. By revealing both methodological and cell type-dependent variability, FIDA underscores the need for standardized EV analytics and supports its integration into preclinical and therapeutic workflows.
Keywords
Extracellular Vesicles, Antibody Binding, Epitope Availability, FIDA, Therapeutic EV Standardization
Funding/Acknowledgments
We thank PD Dr. Philip von Hundelshausen, Dr. Xavier Blanchet and Dr. Henrik Jensen for their fruitful discussions. We thank PD Dr. Stefanie Fenske for the support and discussion of the project. For assistance in TEM sample preparation, we thank Ms. Heidrun Schöl.
