CD9/CD63 Exosome ELISA Kit

The CD9/CD63 Exosome ELISA Kit is a sandwich ELISA kit that utilizes high-performance anti-CD9 (clone 12A12) capture and anti-CD63 (clone 8A12) detection antibodies. It is designed for use on human body fluids or cell culture supernatants to sensitively and quantitatively detect targets that co-express exosome surface markers CD9 and CD63.

Extracellular vesicles (EVs; including classical-exosomes, non-classical-exosomes, microvesicles, large oncosomes, apoptotic bodies, apoptotic vesicles, autophagic extracellular vesicles, amphisomes and ARRMs) are membrane vesicles of 40-1000 nm that are released into the extracellular milieu and body fluids from most cell types, including red blood cells, platelets, lymphocytes, dendritic cells, endothelial cells and tumor cells. These vesicles are classified into 2 types according to their secretory mechanism. Thus, classical exosomes are formed in multivesicular endosomes, whereas microvesicles originate by direct budding from the plasma membrane. Although classical-exosome components vary by their originating cell type, a certain set of molecules appears likely to be shared, regardless of their origin. These molecules include the tetraspanin proteins (CD9, CD63 and CD81) that are thought to be essential components of the biogenesis mechanism of classical exosomes. Accordingly, researchers have used CD9, CD63 and CD81 to isolate and characterize the purity of classical-exosome preparations.

Over the past decade, exosomes have been the focus of intense interest as microRNA (miRNA) carriers, disease biomarkers, and potential therapeutic drug delivery vehicles. Despite the importance of exosomes, their isolation and characterization are still considered major scientific challenges, especially when translating to the demands of the clinic. Classically, exosomes and other EVs have been purified by ultracentrifugation. Technical challenges, time and cost have led to a proliferation of alternative purification approaches, many of them now commercially available. While exosomal content has been reported to include genomic DNA, RNA, proteins, and lipids, recent studies have cast doubt as to whether DNA and many so-called “exosomal” proteins are actual exosomal constituents or simply non-vesicular contaminants co-isolated and comingled by ultracentrifugation forces at the bottom of centrifuge tubes.

The conventional way to quantify exosomes is indirect. For example, it measures encapsulated protein abundance or performs Nanoparticle Tracking Analysis to determine the size distribution profile of small particles (8). The drawback for these methods is that they require exosome purification utilizing ultracentrifugation, for instance. Direct methods to quantitate exosomes in body fluids or culture supernatant is extremely limited, and there were no common methods available until now.

Assay Principle

First, exosomes are captured by sample addition to anti-CD9 antibody-immobilized microtiter plates. Following washing, exosomes expressing both CD9 and CD63 are detected by addition of HRP-conjugated anti-CD63 antibody. Finally, enzymatic substrate is added and HRP activity measured by microplate reader to quantitate exosome content in samples.

Features


• Capture exosomes with solid phase anti-CD9 antibody (12A12), then detect using HRP-conjugated anti-CD63 secondary antibody

• Directly quantitate exosomes in human blood samples or cell culture supernatants

• No special equipment required, apart from a 450nm microplate reader

• To implement stability and reproducibility, kit utilizes a CD9/CD63 fusion protein standard instead of unstable/hard-to-store exosomes themselves

• Normalization with CD9/CD63 fusion protein standard enables relative quantitation across a set of samples


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