Nds; 2Department of Biomedical Engineering and Physics, and Vesicle Observation Center, Academic Health-related Centre of the University of Amsterdam, Amsterdam, The Netherlands; 3Department of Biochemistry and Food Chemistry University of Turku, Turku, Finland; 4Department of Urology Erasmus Medical Center, Rotterdam, The Netherlands; 5Laboratory of Experimental Clinical Chemistry, and Vesicle Observation Center, Academic Health-related Center, University of Amsterdam, Amsterdam, The NetherlandsBackground: Detection of transmembrane proteins on extracellular vesicles (EVs) is typically performed employing Western blot or enzymelinked immunosorbent assay. Even so, both strategies have restricted analytical sensitivity and quantification abilities. Lately, more sensitive and quantitative approaches have come to be obtainable, like surface plasmon resonance imaging (SPRi) and time-resolved fluorecence immunoassay (TRFIA). Methods: Both SPRi and TRFIA capture target-exposing EVs at an antibody-coated surface. SPRi detects a alter in refractive index upon capture of EVs, whereas TRFIA detects captured EVs through labeling with an europium-conjugated antibody. CD9 exposure was determined qualitatively and quantitatively for 16 culture-derived EV samples by SPRi and TRFIA. Outcomes: For 11 EV samples (69), qualitative detection of CD9 with SPRi and TRFIA was in agreement. The quantitative signal amplitudes of all EV samples showed, however, a R2-correlation of 0.09. A cause of discrepancy may be the 80 5 reduction in labeling intensity, when capture and labeling are performed in TRFIA with all the similar antibody (CD9, CD63 and EpCAM), which was confirmed with fluorescence microscopy for EpCAM. An additional cause of discrepancy occurs in the course of labeling of captured EVs by TRFIA. This labeling depends on the antigen density whereas detection by SPRi will not. Hence, samples containing a subpopulation of EVs with higher numbers of antigens were good in TRFIA but not in SPRi. Summary/Conclusion: To conclude, SPRi and TRFIA gave comparable qualitative Angiotensin-Converting Enzyme 2 (ACE2) Proteins Recombinant Proteins phenotyping final results, but incomparable quantitative resultsBackground: In spite of the massive number of technologies currently used to detect and characterize exosomes in biofluids, the need remains for improved solutions. The flow cytometry-based methods for quantitative and qualitative characterization of exosomes, for instance, meet challenges for example the small size of the exosomes, paucity of antigen molecules present on the surface of the exosomes, making it difficult or not possible to distinguish individual exosomes from background by standard flow cytometry. Approaches: We have applied the proximity ligation assay in combination with flow cytometry readout for sensitive and certain detection of individual exosomes. Here, the exosomes are initial enriched on a strong support utilizing a capture antibody – immobilized by means of a cleavable DNA molecule. Subsequently, the exosomes are probed using a set of proximity probes, every consisting of an affinity binder conjugated to a ssDNA molecule. TLK2 Proteins Accession Before the signal amplification by means of rolling signal amplification, the exosomes are released from the strong assistance by DNA cleavage, enabling multicolour detection and measurement of individual exosomes inside a flow cytometer. Results: The use of up to seven antibodies in combination with signal amplification enables detection of exosomes with higher specificity and sensitivity. By using unique reporting fluorophores for every single pair of probes, a distinct exosome popula.