CD9/CD63 Exosome ELISA Kit

Cosmo Bio

Catalog No.: CSR-EXH0102EL

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).
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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 – 21 Found

Premachandran, Srilakshmi; Haldavnekar, Rupa; Ganesh, Swarna; Das, Sunit; Venkatakrishnan, Krishnan; Tan, Bo. Self-Functionalized Superlattice Nanosensor Enables Glioblastoma Diagnosis Using Liquid Biopsy. Acs Nano. 2023;17(20):19832-19852. 

Domon, Hisanori; Maekawa, Tomoki; Isono, Toshihito; Furuta, Kazuyuki; Kaito, Chikara; Terao, Yutaka. Proteolytic cleavage of HLA class II by human neutrophil elastase in pneumococcal pneumonia. Scientific Reports. 2021;11(1):2432. 

Tsunedomi, Ryouichi; Yoshimura, Kiyoshi; Kimura, Yuta; Nishiyama, Mitsuo; Fujiwara, Nobuyuki; Matsukuma, Satoshi; Kanekiyo, Shinsuke; Matsui, Hiroto; Shindo, Yoshitaro; Watanabe, Yusaku; Tokumitsu, Yukio; Yoshida, Shin; Iida, Michihisa; Suzuki, Nobuaki; Takeda, Shigeru; Ioka, Tatsuya; Hazama, Shoichi; Nagano, Hiroaki. Elevated expression of RAB3B plays important roles in chemoresistance and metastatic potential of hepatoma cells. Bmc Cancer. 2022;22(1):260. 

Takaya, Junji; Tanabe, Yuko; Kaneko, Kazunari. Sonic hedgehog N-terminal level correlates with adiponectin level and insulin resistance in adolescents. Journal Of Pediatric Endocrinology & Metabolism : Jpem. 2023;36(2):126-131. 

Yamana, Keisuke; Inoue, Junki; Yoshida, Ryoji; Sakata, Junki; Nakashima, Hikaru; Arita, Hidetaka; Kawaguchi, Sho; Gohara, Shunsuke; Nagao, Yuka; Takeshita, Hisashi; Maeshiro, Manabu; Liu, Rin; Matsuoka, Yuichiro; Hirayama, Masatoshi; Kawahara, Kenta; Nagata, Masashi; Hirosue, Akiyuki; Toya, Ryo; Murakami, Ryuji; Kuwahara, Yoshikazu; Fukumoto, Manabu; Nakayama, Hideki. Extracellular vesicles derived from radioresistant oral squamous cell carcinoma cells contribute to the acquisition of radioresistance via the miR-503-3p-BAK axis. Journal Of Extracellular Vesicles. 2021;10(14):e12169. 

Abdullah, Mohammad; Nakamura, Tomohisa; Ferdous, Taslima; Gao, Yuan; Chen, Yuxin; Zou, Kun; Michikawa, Makoto. Cholesterol Regulates Exosome Release in Cultured Astrocytes. Frontiers In Immunology. 12( 34721384):722581. 

Masaki, Kanako; Ahmed, Abo Bakr F; Ishida, Takenori; Mikami, Yuuki; Funabashi, Hisakage; Hirota, Ryuichi; Ikeda, Takeshi; Kuroda, Akio. Chromatographic purification of small extracellular vesicles using an affinity column for phospholipid membranes. Biotechnology Letters. 2023;45(11-12):1457-1466. 

Ueno, Koji; Kurazumi, Hiroshi; Murakami, Junichi; Mizoguchi, Takahiro; Suzuki, Ryo; Tanaka, Toshiki; Hamano, Kimikazu. miR-765 induces angiogenesis by inhibiting dipeptidyl peptidase 4 and increasing fibroblast growth factor 2. Bba Advances. 9:100193. 

Obuchi, Satoe; Morita, Masamune; Kuno, Goshi; Mogami, Toshifumi; Shuno, Tomohiro; Ito, Yuto; Miyagishi, Makoto; Ohba, Yoshio; Kuramochi, Akiko; Teramura, Yuji. Isolation of small extracellular vesicles by interacting with inorganic surface. Regenerative Therapy. 2026;31:101063. 

Tsubaki, Toshiya; Chijimatsu, Ryota; Takeda, Taiga; Abe, Maki; Ochiya, Takahiro; Tsuji, Shinsaku; Inoue, Keita; Matsuzaki, Tokio; Iwanaga, Yasuhide; Omata, Yasunori; Tanaka, Sakae; Saito, Taku. Aging and cell expansion enhance microRNA diversity in small extracellular vesicles produced from human adipose-derived stem cells. Cytotechnology. 2025;77(1):15. 

Nishimura, Hirotaka; Hashii, Noritaka; Yamamoto, Tomofumi; Sun, Yuchen; Miura, Takumi; Sato, Yoji; Ishii-Watabe, Akiko. Usefulness of Size-Exclusion Chromatography-Multi-Angle Light Scattering to Assess Particle Composition and Protein Impurities for Quality Control of Therapeutic Exosome Preparations. Pharmaceutics. 2024;16(12) 

Ueno, Koji; Kurazumi, Hiroshi; Suzuki, Ryo; Yanagihara, Masashi; Mizoguchi, Takahiro; Harada, Takasuke; Morikage, Noriyasu; Hamano, Kimikazu. miR-709 exerts an angiogenic effect through a FGF2 upregulation induced by a GSK3B downregulation. Scientific Reports. 2024;14(1):11372. 

Hanai, Hiroto; Hart, David A; Jacob, George; Shimomura, Kazunori; Ando, Wataru; Yoshioka, Yusuke; Ochiya, Takahiro; Nakagawa, Shinicihi; Nakamura, Masato; Okada, Seiji; Nakamura, Norimasa. 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. Journal Of Extracellular Vesicles. 2023;12(7):e12337. 

Takeuchi, Ryoko; Katagiri, Wataru; Endo, Satoshi; Kobayashi, Tadaharu. Exosomes from conditioned media of bone marrow-derived mesenchymal stem cells promote bone regeneration by enhancing angiogenesis. Plos One. 14(11):e0225472. 

Rina Kato et., al. . Detection of exosomal miRNA in GCF with chronic periodontitis. J Osaka Dent Univ. 2021;55(1):73-81. 

M Yoshikawa et., al.. Electric Field Stimulation of Stem Cell Culture Media Increases Oxygen Radical Uptake Capacity. The Indonesian Biomedical Journal. 2023;15(4):296-302. 

García-Manrique, Pablo; Serrano-Pertierra, Esther; Lozano-Andrés, Estefanía; López-Martín, Soraya; Matos, María; Gutiérrez, Gemma; Yáñez-Mó, María; Blanco-López, María Carmen. Selected Tetraspanins Functionalized Niosomes as Potential Standards for Exosome Immunoassays. Nanomaterials (Basel, Switzerland). 2020;10(5) 

Ishida, Takenori; Hashimoto, Takuma; Masaki, Kanako; Funabashi, Hisakage; Hirota, Ryuichi; Ikeda, Takeshi; Tajima, Hideji; Kuroda, Akio. Application of peptides with an affinity for phospholipid membranes during the automated purification of extracellular vesicles. Scientific Reports. 2020;10(1):18718. 

Yukawa, Hiroshi; Yamazaki, Shuji; Aoki, Keita; Muto, Kengo; Kihara, Naoto; Sato, Kazuhide; Onoshima, Daisuke; Ochiya, Takahiro; Tanaka, Yasuhito; Baba, Yoshinobu. Co-continuous structural effect of size-controlled macro-porous glass membrane on extracellular vesicle collection for the analysis of miRNA. Scientific Reports. 2021;11(1):8672. 

Iida, Takuya; Hamatani, Shota; Takagi, Yumiko; Fujiwara, Kana; Tamura, Mamoru; Tokonami, Shiho. Attogram-level light-induced antigen-antibody binding confined in microflow. Communications Biology. 2022;5(1):1053. 

Kajihara, Ryota; Tobinaga, Kyohei; Tanigaki, Riho; Ute, Koichi; Tominaga, Tatsuya. Hydrogel-Based Extracellular Vesicle Isolation Method from Various Biological Solutions. Acs Applied Bio Materials. 2026;9(1):287-297. 

CD9/CD63 Exosome ELISA Kit FAQs

Do exosomes need to express both CD9 and CD63 to be detected with CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL)?
Yes. If CD9 is not expressed on the exosome surface, particles are removed during the wash step. If CD63 is not expressed, the detection antibody cannot bind. Therefore, only exosomes expressing both CD9 and CD63 are detected.

Are soluble CD9 or CD63 molecules present in a sample likely to affect measurements with CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL)?
No. Only structures expressing both CD9 and CD63 are detected. Therefore, the presence of soluble CD9 or CD63 in the sample does not affect the assay.

What is the CD9/CD63 fusion protein used as the standard in CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL)?
The standard protein is a recombinant fusion protein consisting of tandemly linked sequences containing the antibody epitope regions of CD9 and CD63.

How is the standard protein in CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL) manufactured?
The standard protein is a recombinant protein expressed in HEK293 cells. A transfection reagent is used for gene delivery.

Can the standard protein in CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL) be frozen?
The standard protein has been confirmed to withstand one freeze-thaw cycle at the original stock concentration.

Can the standard protein in CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL) be stored after dilution in the assay buffer?
We do not recommend storing the standard protein after dilution. The standard protein supplied with the kit can be used for multiple assays and should be aliquoted before use.

Can the number of exosomes be estimated from results obtained with CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL)?
No. The colorimetric signal generated by CD9/CD63 Exosome ELISA Kit is proportional to the amount of detection antibody bound and cannot be used to estimate the number of exosomes.

Can I use human serum directly with CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL) without exosome purification?
Yes. Human serum can be used directly after dilution of at least fourfold. Please refer to the datasheet for examples.

Can I use human plasma with CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL)?
We do not recommend using unpurified plasma, although measurable results can be obtained. Compared with serum, plasma tends to show lower reproducibility, and the cause of this variability has not yet been identified. If you use plasma samples with CD9/CD63 Exosome ELISA Kit, we recommend evaluating purification methods, anticoagulant selection, and sample dilution conditions in advance.

Can I measure cell culture supernatants containing bovine serum such as FBS with CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL)?
Yes. This kit does not react with bovine-derived exosomes and can therefore be used with culture supernatants containing FBS.

Can I use samples from species other than humans with CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL)?
No. The antibodies used in this kit do not react with species other than human.

Is the plate in CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL) a strip-type?
Yes. The plate consists of strip wells arranged as 8 wells × 12 strips, allowing up to 12 separate uses.

What should I do if I run out of a kit component, such as the standard protein or detection antibody?
Individual kit components are available for purchase separately. Please contact us for details.

Are the CD9 and CD63 antibodies used in CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL) the same antibodies as the exosome monoclonal antibodies?
Yes. This kit uses Anti CD9 Antigen (MRP-1/Tspan-29) mAb (Clone 12A12) (Cat. No. CAC-SHI-EXO-M01) and Anti CD63 Antigen (LAMP-3/Tspan-30) mAb (Clone 8A12) (Cat. No. CAC-SHI-EXO-M02).
The detection antibody, Anti CD63 Antigen (LAMP-3/Tspan-30) mAb (Clone 8A12) (Cat. No. CAC-SHI-EXO-M02), is HRP-conjugated.

How many kits from the same lot can be purchased for continuous experiments using CD9/CD63 Exosome ELISA Kit (Cat. No. CSR-EXH0102EL)?
Up to 30 kits can be supplied from the same lot. For quantities exceeding 30 kits, please contact us regarding made-to-order production.