Tetraspanin-decorated extracellular vesicle-mimetics as a novel adaptable reference material

Features like small size, low refractive index and polydispersity pose challenges to the currently available detection methods for Extracellular Vesicles (EVs). In addition, the lack of appropriate standards to set up the experimental conditions makes it difficult to compare analyses obtained by different technical approaches. By modifying synthetic nanovesicles with recombinant antigenic regions of EV-enriched tetraspanins, we aimed to construct an EV-mimetic that can be used as a suitable standard for EV analyses. To this end, the sequences of the large extracellular loops of the tetraspanins CD9, CD63 and CD81 were tagged with a target sequence for the biotin ligase BirA, and co-transformed with a BirA expression plasmid into Escherichia coli. GST fusion proteins were then isolated by affinity chromatography and released using thrombin. Biotinylated recombinant tetraspanin-loops were then coupled to (strept)avidin-coated synthetic nanovesicles and analysed and characterised by Dot-blot, Western-blot, Nanoparticle Tracking Analysis, Flow Cytometry and Transmission Electron Microscopy. With this method, we were able to efficiently produce tetraspanin-domain decorated nanovesicles that share biophysical properties with natural EVs, can be detected using specific antibodies against common EV markers such as tetraspanins, and can be used as robust reference materials for detection techniques that are often used in the EV field.