EXOSOME Dashboard

Isolation and Characterization with

Anti-Human Tetraspanin mAbs and OptiPrep™

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What are exosomes?

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.

A recent landmark paper (Jeppesen, D., Fenix, A., Franklin, J., Higginbotham, J., Zhang, Q., Zimmerman, L., Liebler, D., Ping, J., Liu, Q., Evans, R., Fissell, W., Patton, J., Rome, L., Burnette, D., Coffey, R. (2019). Reassessment of Exosome Composition Cell 177(2), 428-445.e18.) showed that the majority of exosome isolation approaches, including classical ultracentrifugation, suffer from significant protein and DNA contamination from non-vesicular extracellular sources. The methods used to reveal this surprising level of contamination include high resolution iodixanol density gradient ultracentrifugation [to separate small EVs from non-vesicular components (such as vaults and exomeres)] and direct immunocapture (DIC) from human plasma and cell conditioned media using antibodies to tetraspanin molecules CD63, CD9 or CD81.

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High Quality anti-Human Tetraspanin Monoclonal Antibodies

Cryo-electron microscopic detection of exosomes bound by anti-human CD63 antibody (clone 8A12).
Exosomes and anti-human CD63 antibody (clone 8A12) were mixed and imaged with cryo-electron microscopy (Titan Krios). Shown is a cryo-electron micrograph of an exosome (A), a three-dimensional reconstruction of an exosome (green) with a single bound antibody (cyan) (B) and a rotating 3D tomographic reconstruction of an exosome and a bound antibody (Top). Images courtesy of Okinawa Protein Tomography Ltd.

High specificity anti-human tetraspanin antibodies from CosmoBio USA are ideal reagents for characterizing and isolating classical exosomes using new “ultraclean” methods.

Raised against tetraspanin extracellular loops
Target	Clone	Immunogen	isotype	purification
CD9	12A12	Amino acids 113-195: the large second extracellular loop between the 3^rd and 4^th transmembrane domains	IgG2b	Protein G
CD63	8A12	Amino acids 104-202: the large second extracellular loop between the 3^rd and 4^th transmembrane domains	IgG2a	Protein G
CD81	12C4	Amino acids 36-54: the small first extracellular loop between the 3^rdand 4^thtransmembrane domains	IgG2a	Protein G

Human reactive
Target	Clone	Reactive	Non-reactive
CD9	12A12	Human	Mouse, Rat
CD63	8A12	Human	Mouse, Rat
CD81	12C4	Human, Cow	Mouse, Rat

High performance in multiple applications
Application	anti-CD9 (clone 12A12)	anti-CD63 (clone 8A12)	anti-CD81 (clone 12C4)
IP	+	+	+
WB	+	+	+
FC	+	+	+
ELISA	+	+	+
IF	+	+	+
IHC	+	+	+
IEM	+	+	+
ExoTrap™ Exosome Isolation Spin Column	+	-	-
CD9/CD63 Exosome ELISA Kit	+	+	-
ExoScreen assay	+	+	not tested
In vivo metastasis assay	+	not tested	not tested
Mass spectrometric immunoassay with monolith pipette tips	+	not tested	not tested
Membrane fusion assay	not tested	+	not tested
In vitro opsonization assay	+	+	not tested

Effective in multiple human biofluids
Biofluid	anti-CD9 (clone 12A12)	anti-CD63 (clone 8A12)	anti-CD81 (clone 12C4)
plasma	+	+	+
culture supernatant	+	+	+
urine	+	+	not tested
cerebrospinal fluid	+	not tested	not tested
pleural lavage	not tested	+	not tested
milk	not tested	not tested	+
liver tissue	not tested	not tested	+

*Tide Fluor™ 2 (TF2) has spectral properties similar to those of fluorescein and Alexa Fluor® 488 (Invitrogen). Compared to fluorescein, TF2 has much stronger fluorescence and higher photostability. Additionally, its fluorescence is pH-independent from pH 3 to 11. TF2-labeled antibodies exhibit much stronger fluorescence and higher photostability than those labeled with FITC. These characteristics make TF2 a superior alternative to fluorescein.
#Tide Fluor™ 5 (TF5) has spectral properties essentially identical to those of Cy5. Compared to Cy5, TF5 has much stronger fluorescence and higher photostability. Additionally, its fluorescence is pH-independent from pH 3 to 11. TF5-labeled antibodies exhibit much stronger fluorescence and higher photostability than those labeled with Cy5. These characteristics make TF5 a superior alternative to Cy5. 

Conjugated to biotin, Tide Fluor-2^* and Tide Fluor-4^#
anti-CD9 (clone 12A12)	unconjugated
	biotin-conjugated
	Tide Fluor™ 2-conjugated
	Tide Fluor™ 4-conjugated
anti-CD63 (clone 8A12)	unconjugated
	biotin-conjugated
	Tide Fluor™ 2-conjugated
	Tide Fluor™ 4-conjugated
anti-CD81 (clone 4C12)	unconjugated
	biotin-conjugated
	Tide Fluor™ 2-conjugated
	Tide Fluor™ 4-conjugated

Ultraclean direct immunoaffinity capture (DIC) of classical-exosomes with tetraspanin antibodies

Immediately after collection, cell-conditioned medium was subjected to differential centrifugation at 400 x g, 2,000 x g and 15,000 x g at 4°C. The supernatant was filtered through a 0.22mm pore PES filter (Millipore) to generate pre-cleared medium. All following steps were also performed at 4°C. DIC of exosomes directly from human plasma was performed as described above except that the supernatant in these cases was raw, undiluted and unfiltered, plasma (generated by two rounds of 3000 x g centrifugation for 15 min). Pre-cleared medium was split three ways: one portion was incubated with magnetic beads directly conjugated to antibodies directed at CD63, CD81 or CD9; a second portion was incubated with magnetic beads conjugated to mouse IgG, and incubation was allowed to proceed with nutation and rotation for 16h; the third portion was subjected to ultracentrifugation and washing to generate a crude sEV pellet (P120). After incubation, the beads were washed four times in ice-cold 0.1% BSA-PBS (pH 7.4, filtered through a 0.22mm membrane), and finally, washed one time in PBS pH 7.4 (pH 7.4, filtered through a 0.22mm membrane). Immediately following the last wash, the exosome-loaded beads were resuspended first in cell lysis buffer and LDS sample buffer for 30 min on ice, and then resuspended in LDS sample buffer followed by heating to 70°C for 10 min to release proteins. The beads were removed from the suspension by using a magnet and the clarified lysate used for immunoblot analysis. In some cases, immediately following the last wash step, the exosome-loaded beads were lysed for DNA extraction.

(from: Jeppesen, D., Fenix, A., Franklin, J., Higginbotham, J., Zhang, Q., Zimmerman, L., Liebler, D., Ping, J., Liu, Q., Evans, R., Fissell, W., Patton, J., Rome, L., Burnette, D., Coffey, R. (2019). Reassessment of Exosome Composition Cell 177(2), 428-445.e18.)

Click for Tetraspanin mAb User Reports

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Ordering Information

Product	Clone	Label	Unit size	Catalog Number
Tetraspanin antibodies for exosome purification and characterization
Anti CD9 Antigen (MRP-1/Tspan-29) mAb	12A12	-	50 UL	CAC-SHI-EXO-M01-50
Anti CD9 Antigen (MRP-1/Tspan-29) mAb	12A12	-	100 UL	CAC-SHI-EXO-M01-100
Anti CD9 Antigen (MRP-1/Tspan-29) mAb	12A12	biotin	100 UL	CAC-SHI-EXO-M01-B
Anti CD9 Antigen (MRP-1/Tspan-29) mAb	12A12	TF	100 UL	CAC-SHI-EXO-M01-TF2
Anti CD9 Antigen (MRP-1/Tspan-29) mAb	12A12	TF5	100 UL	CAC-SHI-EXO-M01-TF5
Anti CD63 Antigen (LAMP-3/Tspan-30) mAb	8A12	-	50 UL	CAC-SHI-EXO-M02-50
Anti CD63 Antigen (LAMP-3/Tspan-30) mAb	8A12	-	100 UL	CAC-SHI-EXO-M02-100
Anti CD63 Antigen (LAMP-3/Tspan-30) mAb	8A12	biotin	100 UL	CAC-SHI-EXO-M02-B
Anti CD63 Antigen (LAMP-3/Tspan-30) mAb	8A12	TF2	100 UL	CAC-SHI-EXO-M02-TF2
Anti CD63 Antigen (LAMP-3/Tspan-30) mAb	8A12	TF5	100 UL	CAC-SHI-EXO-M02-TF5
Anti CD81 Antigen (TAPA-1/Tspan-28) mAb	12C4	-	50 UL	CAC-SHI-EXO-M03-50
Anti CD81 Antigen (TAPA-1/Tspan-28) mAb	12C4	-	100 UL	CAC-SHI-EXO-M03-100
Anti CD81 Antigen (TAPA-1/Tspan-28) mAb	12C4	biotin	100 UL	CAC-SHI-EXO-M03-B
Anti CD81 Antigen (TAPA-1/Tspan-28) mAb	12C4	TF2	100 UL	CAC-SHI-EXO-M03-TF2
Anti CD81 Antigen (TAPA-1/Tspan-28) mAb	12C4	TF5	100 UL	CAC-SHI-EXO-M03-TF5
Tetraspanin antibody-based exosome purification and ELISA kit
ExoTrap™ Exosome Isolation Spin Column Kit	12A12	-	10 preps	CSR-SHI-EXO-K010
CD9/CD63 Exosome ELISA Kit	12A12/8A12	-	1 Kit	CSR-EXH0102EL
Ultracentrifugation media for exosome purification
OptiPrep™-Density Gradient Media (Iodixanol)	-	-	1 x 250 ML	AXS-1114542
OptiPrep™-Density Gradient Media (Iodixanol)	-	-	5 x 250 ML	AXS-1114542-5
Milk exosome reagents
Anti-Bovine Milk Exosome pAb	-	-	100 UL	CAC-EXO-AB-01
Human milk-derived exosomes	-	-	1 ML	CSR-EXHM100L
Antibodies and ELISAs for putative exosome markers and negative controls
Human TSG101 ELISA Kit	-	-	1 x 96 Rxns	CSB-EL025125HU
Human ALIX ELISA Kit	-	-	1 x 96 Rxns	CSB-EL017673HU
Human Flotillin-1 (FLOT1) ELISA Kit	-	-	1 x 96 Rxns	CSB-EL008727HU
Human HSP70 ELISA Kit	-	-	1 x 96 Rxns	CSB-E08297h
Human GAPDH ELISA Kit	-	-	1 x 96 Rxns	CSB-E13911h
Anti Human EPCAM mAb	hrk29	-	50 UG	CAC-HT-MAB1
Human EPCAM ELISA kit	-	-	1 x 96 Rxns	CSB-EL007717HU
Anti Vimentin pAb (Rabbit, Antiserum)	-	-	100 UL	LSL-LB-3010
Human Vimentin ELISA Kit	-	-	1 x 96 Rxns	CSB-E08982h
Human CETP ELISA Kit	-	-	1 x 96 Rxns	CSB-E08567h
Human CXCR4 ELISA Kit	-	-	1 x 96 Rxns	CSB-E12825h
Anti CD68 mAb	PM-1K	-	50 UG	KAL-KT117
Human CD68 ELISA Kit	-	-	1 x 96 Rxns	CSB-E15979h
Anti CD44 Antigen v9 mAb	RV3	-	50 UG	CAC-LKG-M003
Anti CD44 Antigen v9 mAb	RV3	-	100 UG	CAC-LKG-M001
Human CD44 ELISA Kit	-	-	1 x 96 Rxns	CSB-E11846h
Human HLA-G ELISA Kit	-	-	1 x 96 Rxns	CSB-E09401h
Human HSPA8 ELISA Kit	-	-	1 x 96 Rxns	CSB-EL010829HU

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