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Home > Products > Life Science > Genetic Research > PS Capture™ Exosome ELISA Kit
Home > Products > Life Science > Immunochemistry > PS Capture™ Exosome ELISA Kit

A novel tool for qualitative and quantitative analysis of extracellular vesicles in sample of cell culture supernatantimage
PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD)


1. Link

2. Product Outline

  The kit includes reagents for enzyme-linked immunosorbent assay (ELISA) available for a Qualitative Analysis of extracellular vesicles purified from cell culture supernatant or body fluid as well as a Quantitative Analysis of extracellular vesicles in cell culture supernatant.
  It can detect extracellular vesicles, which have any surface marker protein, with high sensitivity by using a mouse monoclonal antibody against any surface marker proteins of extracellular vesicles as a primary detection antibody and HRP-conjugated anti mouse IgG antibody of the kit as a secondary detection antibody after extracellular vesicles are captured by a plate on which proteins that specifically bind with phosphatidylserine (PS) on the surface of extracellular vesicles are immobilized.

[Reference]
“A novel affinity-based method for the isolation of highly purified extracellular vesicles”, W. Nakai, T. Yoshida, D. Diez, Y. Miyatake, T. Nishibu, N. Imawaka, K. Naruse, Y. Sadamura & R. Hanayama, Sci Rep 6, 33935 (2016).

3. Features ~ PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) ~

PS-binding molecule, Tim4,
immobilized plate is adopted

  • Capturing by PS-binding molecule※1
  • Can detect extracellular vesicles in cell culture supernatant corresponding to 0.1 μL
  • Quantitative analysis with reference standard of purified extracellular vesicles※2
  • Can detect with 50-1,000 times higher sensitivity than WB※3

High-sensitive qualitative and quantitative analysis

  ※1 Available for high-sensitive qualitative and quantitative analysis than ELISA kit based on antibody-immobilized plate
※2 Use reference standard of purified extracellular vesicles from a sample
      with MagCapture™ Exosome Isolation Kit PS (Code No. 293-77601).
※3 WB: western blot.

Optimized reagent is kitted

  • Optimized control primary antibody※4
  • Optimized secondary antibody, HRP-conjugated※5
  • Chromogenic method is adopted

Easy operation
High reproducibility

  ※4 Anti-human CD63 mouse antibody is contained in the kit.
      When intending to detect other surface marker proteins, use an optional mouse monoclonal antibody.
※5 Anti-mouse IgG antibody, HRP-conjugated is contained in the kit.

4. Principle / Components

Principle

Principle

Components

Kit component Amount
(for 96 tests)
Exosome Capture 96 Well Plate 1 plate
(8 well ×12 strips)
Reaction / Washing Buffer (10×) 50 mL × 2 vials
Exosome Binding Enhancer (100×) 10 mL × 1 vial
Control Primary Antibody Anti-CD63 (100×) 120 μL × 1 vial
Secondary Antibody HRP-conjugated Anti-mouse IgG (100×) 120 μL × 1 vial
TMB Solution 12 mL × 1 vial
Stop Solution 12 mL × 1 vial
Plate Seal 4 sheets
Instruction manual 1 copy

5. Product List

Product Name Package Size Wako Catalog No. Storage Condition
PS Capture™ Exosome ELISA Kit (Anti Mouse IgG POD) 96 tests 297-79201 Keep at 2-10°C.

Note: when intending to analyze extracellular vesicles of body fluids, qualitative analysis is available by using purified extracellular vesicles from body fluids with MagCapture™ Exosome Isolation Kit PS (Code No. 293-77601).

6. Purpose

(1) A qualitative analysis of extracellular vesicles purified from cell culture supernatant or body fluids  
  The kit provides a highly sensitive qualitative analysis of any surface marker protein of extracellular vesicles purified from cell culture supernatant or body fluids with MagCapture™ Exosome Isolation Kit PS by using a mouse monoclonal antibody against any surface marker protein of extracellular vesicles as a primary detection antibody.

(2) A quantitative analysis of extracellular vesicles in cell culture supernatant
  Extracellular vesicles which are positive for any marker protein in cell culture supernatant can be quantitatively analyzed by using extracellular vesicles purified from cell culture supernatant with MagCapture™ Exosome Isolation Kit PS as a reference standard and using a mouse monoclonal antibody against any surface marker protein of extracellular vesicles as a primary detection antibody.

Note: While Anti CD63 antibody as a control primary detection antibody in the kit can detect human CD63, it cannot detect mouse, rat, and bovine CD63. When a surface marker protein other than human CD63 is required to be detected, use a mouse monoclonal antibody of interest.
Note: Since HRP-conjugated Anti mouse IgG as a secondary detection antibody of the kit can strongly react non-specifically with mouse IgG in a sample and weakly react non-specifically with human IgG and rat IgG, a quantitative analysis of serum or plasma samples including these IgGs should be avoided.

7. Qualitative analysis of extracellular vesicles purified from various cell culture supernatants

  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.

Comparison of qualitative data per 1 ng of purified exosomes

Reference comparative data

Purified exosome: 150 ng


Expression pattern of marker proteins between ELISA and WB have a correlation.
Reference data: Qualitative analysis of EVs purified from various cell culture supernatants

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.

■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

8. Qualitative analysis of extracellular vesicles purified from human normal serum

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.

Qualitative analysis of extracellular vesicles


The results showed properly linear curves in each samples.

9. Reference data: Detection sensitivity of extracellular vesicles purified from cell culture supernatant

Comparing the detection sensitivities of western blot and PS ELISA

Extracellular vesicles were purified from cell culture supernatant of COLO201, and then detection sensitivities of the kit and western blot were compared.


(a), (b) Result of sensitivity by Western Blot with each of anti-CD63 antibody (supplier A and Wako: Code No. 012-27063).
 Sample: purified extracellular vesicles from cell culture supernatant of COLO201 with MagCapture™ Exosome
               Isolation Kit PS (Code No. 293-77601)
:detection limit by western blot

(c) Data of a detection sensitivity by Exosome ELISA Kit
A standard curve was prepared using blank value of buffer and absorbance value of 2-fold serial dilution samples of extracellular vesicles purified from cell culture supernatant of COLO201 cells with MagCapture™ Exosome Isolation Kit PS. Then, the detection limit of extracellular vesicles purified from cell culture supernatant of COLO201 was calculated using its standard curve. (each dilution point: n=6, blank: n=12)

Exosome ELISA Kit detected the marker proteins with higher sensitivoty than WB.

10. Reference data: Dilution linearity of cell culture supernatant samples

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

Dilution linearity of cell culture supernatant samples of COLO201 cells
(Detected by anti CD63 antibody)
CM volume(μL) Dilution Assay value Expected value % of expected
Ratio Factor (×) ng/mL ng/mL
0.0625 1:1600 0.000625 0.89 0.91 98.4
0.125 1:800 0.00125 1.82 1.72 105.6
0.25 1:400 0.0025 3.44 3.52 97.8
0.5 1:200 0.005 7.04 6.78 103.9
1 1:100 0.01 13.6 - -
 
Reference standard: extracellular vesicles purified from cell culture supernatant of COLO201 with MagCapture™ Exosome Isolation Kit PS
Measured sample: cell culture supernatant of COLO201
Primary antibody: anti-CD63 antibody in the kit

11. Monitoring changes of the amount of extracellular vesicles over time by the number of seeding cell and culture day

1 × 107 , 2 × 107 , and 3 × 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 × ) 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
      (4 μL → diluted to 100 μL)
  • Number of seeding cells: 1 × 107 , 2 × 107 , 3 × 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.

12. Changes in extracellular vesicle production induced by addition of chemical agent

  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 × ) 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 × ) 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.

13. Comparison of detection sensitivity of various ELISA kits using exosomes purified from COLO201 cell culture supernatant and human serum

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
* Data under the detection limit are not indicated.

Results

  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).

14. Exosome Related Products Q&A

15. Related Products

Isolation of high purity exosomes by a novel affinity molecule

Product Name Package Size Wako Catalog No. Storage Condition
MagCapture™ Exosome Isolation Kit PS 2 purifications*1 299-77603 Keep at 2-10°C
10 purifications*1 293-77601

*1 Used Exosome Capture-immobilized beads can be recycled up to 4 times and buffers of kit component also be contained enough for the case of recycling. When repeated isolations of extracellular vesicles from same sample are required, please try the recycling. However, when repeated isolations of extracellular vesicles from different kinds of samples are required, please don't try the recycling for preventing a contamination.

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Home > Products > Life Science > Immunochemistry > PS Capture™ Exosome ELISA Kit