Exosome ELISA Kits

Features

• 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)

　

Application Data

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

  

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.

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.

　

Reference data: dilution linearity of cell culture supernatant sample

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.

　

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

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.

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 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.

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

	(1)	(2)	(3)	(4)	(5)	(6)
x 1	1/16	1/1000	40 ng/mL	160 ng/mL	800 ng/mL	2000 µg/mL
x 0.5	1/32	1/2000	20 ng/mL	80 ng/mL	400 ng/mL	1000 µg/mL
x 0.25	1/64	1/4000	10 ng/mL	40 ng/mL	200 ng/mL	500 µg/mL
x 0.125	1/128	1/8000	5 ng/mL	20 ng/mL	100 ng/mL	250 µg/mL
x 0.0625	1/256	1/16000	2.5 ng/mL	10 ng/mL	50 ng/mL	125 µg/mL
0 (BLANK)	0	0	0	0	0	0

Comparison of detection sensitivity of individual exosome ELISA kits

• Features

  • Sensitive qualitative analysis (50 to 1000 folds more sensitive than WB in detection)
  • Direct relative quantitation of extracellular vesicles in conditioned medium and body fluid specimens
  • Application to detection systems using antibodies from various animal species and lectins
  • Microanalysis with small amounts of extracellular vesicles
    (only 1/10 to 1/1000 of the sample amount for WB)

• Assay Principle

  

  Exosome Capture 96 Well Plate which is pre-coated with protein that specifically binds to phosphatidylserine (PS) on the surface of extracellullar vesicles (EVs) capture EVs with Ca²⁺, Then biotinylated antibody for the surface marker protein of EVs is used as a primary detection and HRP-conjugated streptavidin is used as a secondary detection.

Application Data

Dilution Linearity of Cell Culture Supernatant and Blood Samples

Dilution linearity of four specimens, ① human serum diluent ② human heparin plasma diluent ③ human EDTA plasma diluent (2 specimens, 4-step serial dilution each) and ④ COLO201 cells cell culture supernatant diluent (4-step serial dilution), was assessed by measuring the concentration of exosomes (CD63 detection) with standard curve obtained using extracellular vesicles purified from cell culture supernatant of COLO201 cells.

• ①Serum

  

• ②Heparin plasma

  

• ③EDTA plasma

  

• ④COLO201 cell culture supernatant

  

Comparison of Capture Ability between PS Affinity and Antibody

Microplate wells, pre-coated with anti-CD antibodies (CD9, CD63, CD81) and PS Affinity (Tim4), were incubated with pretreated ※ 1 cell culture supernatants of iPSC, COLO201 and HEK293T cells. Captured exosomes on the plate were detected using biotinylated anti-CD antibodies (CD9, CD63, CD81).

※ 1 Pretreatment condition: 10,000 x g, 30min.

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Spike and Recovery Assay with Blood Samples【EpCAM】

Pretreated^※2 pooled normal human serum, EDTA-treated plasma, and heparinized plasma were spiked with exosomes isolated and purified from COLO201 cell conditioned medium using MagCapture™ Exosome Isolation Kit PS (Code No. 293-77601) at various concentrations. The spiked exosomes in a sample were detected using biotin-labeled anti-EpCAM^※3 antibody (MBL), and the recovery rate of exosomes from the sample was calculated based on the standard curve prepared using exosomes purified from COLO201 cell conditioned medium.

※ 2 Pretreatment condition: 10,000 x g, 30min.　　※ 3 EpCAM: Epithelial cell adhesion molecule

Test of Residual Extracellular Vesicles in EV-depleted FBS

Extracellular vesicles in untreated FBS, commercial EV-depleted FBS and ultracentrifugation (UC)-treated FBS ^{※ 4} were detected using Biotinylated anti-CD9 antibody ^{※ 5}.

Sugar Chain Analysis by Sandwich ELISA with rBC2LCN Lectin and Tim4

Exosomes in five-fold dilution of pretreated^※6 cell culture supernatants of iPS cells were detected using Biotinylated anti-CD63 antibody (Code No. 019-27713) and Biotinylated rBC2LCN lectin^※7.

Comparison of recovery efficiency with Polymer Precipitation

From 1 mL of pretreated ^{※ 8} COLO201 cell conditioned medium, exosomes were isolated and purified by PS affinity method or polymer precipitation method (competiter A) and then measured for signals of CD63, an exosome marker, using PS Capture™ Exosome ELISA Kit (streptavidin HRP). From absorbance of each sample, the amount of residual exosomes in a post-purification sample and the recovery rate of exosomes were determined for comparison.

Features

• Specific to human extracellular vesicles.
• Direct analysis from cell culture sup. or blood samples. No need for isolation.
• Qualitative and quantitative analysis.
  Note: Purified extracellular vesicle standard is required for quantification.
• Simple operation and high reproducibility

Application Data

Spike and recovery assay of human derived-extracellular vesicles from mouse EDTA plasma.

Evaluate the assay linearity using mouse EDTA plasma (2-fold diluted) spiked with Exosome, from COLO201 cells, Purified [code No. 052-09301].

• 1. CD9-Capture Human Exosome ELISA Kit

  

• 2. CD63-Capture Human Exosome ELISA Kit

  

• 3. CD81-Capture Human Exosome ELISA Kit

  

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  Assay Sample	2-fold diluted mouse EDTA plasma spiked with exosomes purified from COLO201 cells at each concentration.
  Assay Kit	CD9/63/81-Capture Human Exosome ELISA Kit

Comparison of extracellular vesicles expression in various human cell lines culture supernatant.

Culture supernatants of 12 types of human-derived cell lines were diluted 2, 10, 25, and 100 fold and measured with this kit.

1. CD9-Capture Human Exosome ELISA Kit

2. CD63-Capture Human Exosome ELISA Kit

3. CD81-Capture Human Exosome ELISA Kit

The reason of some cell lines showed no signal at 2 and 10-fold dilution is that the signal was over the detection limit.