Implementation of a Novel Nanobody Panel for the Efficient Capture of Extracellular Vesicles from Human Plasma

Extracellular vesicles (EVs) are nanoscale particles released by cells and are significant components in intercellular communication. Their ability to reflect the molecular state of parental cells and their presence in body fluids make them increasingly recognized as promising non-invasive biomarkers for different pathological conditions. However, the existence of different EV populations and frequent co-isolation of contaminants present challenges for EV purification and downstream analyses. In this study, we used three novel nanobodies (VHH) for selective isolation of EVs from human plasma. Nanobodies were obtained by direct panning on EVs. All examined nanobodies have excellent physicochemical properties resulting in excellent expression and solubility. The three nanobodies being studied-NA8, ND10₁, and ND10₂-share a conserved VHH scaffold but exhibit different loop architectures. The Biopython ProtParam module was used for calculation of VHH physicochemical properties, while sequence alignments for evaluation of variations were performed with the Biopython pairwise2 module. In addition, structural modeling of nanobodies with AlphaFold revealed notable differences in CDR3 conformations. VHH were produced in <i>E. coli</i>, and upon immobilization onto a solid carrier, they were used for immunoaffinity-based capture of EVs from human plasma. Combined characterization of isolated EVs supports efficient application of an immunoaffinity-based system based on such nanobodies for the isolation of EVs from human plasma to be used for downstream analyses.