1House Dust Mite (Der p 10) Detection ELISA Kit
Catalog # 6031
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA Kit to quantify house dust mite (Der p 10)

FORMAT:

Pre-coated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

4 hours

STANDARD RANGE:

200 - 3.1 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Culture media, serum, plasma, and solubilized samples (extracts)

RECOMMENDED SAMPLE DILUTIONS:

1:1 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

Intra-Assay (3.8-6.2%)/Inter-Assay (2.4-8.9%)/Spiking Test (93-105%)

NOTES:© 2020 Chondrex, Inc. All Rights Reserved, 6031 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

House Dust Mite (Der p 10) Detection ELISA Kit
Catalog # 6031
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Allergic diseases and symptoms arise from an active immune response to antigens which are usually harmless, such as pollen, pet dander,or food. Specifically, food allergies and asthma are two common childhood autoimmune diseases that have affected more individuals recently.
A food allergy is an immune response to foods or food ingredients that most other people can tolerate with no problem. Chicken egg allergy
is the second most common food allergy (the first being bovine milk allergy) and is observed in 0.5 to 2.5% of young children. Egg allergy is
an immunological reaction induced by egg proteins and is defined as an allergen-specific IgE antibody-mediated allergy, also known as atype I food allergy. Among egg proteins, ovalbumin (OVA) is one of the identified allergenic proteins (1) (2).
Asthma is a chronic inflammatory disease that affects 300 million people of all ages worldwide (3). It is caused by exposure to allergens suchas dust mites, pet dander, pollen, or mold, and characterized by airflow obstruction and bronchospasm. House dust mite (HDM) allergens
are the most important indoor allergens in humans (3). Approximately 10% of asthma patients sensitized to HDM demonstrate IgE antibodies
to the tropomyosin protein (Der p10) from dust mite, Dermatophagoides pteronyssinus (2). Der p10 is a 32 kDa, group 10 allergen(tropomyosins). Tropomyosins of a similar structure are found in invertebrates such as crustaceans (shrimp, lobster, crawfish, and crab),arachnids (house dust mites), insects (cockroaches), and mollusks. Tropomyosin is a heat-stable protein and sensitization may lead tosevere reactions to the allergenic source. Therefore, the Der p10 antigen may play a role in the cross-induction of allergic reactions to many
allergens. (4)To analyze the pathogenesis of these allergens, OVA and HDM (Der p 10), in allergic reactions in patients or animal disease models,Chondrex, Inc. provides allergen detection ELISA kits. For more information, please visit www.chondrex.com or contact
support@chondrex.com.
NOTE: This kit does not detect the tropomyosin protein (Group 10 allergen) from Dermatophagoides farinae (Der f 10).

KIT COMPONENTS
Item

Quantity

AmountStorageStandard (60311)

1 vial200 ng, Lyophilized-20°CDetection Antibody (60313)

1 vial100 µl-20°C

Solution B - Sample/Standard/Detection Antibody Dilution Buffer (67015)

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

200 µl-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 (Gray)

1 each8-well Strips x12-20°C© 2020 Chondrex, Inc. All Rights Reserved, 6031 2.016928 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

3

ASSAY OUTLINE

PLATE MAPPING
123456789101112A

200200

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

100100

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C500

50

S3S3S11

S11

S19

S19

S27

S27

S35

S35

D2525

S4S4S12S12

S20

S20

S28

S28

S36

S36

E

12.512.5

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

6.36.3

S6S6

S14

S14

S22

S22

S30

S30

S38

S38

G

3.1

3.1

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

H

B

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples© 2020 Chondrex, Inc. All Rights Reserved, 6031 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

4

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 placing
the 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 for
disposal.

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: The recommended standard range is 3.1 - 200 ng/ml. Dissolve one vial of HDM standard with 1 ml ofSample/Standard/Detection Antibody Dilution Buffer (Solution B) for the 200 ng/ml standard. Then serially dilute it with Solution B. For
example, mix 250 µl of the standard (200 ng/ml) with an equal volume of Solution B to make a 100 ng/ml solution, and then repeat it
five more times for 50, 25, 12.5, 6.3, and 3.1 ng/ml solutions. The remaining 200 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 ml

250 ml2345

6781000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml200 pg/ml

100 pg/ml

50 pg/ml

25 pg/ml

12.5 pg/ml

6.3 pg/ml

3.1 pg/ml

Blank

1

2.

Prepare Samples: Dilute samples at least 1:1 with Solution B depending on the estimated HDM level in the samples. Two to threedifferent sample dilutions are recommended if the HDM levels in the samples are unknown.
NOTE: Samples must be diluted with Solution B to maintain optimal assay conditions.

3.

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.

4.

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.

© 2020 Chondrex, Inc. All Rights Reserved, 6031 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

55.

Add Detection Antibody Solution: Prepare the detection antibody solution with Sample/Standard/Detection Antibody Dilution Buffer(Solution B) as shown in the following table. Add 100 µl of detection antibody solution to each well and incubate at room temperaturefor 1 hour.
Strip #24681012Detection Antibody (µl)
1725506682100

Solution B (ml)
1.7
3.3
5.0
6.6
8.2
10.0

6.

Wash: 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.

7.

Add Streptavidin Peroxidase Solution: Prepare streptavidin peroxidase solution with Streptavidin Peroxidase Dilution Buffer(Solution D) as shown in the following table. Add 100 µl of streptavidin peroxidase solution to each well and incubate at room
temperature for 30 minutes.
Strip #24681012Streptavidin Peroxidase (µl)
81725334250

Solution D (ml)
1.7
3.3
5.0
6.6
8.2
10.0

8.

Wash: 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.

9.

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

10. Stop: Stop the reaction with 50 µl of 2N Sulfuric Acid (Stop Solution) to each well.
11. 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, 6031 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 HDM (ng/ml). Using a log/log plot will linearize the data. Figure 1 shows arepresentative experiment where the standard range is 3.1 - 200 ng/ml.

4.

The ng/ml of HDM (Der p 10) in test samples can be calculated using regression analysis.

Figure 1 - A Typical Standard Curve for the HDM (Der p 10) Detection ELISA Kit
10.00
R² = 0.9993

OD 450

1.00

0.10

0.01
110

100

1000

HDM Concentration (ng /ml)

ASSAY VALIDATION
Table 1 – Reproducibility Data for the HDM (Der p 10) Detection ELISA Kit
Test

6.3 ng/ml

25 ng/ml100 ng/ml

Intra-Assay CV (%)

5.4

3.8

6.2

Inter-Assay CV (%)

8.9

4.4

2.4

Spike Test* (%)
96%
105%
93%
* Known amounts of HDM were added to samples and 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.

REFERENCES
1. J. Caubet, J. Wang, Current Understanding of Egg Allergy. Pediatr Clin North Am 58, 427-43, xi (2011).
2. M. Lloyd, Advances in Allergy Testing: Component Resolved Diagnostics. Journal for Clinical Studies, 1–4 (2017).
3. T. Buday, J. Plevkova, House Dust Mite Allergy Models - Reliability for Research of Airway Defensive Mechanisms. OJMIP 04, 27-35(2014).
4. R. H. Shafique, M. Inam, M. Ismail, F. R. Chaudhary, Group 10 allergens (tropomyosins) from house-dust mites may cause covariationof sensitization to allergens from other invertebrates. Allergy Rhinol (Providence) 3, e74–90 (2012).

© 2020 Chondrex, Inc. All Rights Reserved, 6031 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