• High sensitivity (detectable at a sensitivity 50-1,000 times higher than that of WB)
• Direct qualitative/quantitative analysis of exosomes in the culture supernatant
• Capable of saving the number of exosomes used for analysis (less than 1/10-1/1,000 of the number required for WB)

Add 1 ng of extracellular vesicles purified from various cell culture supernatants to each well, and the expression level of surface marker proteins was compared by a qualitative analysis with three primary detection antibodies.

In addition, as reference comparative data, 150 ng of extracellular vesicles were purified from various cell culture supernatants and expression levels of their each surface markers were detected by Western Blot similarly. Then, qualitative analysis was conducted.

Expression pattern of marker proteins between ELISA and WB have a correlation.

Comparison of qualitative data per 1 ng of purified exosomes

Reference comparative data

Comparison of qualitative data per 1 ng of purified exosomes

Detection antibody

ELISA
Anti-CD9 mouse mAb (M-L 13), BD Bioscience

Anti-CD63 mouse mAb (H5C6), BD Bioscience

Anti-CD81 mouse mAb (JS-81), BD Bioscience

WB
Anti-CD9 rabbit pAb, System Bioscience

Anti-CD63 mouse mAb (8A12), CosmoBio

Anti-CD81 mouse mAb (1D6), Novus Biologicals

With 1 ng of extracellular vesicles purified from various cell culture supernatants to each well, expression level of surface marker proteins were detected by using three primary detection antibodies for CD9, CD63, and CD81.

It is confirmed that expression levels of particular markers on exosomes is different between cell strains.

Comparison of qualitative data of each sample

Each of 40, 20, and 10 ng of extracellular vesicles purified from six human normal serum samples was added to a well and qualitative analysis was conducted using a control primary detection antibody against CD63 in the Kit.

The results showed properly linear curves in each samples.

A standard curve was prepared using extracellular vesicles purified from cell culture supernatant of COLO201 cells, and then the dilution linearity of 5-step dilution samples of cell culture supernatant of COLO201 cells (1:100 to 1:1600) was evaluated.

Comparison of qualitative data of each sample

Reference standard: extracellular vesicles purified from cell culture supernatant of COLO201 cells with MagCapture Exosome Isolation Kit PS
Measured sample: cell culture supernatant of COLO201 cells
Primary antibody: anti-CD63 antibody in the kit

1 x 10⁷, 2 x 10⁷, and 3 x 10⁷ COLO201 cells were separately seeded into T75 flasks and cultured for 72 hours. The small amount of culture supernatant samples were collected every 24 hours and subjected to spectrophotometric assay of CD63 using PS Capture Exosome ELISA Kit (Anti Mouse IgG POD) and Control Primary Antibody Anti-CD63 (100 x ) included in the kit.

For the CD63 assay, 4 μL cell culture supernatant was diluted to 100 μL with Reaction/Washing Buffer (1×) supplemented with Exosome Binding Enhancer.

• Assay sample: 25-fold diluted COLO201 cell culture supernatant (4μL → diluted to 100μL)
• Number of seeding cells: 1 x 10⁷, 2 x 10⁷, 3 x 10⁷ cells/T75 flask
• Culture duration: 24, 48, 72 hours
• Primary antibody: anti-CD63 antibody

Since it is possible to directly measure the amount of extracellular vesicles in the medium using only 4 μL of medium, this assay is recommended because an optimization of culture condition for newly culturing a cell line takes time and effort. By this method, the conditions under which the most extracellular vesicles are secreted can be easily examind.

K562 cells were seeded into T225 flasks and cultured in serum-free medium for 72 hours. Then, the culture medium was changed to serum-free medium supplemented either with or without monensin sodium salt whose final concentration is 10 μM and cultured for 24 hours. After the end of culture, culture supernatant samples were collected and subjected to spectrophotometric assay of CD63 using PS Capture Exosome ELISA Kit (Anti Mouse IgG POD) and Control Primary Antibody Anti-CD63 (100 x ) included in the kit.

10-fold and 100-fold diluted cell culture supernatant samples were prepared by dilution of collected cell culture supernatant with Reaction/Washing Buffer (1 x ) supplemented with Exosome Binding Enhancer.

• Assay sample: K562 cell culture supernatant (cultured for 24 hours after changing to culture medium supplemented with monensin sodium salt or control culture medium)
• Primary antibody: anti-CD63 antibody

This assay requires no sample purification and just a small aliquot of culture medium collected during culture is sufficient for assay.
Since changes of the amount of exosomes in culture medium over time can be assayed and quantified, comparative assay of them is much easier than WB analysis. It's very convenient.

The following samples (1) to (6) were prepared and used for comparison of detection sensitivity of PS Capture Exosome ELISA Kit, Competitor A ELISA kit, and Competitor A ELISA kit (high-sensitivity type) with detection of CD63, an exosome marker protein.

Samples used for comparison

(1) Standard included in Competitor A ELISA kit
(2) Standard included in Competitor A ELISA kit (high-sensitivity type)
(3) Exosomes purified from COLO201 cell culture supernatant using MagCapture Exosome Isolation Kit PS
(4) Exosomes purified from COLO201 cell culture supernatant by polymer precipitation
(5) Exosomes purified from human serum using MagCapture™ Exosome Isolation Kit PS
(6) Exosomes purified from human serum by polymer precipitation

Dilution rates and protein concentrations

Comparison of detection sensitivity of individual exosome ELISA kits

PS Capture Exosome ELISA Kit detected CD63 at a sensitivity higher than those of Competitor A ELISA Kit and Competitor A ELISA Kit (high-sensitivity type). While both Competitor A ELISA Kit and Competitor A ELISA Kit (high-sensitivity type) strongly reacted with standards in their kit, their reactivity to exosomes purified by the PS affinity method was low.

These results suggested that PS Capture Exosome ELISA Kit was capable of detecting CD63 on the surface of exosomes more specifically and at a higher sensitivity than Competitor A ELISA Kit and Competitor A ELISA Kit (high-sensitivity type).