o:36027 Urinary extracellular vesicles as a readily available biomarker source: a simplified stratification method en ABSTRACT Urine, a common source of biological markers in biomedical research and clinical diagnosis, has recently generated a new wave of interest. It has recently become a focus of study due to the presence of its content of extracellular vesicles (EVs). These uEVs have been found to reflectphysiological and pathological conditions in kidney, urothelial, and prostate tissue and can illustrate further molecular processes, leading to a rapid expansion of research in this field In this work,we present the advantages of an immunoaffinity-based method for uEVs’ isolation with respect to the gold standard purification approach performed by differential ultracentrifugation [in terms of purity and antigen presence. The immunoaffinity method was made feasible by combining specific antibodies with a functionalized polymethacrylate polymer. Flow cytometry indicated a significant fluorescence shift, validating the presence of the markers (CD9, CD63, CD81) and confirming the effectiveness of the isolation method. Microscopy evaluations have shown that the morphology of the vesicles remained intact and corresponded to the expected shapes and dimensions of uEVs. The described protocol is inexpensive, fast, easy to process, has good reproducibility, and can be applied to further biological samples. extracellular vesicles; nanobodies; immunoaffinity chromatography; ultracentrifugation 1552099 10.3390/ijms25158004 2025-03-19T13:50:44.781Z 44 yes 46 Lidija Filipović Innovative Centre of the Faculty of Chemistry, Belgrade, Serbia Milica Spasojević Savković Innovative Centre of the Faculty of Chemistry, Belgrade, Serbia person Radivoje Prodanović Faculty of Chemistry, University of Belgrade, Belgrade, Serbia person Suzana Matijašević Joković Faculty of Biology, University of Belgrade, Belgrade, Serbia person Sanja Stevanović Center for Chemistry, Institute for Chemistry, Technology, and Metallurgy, National Institute of Republic of Serbia, Belgrade, Serbia person Ario de Marco Laboratory for Environmental and Life Sciences, University of Nova Gorica, Nova Gorica, Slovenia person Maja Kosanović University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia doktor bioloških nauka person 0000-0002-1143-1805 Goran Brajušković Faculty of Biology, University of Belgrade, Belgrade, Serbia person Milica Popović Faculty of Chemistry, University of Belgrade, Belgrade, Serbia person application/pdf 3692297 https://phaidrabg.bg.ac.rs/o:36027 no yes 16 70 92000001 11A19 11A1907 11A02 11A24 11A29 11A2904 International Journal of Molecular Sciences 25 15 MDPI, Basel, Switzerland 2024