Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells.However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs -in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification.

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes –UnFold probes – where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic “unfolding” step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.