Exosome Marker Antibodies

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Webinar – Flow Analysis of Extracellular Vesicles

Protein Markers for Exosome Characterization

Members of the International Society for Extracellular Vesicles (ISEV) delineated minimum requirements for the reproducible and accurate investigation of exosomes in human health and disease. These requirements include suggested proteins to discriminate extracellular vesicles (EVs) from contaminating components in culture media or biological fluids such as serum, plasma, and urine. The presence of exosomes is confirmed by detection of at least one transmembrane or GPI-anchored protein and one cytosolic lipid or membrane associated protein. To assess the degree of contamination of an exosome preparation with cellular components, the assay should also include targets that rarely associate with EVs like albumin, ApoA1, Calnexin, or ribosomal protein S6/RPS6.

Enlarged image of exosome with example intravesicular and transmembrane proteins.

Bio-Techne antibodies are highly specific and rigorously tested using the 5 Pillars of Antibody Validation.

General Exosome Markers: Tetraspanins

The most common transmembrane markers to identify exosomes come from the tetraspanin family of proteins (CD63 ^a, CD81 ^b,c, and CD9 ^c,d). Proteins in this family have four membrane spanning regions and are localized to tetraspanin enriched microdomains (TEM) in the plasma membrane.

Microscopy image with fluorescent tags showing expression of CD9, CD81, and CD63 on extracellular vesicles from two cell lines.

Markers

Function

Associate with adhesion molecules, transmembrane receptors and intracellular signaling proteins to facilitate and regulate signaling events.

Western Blot Exosome Marker Antibody Pack

When examined by single-vesicle imaging, using total internal reflection fluorescence (TIRF) microscopy, researchers found distinct co-localization of tetraspanins depending on cellular source. Their data, along with others, suggest tetraspanin expression is not uniform and there are distinct subpopulations of EVs secreted from each cell type. CD9 and CD81 tended to co-localize on the same vesicle, while CD63 was more often found alone.

Single vesicle imaging by TRIF of EVs isolated from the human breast cancer cell line MCF-7 and the mouse melanoma cell line B16BL6. Immobilized vesicles were stained with Mouse Anti-Human CD9 (MEM-61) (NB500-327) in green in merged image. Merged image shows co-localization of CD81 and CD9 (blue) on individual vesicles. Image was adapted from Han C, et al. Single-vesicle imaging and co-localization analysis for tetraspanin profiling of individual extracellular vesicles. J Extracell Vesicles. 2021 Jan;10(3):e12047. Access provided by Creative Commons License.

General Exosome Markers: Multivesicular Body (MVB)-Associated Proteins

In the endosomal maturation pathway, very specific cellular machinery is required for formation of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs). The formation of MVBs is regulated by complexes of proteins, collectively referred to as the endosomal sorting complex required for transport (ESCRT). Consequently, ESCRT-related proteins can be useful markers for exosome characterization, as they suggest, but do not prove, the intracellular pathway of vesicle biogenesis.

Simple western lane view with two lanes. One lane shows signal band for TSG101 and the other showing signal band for HSP90

Markers	Function
ALIX TSG101	Interact with proteins of the midbody involved in cytokinesis and membrane scission
Flotillin-1	Scaffolding protein within caveolar membranes, participate in formation of calveolae or calveolae-like vesicles
Clathrin	Involved in receptor-mediated endocytosis and formation of early endosomes

Simple western lane view (acquired with Wes) showing Rabbit Anti-Human TSG101 (NBP-267884), and Rabbit Anti-Human HSP90 (NBP2-67395) on EVs isolated by ultracentrifugation. Image adapted from Haney MJ, Zhao Y, Jin YS, Batrakova EV. Extracellular Vesicles as Drug Carriers for Enzyme Replacement Therapy to Treat CLN2 Batten Disease: Optimization of Drug Administration Routes. Cells. 2020;9(5):1273. doi:10.3390/cells9051273. Access provided by Creative Commons License.

Role of Chaperone Proteins in Exosomes

Heat-shock proteins (HSPs) are a family of intracellular molecular chaperones that are produced in response to stress. The most well-studied are HSP60, HSP70, and HSP90. Because secretion of exosomes often increases under conditions of stress, such as cancer, inflammation, and hypoxia, HSPs are often found incorporated into exosomes. HSP70 is expressed in a majority of cancers, and research has shown that HSP70-associated EVs are immunosuppressive in the tumor microenvironment.

Markers

Function

Ensure proper protein-folding and prevent abberant protein aggregates from building up within a cell.

Western blot showing expression of protein (CD147/EMMPRIN) in one lane but not the other lane (knockout cell). Loading control GAPDH shown below and DNA double helix showing knockout validation badge.

Simple Western lane view showing lysates of Jurkat human acute T cell leukemia cell line untreated (-) or treated (+) by heat shock (HS) and stained with Mouse Anti-Human HSP70/HSPA1A (MAB1663). Band appears around 70 kDa in HS but not untreated cells. The biological strategies badge shows antibody specificity using biological strategies (heat-shock) and detection of protein only in treated cells.”

Simple Western Exosome Marker Antibody Pack

Tissue/Cell Type-Specific Exosome Markers

Protein markers are also important in identifying the cellular origin of EVs. Just as individual cell types express identifying markers, exosomes derived from those cells often express cell-type characteristic proteins. For example, CD147/EMMPRIN is enriched on EVs produced from tumor cells in ovarian cancer.

Western blot lysates of HEK293T human embryonic kidney parental cell line and EMMPRIN/CD147 knockout (KO) HEK293T cell line. PVDF membrane was probed with Goat Anti-Human EMMPRIN/CD147 (AF972), followed by HRP-conjugated Rabbit Anti-Goat IgG Secondary Antibody (HAF017). GAPDH (AF5718) is shown as a loading control. The Genetic strategies badge shows antibody specificity by comparing parental cell line to KO cell line.

Cell Type	Exosome Marker
	EpCAM ^e CD147/EMMPRIN EGFR
	CD3 CD19 CD20 CD37 NKG2D CD56
	MMP-9 CD44 CD73 HGF
	CD31/PECAM-1 CD41 CD62p PF4 GP-IX(CD42a)
	L1CAM GluR2 NF-L TDP43

Markers of Exosome Function

EVs carry out a wide range of functions within the body and their cargo is equally varied. Specific content depends on host species, cellular origin, biofluid, and disease state. Some common markers of signaling, metabolism, cell death, and immunomodulation are listed below:

Function	Markers
Signaling	HIF-1α ^f β-catenin VEGF CDC42 ARF1
Metabolic Enzymes	GAPDH PGK1
Cell death/cytotoxicity	FasL Granzyme A Granzyme B
Immunomodulatory	HLA-A/B/C HLA-DR/DP/DQ TNF-α CD86 Galectin-9 TGF-β PD-L1 ^e

Select References

Wang J, Wuethrich A, Sina AAI, Lane RE, Lin LL, Wang Y, et al. Tracking extracellular vesicle phenotypic changes enables treatment monitoring in melanoma. Sci Adv 2020;6:. https://doi.org/10.1126/sciadv.aax3223. Anti-CD63 (NBP2-42225) Application: WB, capture

Kibria G, Ramos EK, Lee KE, Bedoyan S, Huang S, Samaeekia R, et al. A rapid, automated surface protein profiling of single circulating exosomes in human blood. Sci Rep 2016;6:1–9. https://doi.org/10.1038/srep36502. Anti-CD81 (NB100–65805) Application: WB

Ren W, Hou J, Yang C, Wang H, Wu S, Wu Y, et al. Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery. J Exp Clin Cancer Res 2019;38:. https://doi.org/10.1186/s13046-019-1027-0. Anti-CD81 (NB100–65805); Anti-CD9 (NBP2-22187) Application: WB

Han C, Kang H, Yi J, Kang M, Lee H, Kwon Y, et al. Single‐vesicle imaging and co‐localization analysis for tetraspanin profiling of individual extracellular vesicles. J Extracell Vesicles 2021;10:e12047. https://doi.org/10.1002/jev2.12047. Anti-CD9 (NB500-327) Application: Imaging

Gieri R, Piva F, Occhipinti G, et al. Clinical impact of different exosomes' protein expression in pancreatic ductal carcinoma patients treated with standard first line palliative chemotherapy. PLoS One. 2019;14(5):e0215990. EpCAM (MAB9601) ; PD-L1 (MAB1561) Application: ELISA

Feng Q, Zhang C, Lum D, Druso JE, Blank B, Wilson KF, et al. A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis. Nat Commun 2017;8:. https://doi.org/10.1038/ncomms14450. Anti-HIF-1α (NB100-105) Application: WB

Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 2018;7:. https://doi.org/10.1080/20013078.2018.1535750

Termini CM, Gillette JM. Tetraspanins function as regulators of cellular signaling. Front Cell Dev Biol 2017:34. https://doi.org/10.3389/fcell.2017.00034.

Bellin G, Gardin C, Ferroni L, Chachques J, Rogante M, Mitrečić D, et al. Exosome in Cardiovascular Diseases: A Complex World Full of Hope. Cells 2019;8:166. https://doi.org/10.3390/cells8020166.

Soria FN, Pampliega O, Bourdenx M, Meissner WG, Bezard E, Dehay B. Exosomes, an unmasked culprit in neurodegenerative diseases. Front Neurosci 2017:26. https://doi.org/10.3389/fnins.2017.00026.

Tao SC, Guo SC, Zhang CQ. Platelet-derived extracellular vesicles: An emerging therapeutic approach. Int J Biol Sci 2017:828–34. https://doi.org/10.7150/ijbs.19776.

Frühbeis C, Fröhlich D, Krämer-Albers EM. Emerging roles of exosomes in neuron-glia communication. Front Physiol 2012;3 APR:119. https://doi.org/10.3389/fphys.2012.00119.

Murphy ME. The HSP70 family and cancer. Carcinogenesis 2013;34:1181–8. https://doi.org/10.1093/carcin/bgt111