biostudies-literatureTerzic JEuropean UnionScience Fund of the Republic of Serbia4337https://www.ebi.ac.uk/biostudies/studies/S-EPMC12654627biostudies-literatureUnknown30(22)Extracellular vesicles (EVs) are nanosized structures involved in intercellular communication that have high potential as disease biomarkers and for the delivery of therapeutic cargos. However, translation to the clinic is hampered by time-consuming, low-yield, and poorly reproducible EV isolation methods. We describe a cryogel-based immunoaffinity chromatography system that exploits single-domain VHH antibodies as capture elements for the selective isolation of EVs from human plasma. Supermacroporous cryogels functionalized with five unique anti-EV VHHs (total immobilization capacity ~500 µg/g) were prepared, yielding a highly permeable and hydrophilic support. They were captured and eluted under mild conditions, and their morphology and identity were confirmed by SEM, AFM, NTA, and flow Molecules (Basel, Switzerland)Application of VHH-Immobilized Cryogel-Based Immunoaffinity Chromatography for Isolation of Extracellular Vesicles.PMC12654627Grant Agreement No. 101182851Grant PRISMA No. 4747Mitic NKrstic JStevanovic STerzic JPopovic MFilipovic LCourraud Jde Marco AfalseApplication of VHH-Immobilized Cryogel-Based Immunoaffinity Chromatography for Isolation of Extracellular Vesicles.Extracellular vesicles (EVs) are nanosized structures involved in intercellular communication that have high potential as disease biomarkers and for the delivery of therapeutic cargos. However, translation to the clinic is hampered by time-consuming, low-yield, and poorly reproducible EV isolation methods. We describe a cryogel-based immunoaffinity chromatography system that exploits single-domain VHH antibodies as capture elements for the selective isolation of EVs from human plasma. Supermacroporous cryogels functionalized with five unique anti-EV VHHs (total immobilization capacity ~500 µg/g) were prepared, yielding a highly permeable and hydrophilic support. They were captured and eluted under mild conditions, and their morphology and identity were confirmed by SEM, AFM, NTA, and flow 2025-01-01T00:00:00Z2025 Nov2026-05-20T03:19:12.553Z2026-05-20T03:09:03.512ZS-EPMC126546274130239710.3390/molecules30224337