CD9/CD63 Exosome ELISA Kit

Cosmo Bio LTD

Catalog No: CSR-EXH0102EL

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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

References:

1. J. Skog, T. Wurdinger, S. vanRijn, D. H. Meijer, L. Gainche, M. Sena-Esteves, W. T. Jr. Curry, B. S. Carter, A. M. Krichevsky and X. O. Breakefield: Nat Cell Biol., 10, 1470 (2008).
2. T. Pisitkun, R. F. Shen and M. A. Knepper: Proc Natl Acad Sci USA, 101, 13368 (2004).
3. S. Runz, S. Keller, C. Rupp, A. Stoeck, Y. Issa, D. Koensgen, A. Mustea, J. Sehouli, G. Kristiansen and P. Altevogt: GynecolOncol, 107, 563 (2007).
4. S. Keller, A. K. Konig, F. Marme, S. Runz, S. Wolterink, D. Koensgen, A. Mustea, J. Sehouli and P. Altevogt: Cancer Lett, 278, 73 (2009).
5. C. Lasser, V. Seyed Alikh, S. Gabrielsson, J. Lotvall and H. Valadi: J Transl Med, 9, 9 (2011).
6. Y. Naito, Y. Yoshioka, Y. Yamamoto and T. Ochiya: Cell Mol Life Sci, 74, 697 (2017).
   1. Zoraida and M. Yáñez-Mó: Front Immunol, 5, 442 (2014).
7. V. Filipe, A. Hawe and W. Jiskoot: Pharm Res, 27, 796 (2010).

 

Citations for CD9/CD63 Exosome ELISA Kit – 11 Found

Self-Functionalized Superlattice Nanosensor Enables Glioblastoma Diagnosis Using Liquid Biopsy.
ACS Nano. 2023 Oct 24;17(20):19832-19852. doi: 10.1021/acsnano.3c04118. Epub 2023 Oct 12. 

Proteolytic cleavage of HLA class II by human neutrophil elastase in pneumococcal pneumonia.
Sci Rep. 2021 Jan 28;11(1):2432. doi: 10.1038/s41598-021-82212-5. 

Microbial Antigen-Presenting Extracellular Vesicles Derived from Genetically Modified Tumor Cells Promote Antitumor Activity of Dendritic Cells.
Pharmaceutics. 2021 Jan 4;13(1):57. doi: 10.3390/pharmaceutics13010057. 

Elevated expression of RAB3B plays important roles in chemoresistance and metastatic potential of hepatoma cells.
BMC Cancer. 2022 Mar 11;22(1):260. doi: 10.1186/s12885-022-09370-1. 

miR-709 exerts an angiogenic effect through a FGF2 upregulation induced by a GSK3B downregulation.
Sci Rep. 2024 May 18;14(1):11372. doi: 10.1038/s41598-024-62340-4. 

Ultrafast sensitivity-controlled and specific detection of extracellular vesicles using optical force with antibody-modified microparticles in a microflow system.
Nanoscale Horiz. 2023 Jul 24;8(8):1034-1042. doi: 10.1039/d2nh00576j. 

Sonic hedgehog N-terminal level correlates with adiponectin level and insulin resistance in adolescents.
J Pediatr Endocrinol Metab. 2023 Jan 2;36(2):126-131. doi: 10.1515/jpem-2022-0385. Print 2023 Feb 23. 

Small extracellular vesicles derived from human adipose-derived mesenchymal stromal cells cultured in a new chemically-defined contaminate-free media exhibit enhanced biological and therapeutic effects on human chondrocytes in vitro and in a mouse osteoarthritis model.
J Extracell Vesicles. 2023 Jul;12(7):e12337. doi: 10.1002/jev2.12337. 

Extracellular vesicles derived from radioresistant oral squamous cell carcinoma cells contribute to the acquisition of radioresistance via the miR-503-3p-BAK axis.
J Extracell Vesicles. 2021 Dec;10(14):e12169. doi: 10.1002/jev2.12169. 

Exosomes from conditioned media of bone marrow-derived mesenchymal stem cells promote bone regeneration by enhancing angiogenesis.
PLoS One. 2019 Nov 21;14(11):e0225472. doi: 10.1371/journal.pone.0225472. eCollection 2019. 

Exosomes from conditioned media of bone marrow-derived mesenchymal stem cells promote bone regeneration by enhancing angiogenesis.
PLoS One. 2019 Nov 21;14(11):e0225472. doi: 10.1371/journal.pone.0225472. eCollection 2019.