1Human CD63 Expressed Exosome Detection ELISA Kit
Catalog # 6037
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA Kit to quantify human exosomes expressing CD63

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

4 hours

STANDARD RANGE:

50 ng/ml to 0.8 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Culture Media, Serum, Plasma, and Purified Samples

RECOMMENDED SAMPLE DILUTIONS:

1:1 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

Intra-Assay (1.3-8.7%)/Inter-Assay (4.7-7.2%)/Spiking Test (92-94%)

NOTES:© 2020 Chondrex, Inc. All Rights Reserved, 6037 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2

Human CD63 Expressed Exosome Detection ELISA Kit
Catalog # 6037
For Research Use Only - Not Human or Therapeutic Use

Chondrex, Inc. provides a human CD63 expressed exosome quantitative ELISA kit for cell culture media, serum, plasma, and purified
samples.
KIT COMPONENTS
Item

Quantity

AmountStorage

Standard - Human CD63 Peptide Conjugate (60371)

1 vial

50 ng, Lyophilized-20°C

Detection Antibody (60373)

1 vial100 µl-20°C

Solution B - Sample/Standard/Detection Antibody Dilution Buffer (60364)

1 bottle

50 ml

-20°C

Solution D - Streptavidin Peroxidase Dilution Buffer (9055)

1 bottle

20 ml

-20°C

Streptavidin Peroxidase (9029)

2 vials

50 µl-20°C

TMB (90023)

2 vials

0.2 ml-20°C

Chromogen Dilution Buffer (90022)1 bottle

20 ml

-20°C

Stop Solution - 2N Sulfuric Acid (9016)

1 bottle

10 ml

-20°C

Wash Buffer, 20X (9005)

1 bottle

50 ml

-20°C

Capture Antibody Coated 96-Well ELISA Plate (Blue)

1 each8-well strips x 12-20°C

NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placingthe bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.
NOTE 6: For partial reagent use, please see the assay protocol’s corresponding step for the appropriate dilution ratio. For example, if theprotocol dilutes 50 µl of a stock solution in 10 ml of buffer for 12 strips, then for 6 strips, dilute 25 µl of the stock solution in 5 ml of buffer.
Partially used stock reagents may be kept in their original vials and stored at -20⁰C for use in a future assay.
NOTE 7: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and fordisposal.

© 2020 Chondrex, Inc. All Rights Reserved, 6037 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3ASSAY OUTLINE
Add 100 ml of diluted standards and samples into wells

Incubate at room temperature for 2 hours.
Wash plate.
Add 100 ml of diluted Detection Antibody

Incubate at room temperature for 1 hour.
Wash plate.
Add 100 ml of diluted Streptavidin Peroxidase

Incubate at room temperature for 30 minutes.
Wash plate.
Add 100 ml of TMB solution

Incubate at room temperature for 25 minutes.

Add 50 ml of Stop Solution

Read plates at 450 nm/630 nm

PLATE MAPPING
123

4

5

67

8910

1112A

50

50

S1S1S9S9S17

S17S25

S25

S33

S33

B25

25

S2S2S10

S10S18

S18

S26

S26

S34

S34

C

12.5

12.5

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D6.3

6.3

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

3.1

3.1

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

1.6

1.6

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

0.8

0.8

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

HB

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples© 2020 Chondrex, Inc. All Rights Reserved, 6037 2.016928 Woodinville-Redmond Rd NE Suite B-101Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

4

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: The recommended standard range is 0.8 - 50 ng/ml. Dissolve one vial of human CD63 peptide conjugate
standard in 1 ml of Sample/Standard/Detection Antibody Dilution Buffer (Solution B) for the 50 ng/ml standard. Then serially dilute itwith Solution B. For example, mix 250 µl of the standard (50 ng/ml) with an equal volume of Solution B to make a 25 ng/ml solution,and then repeat it five more times for 12.5, 6.3, 3.1, 1.6, and 0.8 ng/ml solutions. The remaining 50 ng/ml standard stock may be stored
at -20°C for use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 ml
250 ml

250 ml

250 ml

250 ml

250 ml2345

6781000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

50 ng/ml

25 ng/ml

12.5 ng/ml

6.3 ng/ml

3.1 ng/ml

1.6 ng/ml

0.8 ng/ml

Blank

1

2.

250 mlPrepare Samples: Exosomes can be stored at 4°C for up to 1 week. For long-term storage, keep at -20°C or -80°C. If multiple assays
are required, Chondrex, Inc. recommends aliquoting samples into tubes which will only undergo one freeze/thaw cycle in order to avoid
damaging the exosomes.
Cell Culture Media: Remove cell debris and insoluble precipitate by centrifuging at 10,000 rpm for 5 minutes. When not in use, store
the supernatant samples at -20°C; avoid repeat freeze-thaw cycles.
Serum*: Clot blood samples by incubating samples at room temperature for 2 hours. Collect serum by centrifuging samples at 10,000rpm for 5 minutes. When not in use, store the serum supernatant samples at -20°C; avoid repeat freeze-thaw cycles.
Plasma*: Collect plasma samples with the use of anticoagulants such as heparin. Collect plasma by centrifuging samples at 10,000rpm for 5 minutes within 30 minutes of blood collection. When not in use, store the plasma supernatant samples at -20°C; avoid repeat
freeze-thaw cycles.
NOTE: Using lipemic (cloudy) samples may affect assay results. The stored samples must be centrifuged at 10,000 rpm for an additional
5 minutes before the assay.
Dilution: Dilute samples at least 1:1 with Solution B depending on the estimated exosome levels in the samples. Two to three different
sample dilutions are recommended if the exosome levels in the samples are unknown.
NOTE: Samples must be diluted with Solution B to maintain optimal assay conditions.

3.

Dilute Wash Buffer: Dilute 50 ml of Wash Buffer, 20X in 950 ml of distilled water (1X wash buffer). Wash the plate with 1X wash buffer
at least 3 times using a wash bottle with manifold or an automated plate washer. Empty the plate by inverting it and blotting on a paper
towel to remove excess liquid. Do not allow the plate to dry out.

4.

Add Standards and Samples: Add 100 µl of Solution B (blank), standards, and samples to designated wells in duplicate and incubate
at room temperature for 2 hours.

5.

Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty theplate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

© 2020 Chondrex, Inc. All Rights Reserved, 6037 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

56.

Add Detection Antibody Solution: Dilute one vial of detection antibody with 10 ml of Sample/Standard/Detection Antibody Dilution
Buffer (Solution B). Add 100 µl of detection antibody solution to each well and incubate at room temperature for 1 hour.
Strip #24681012Detection Antibody (µl)
17255066
82100Solution B (ml)
1.7
3.3
5.0
6.6
8.210.0

7.

Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty theplate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

8.

Add Streptavidin Peroxidase Solution: Dilute one vial of streptavidin peroxidase solution with 10 ml of Streptavidin Peroxidase
Dilution Buffer (Solution D). Add 100 µl of streptavidin peroxidase solution to each well and incubate at room temperature for 30 minutes.
Strip #246810129.

Streptavidin Peroxidase (µl)
81725334250

Solution D (ml)
1.7
3.3
5.0
6.6
8.2
10.0

Wash: Wash the plate with 1X wash buffer at least 3 times using a wash bottle with manifold or an automated plate washer. Empty theplate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

10. Add TMB Solution: Dilute one vial of TMB in 10 ml of Chromogen Dilution Buffer just prior to use. Add 100 µl of TMB solution to allwells immediately after washing the plate and incubate for 25 minutes at room temperature.
Strip #24681012TMB (µl)
3466100132164200Chromogen Dilution Buffer (ml)
1.7
3.3
5.0
6.6
8.2
10.0

11. Stop: Stop the reaction with 50 µl of 2N Sulfuric Acid (Stop Solution) to each well.
12. Read Plate: Read the OD values at 450 nm. If the OD values of samples are greater than the OD values of the highest standard, reassay the samples at a higher dilution. A 630 nm filter can be used as a reference.

© 2020 Chondrex, Inc. All Rights Reserved, 6037 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

6

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank, standards, and test samples.

2.

Subtract the “blank” (B) values from the averaged OD values in step 1.

3.

Plot the OD values of standards against the concentration of CD63 peptide conjugate (ng/ml). Using a log/log plot will linearize the data.
Figure 1 shows a representative experiment where the standard range is 0.8 - 50 ng/ml.

4.

The ng/ml of CD63 expressed exosomes in test samples can be calculated using regression analysis.

Figure 1 - A Typical Standard Curve for the Human CD63 Expressed Exosome Detection ELISA Kit
10.00

R² = 0.9997

OD 450 nm

1.00

0.10

0.01
0.1

110
CD63 Concentration (ng/ml)

100

ASSAY VALIDATION
Table 1 - Reproducibility Data for the Human CD63 Expressed Exosome Detection ELISA Kit
Test

1.6 n/ml

6.3 ng/ml

25 ng/ml

Intra-Assay CV (%)

8.7

1.5

1.3

Inter-Assay CV (%)

5.2

7.2

4.7

Spike Test* (%)
94%
92%
93%
* Known amounts of CD63 peptide were added to normal mouse serum and then diluted with Sample/Standard/Detection Antibody
Dilution Buffer (Solution B).

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s ELISA FAQ for more information.

© 2020 Chondrex, Inc. All Rights Reserved, 6037 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com