Quantitation of EVs in Human Plasma: Applying the ExoView® Tetraspanin Assay in a Physiologically Relevant System The ExoView® Tetraspanin Assay uses human tetraspanin antibodies arrayed on a chip to capture specific extracellular vesicles (EVs) that are detected using specific antibodies conjugated to fluorescent readouts. The data from 3 fluorescent channels and UV-based interferometric images are analyzed to identify the number, size and composition of specific EVs. The ExoView assay chips can be customized to bind custom surface markers. One application is the detection and quantitation of Tspan8 (Tetraspanin 8) positive EVs in plasma. Tspan8 is a biomarker associated with carcinomas such as pancreatic cancer, therefore assaying the number of Tspan8 positive EVs in patient blood samples would be of great clinical importance for therapeutic monitoring and disease diagnostics. Tspan8 positive EVs are present at very low concentrations, so it is necessary to use an assay with high levels of sensitivity and specificity. ASSAY SENSITIVITY Complete conditioned media from HEK293 cells were assayed to determine the concentration range of EV detection. The slope of the EV count vs. dilution was linear down to the lowest concentration tested, 1:25600. Based on an NTA concentration of 3.5x1010 EVs/mL, the limit of detection is less than or equal to 1.37x106 EVs/ mL in the ExoView assay. Given that the assay saturated at a 1:100 dilution of complete conditioned media, the upper concentration range of the ExoView assay in this experiment was 3.5x108 EVs/mL or 2.4 log linear range. Other samples tested (data not shown) show EVs detectable from 1.37x106 EVs/mL to 1x109 EVs/mL or 2.8 log detection range. 10000 HEK293T Complete Medium R2=0.99 8000 CD63 R2=0.99 6000 CD9 CD81 4000 EV Count R2=0.99 2000 0 0.000 0.002 0.004 0.006 0.008 0.010 Dilution The ExoView assay has a lower limit of detection of approximately 1.4x106 EVs/ml - about 10X more sensitive than other assays. 1 NanoView Biosciences, Boston MA For Research Use Only +1-781-365-8439 [email protected] ASSAY SPECIFICITY Three media samples from cultured HEK293 cells were concentrated 45x by UCF before being experimentally tested: • Basal: Conditioned medium without FBS that includes EVs from cells • Complete: Conditioned medium with exosome depleted FBS that contain EVs from cells and serum proteins • Complete NC: Unconditioned medium containing serum (i.e. no cell culture EVs) ExoView NTA Fluorocet Bradford Assay 8 x 10 11 6x10 11 4 x 10 11 2x10 11 0 The Bradford and Fluorocet assays nonspecifically detect proteins in serum containing media while the ExoView assays specifically detect EVs from conditioned serum-free media or media with exosome depleted serum. A clinically relevant application of the TSPAN8 ExoView assay is the detection of disease- A CD9 specific EVs in patient plasma with a high degree of specificity and sensitivity. The ExoView assay was used to determine if a specific EV subpopulation can be CD9 specifically measured when spiked into human plasma at low concentrations. • CD9 positive EVs that are TSPAN8 B counterstained using a Tspan8 antibody CD9 show the expected linear concentration curve. CD63 • EVs captured using Tspan8 on the ExoView TSPAN8 chip show concentration-dependent detection of CD9, CD63 and Tspan8, in fluorescence counterstaining. The ExoView assay offers sensitive and specific detection of EV sub populations and has flexible configurations for capture and detection methods. 2 NanoView Biosciences, Boston MA For Research Use Only +1-781-365-8439 [email protected].