Abstract
Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.
Highlights
Fusion into internal endosomal-derived multivesicular bodies (MVB)
We developed and optimized a protocol based on commercially available TotalSeq-A antibody (BioLegend Inc.) which is the key component in the CITE-seq method for single cell surface protein profiling[14]
TotalSeq-A is the only format with confirmed nucleotide sequence and a poly-A tail, the other two contain sites of random nucleotide incorporation which brings unpredictable results on the following extension and qPCR step
Summary
Fusion into internal endosomal-derived multivesicular bodies (MVB). Exosomes are released from the cell following membrane fusion of the MVB with the plasma membrane. The proximity-dependent barcoding assay (PBA) utilizes single-stranded DNA clusters to barcode individual exosomes. Using this method, the group has successfully profiled 38 different surface proteins simultaneously on individual exosomes. We developed and optimized a protocol based on commercially available TotalSeq-A antibody (BioLegend Inc.) which is the key component in the CITE-seq method for single cell surface protein profiling[14]. Each antibody signal could be amplified with the primer pairs on any real-time PCR machine Combining this approach with micro-SEC, fractions from as little as 10 μL serum sample could be used to profile EV biomarkers. The TotalSeq-A antibody could be used to successfully profile cell surface proteins, greatly enhancing the utility of this method. We developed a fast, sensitive and cost-effective multiplexing protein profiling assay that may serve as an ideal platform for EV biomarker studies and diagnostics
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