[PubMed] [Google Scholar]  Giedt RJ, Pathania D, Carlson JCT, McFarland PJ, Del Castillo AF, Juric D,Weissleder R
[PubMed] [Google Scholar]  Giedt RJ, Pathania D, Carlson JCT, McFarland PJ, Del Castillo AF, Juric D,Weissleder R. EV protein profiling. Antibody-DNA conjugates are used to label EV and labeled single EV are encapsulated into droplets. Using PCR, DNA barcodes are amplified for imaging based readout. It has shown that multiplex protein analysis is possible in single EV, opening the door for future analyses. Introduction Single cell analysis continues to have a major impact on our understanding of cell subtypes, biology, and medicine.[1,2]. The same is likely true for analyses of single exosomes or extracellular vesicles (EV) in general. It has become apparent that EV populations can be even more heterogeneous than the parental cells from which they are derived.[3,4] This is likely due to selective shedding of proteins and nucleic acid Alofanib (RPT835) cargo, the much smaller size and payload capacity of EV, and stochastic cellular effects. In order to use EV more efficiently as biomarkers of disease, we need a better understanding of their composition and heterogeneity. A number of single EV analytical methods have been proposed. These include analysis by microscopic imaging of immobilized vesicles (SEA)[5,6], altered flow cytometry[5,7C10], and digital detection using ELISA or nucleic acid-based amplification. In spite of this progress, it remains challenging to detect rare proteins in single EV, given the inherent signal/background limitations of direct fluorescence imaging and relatively modest enzyme mediated signal amplification in ELISA. Here we describe a new method for ultra-sensitive detection of proteins in single EV that exploits antibody-based immuno-droplet digital PCR (iddPCR). The described method is not only sensitive but also allows multiplexing (currently up to three proteins). We used uniquely designed DNA barcoded antibodies for protein recognition. The labeled EV are encapsulated Alofanib (RPT835) into 70 m droplets in which PCR IL1R1 antibody amplifies the Alofanib (RPT835) message of the DNA barcode. Using different barcode sequences and fluorophores, we show that it is possible to profile proteins in individual EV. We optimized the experimental conditions, validated measurements, and then applied EV profiling to PD-L1 measurements in cancer-cell derived EV. Results Description of the single EV-iddPCR technique To reveal proteins of interest, we used target-specific monoclonal antibodies barcoded with unique and amplifiable DNA sequences. A number of routes to prepare such constructs have been described, including NHS/maleimide chemistry, photoaffinity labeling, and protein adaptors among others.[12C15] Here, we selected bioorthogonal em trans- /em cycloctene/tetrazine (TCO/Tz) conjugation as it is cost effective and allows rapid, titratable, and readily-purified labeling reactions. Barcoded antibodies were incubated with EV for labeling, followed Alofanib (RPT835) by size exclusion chromatography (SEC) to remove unbound antibody-DNA (Ab-DNA) molecules (Physique 1A). The concentration of labeled EV in answer was then determined by nanoparticle tracking analysis (NTA), allowing us to adjust concentrations and flow rates to optimize the statistical likelihood of single EV encapsulation.[16C18] By encapsulating the Ab-DNA barcoded EV with a PCR grasp mix (BioRAD), the signal (if present) can be directly amplified in a given droplet (Physique 1B). Following PCR amplification, individual droplets were imaged by fluorescence microscopy to quantify the fraction of droplets made up of EV with the target of interest Alofanib (RPT835) (Physique 1C). Open in a separate window Physique 1: Schematic of droplet-based single EV detection.Single EV detection is usually achieved via three steps. A. First, EV are labeled with Ab-DNA conjugates for multiplexing and DNA-based detection of protein expressions. Remaining free Ab-DNA conjugates are then removed using size exclusion chromatography (SEC) before proceeding to the next step. B. Second, single EV are encapsulated in droplets by.