1Mouse Anti-Der p2 Antibody Subtype/Subclass ELISA Kits
Catalog # 3065, 3066, 3067, and 3068
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kits to quantify mouse anti-Der p2 antibodies

FORMAT:

Precoated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

3065 (IgG): 12.5 - 0.2 ng/ml

3066 (IgG2a) : 12.5 - 0.2 ng/ml
3067 (IgG2b) : 12.5 - 0.2 ng/ml
3068 (IgM): 100 - 1.6 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum & Plasma (pre-treatment acceptable)

RECOMMENDED SAMPLE DILUTIONS:

1:100 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

3065: Intra-Assay (1.3-4.9%)/Inter-Assay (2.8-7.7%)/Spiking Test (94-104%)
3066: Intra-Assay (1.2-4.1%)/Inter-Assay (2.4-10.1%)/Spiking Test (96-100%)
3067: Intra-Assay (4.7-5.1%)/Inter-Assay (5.5-5.8%)/Spiking Test (92-95%)
3068: Intra-Assay (0.9-2.9%)/Inter-Assay (3.3-6.3%)/Spiking Test (106-110%)NOTES:

If serum samples require a lower dilution than 1:100, please contact support@chondrex.com© 2023 Chondrex, Inc. All Rights Reserved, DP2 Isotypes 1.1
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

2Mouse Anti-Der p2 Antibody Subtype/Subclass ELISA Kits
Catalog # 3065, 3066, 3067, and 3068
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Asthma is a common chronic inflammatory disease that affects 300 million people of all ages worldwide (1, 2). It is caused by exposure toallergens such as dust mites, pet dander, pollen, or mold, and characterized by airflow obstruction and bronchospasm. House dust mite(HDM) is the most common asthma allergen, which affects up to 85% of asthma patients (3, 4). Of the two main mite species,
Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f), more than 20 types of HDM allergens are defined basedon sequential and functional homologies. Among those HDM allergens, group 1 (Der 1) and group 2 (Der 2) dominate overall allergic
responses in patients and are the most commonly researched allergens (5–7).
Der p2 is a myeloid differentiation factor 2-related lipid-recognition protein consisting of 146 amino acids. Der p2 can activate toll-like receptor
4 on respiratory epithelial cells and thus play a role in developing respiratory diseases (8,9). In mice, Der p2 has been utilized as a potential
candidate for allergy vaccine development (10), especially for DNA vaccines (11).
To study the immune response to allergens and allergen-specific pathological effects in mouse asthma models, Chondrex, Inc. provides themouse anti-Der p2 antibody ELISA kits listed below. Chondrex, Inc. also offers ELISA kits for assaying anti-Der p1, anti-HDM, anti-Gliadin,
anti-Crude Peanut Extract, and anti-Ovalbumin antibody subtypes/subclasses as well as total immunoglobulin subtypes/subclasses. Please
visit www.chondrex.com for more information.
Note: Chondrex, Inc. offers both anti-HDM Der p and anti-HDM Der f ELISA kits. It is important to distinguish between these two antigens asthese are two distinct species of house dust mite; there will be some degree of homology between them that can result in cross-reactivity ofelicited antibodies. If HDM Der p is used to induce disease in animal models, then HDM Der p ELISA kits must be used to evaluate thoseanimals. And if HDM Der f is used to induce disease in animal models, then HDM Der f ELISA kits must be used to evaluate those animals
Other antibody subtype ELISA kits against HDM as well as HDM antigen detection kits are currently under development. Please contact
Chondrex, Inc. (support@chondrex.com) for more information.

LIST OF MOUSE ANTI-HDM ANTIBODY SUBTYPE/SUBCLASS ELISA KITS
Kit
Mouse Anti-HDM IgG Antibody ELISA Kit
Mouse Anti-HDM IgG1 Antibody ELISA Kit
Mouse Anti-HDM IgG2b Antibody ELISA Kit
Mouse Anti-HDM IgM Antibody ELISA Kit
Mouse Anti-HDM IgE Antibody ELISA Kit
Mouse Anti-HDM IgG2a Antibody ELISA Kit
Mouse Anti-HDM IgG3 Antibody ELISA Kit
Mouse Anti-HDM IgA Antibody ELISA Kit

Der p Catalog #3030
3034
3035
3036
3037
3038
3039
3046

Der p1 Catalog #
3047
3048
Coming soon!
3049
Coming soon!
Coming soon!
3064
Coming soon!

Der p2 Catalog #3065
Coming soon!
3067
3068
Coming soon!
3066
Coming soon!
Coming soon!

Der f Catalog #
3072
3073
3074
3076
3081
Coming soon!
3075
Coming soon!

© 2023 Chondrex, Inc. All Rights Reserved, DP2 Isotypes 1.1
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

3

KIT COMPONENTS
Item
IgG (30651) – 12.5 ng
IgG2a (30661) – 12.5 ng
Standard
IgG2b (30671) – 12.5 ng
IgM (30681) – 100 ng
IgG (30113)
IgG2a (62102)
Secondary Antibody
IgG2b (30353)
IgM (30173)
Solution B - Sample/Standard Dilution Buffer (30055)
Solution C - Secondary Antibody Dilution Buffer (2073)
TMB Solution (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
Der p2 from Dermatophagoides pteronyssinus coated ELISA Plate (Orange)

QuantityAmountStorage

1 vialLyophilized-20°C

2 vials

50 µl

-20°C

1 bottle
1 bottle
2 vials

50 ml
20 ml
0.2 ml-20°C-20°C-20°C

1 bottle
1 bottle
1 bottle
1 each

20 ml
10 ml
50 ml
96-well (8-well strips x 12)

-20°C
-20°C
-20°C
-20°C

ASSAY OUTLINE
Add 100 ml of blocking buffer into wells

Incubate at room temperature for 1 hour.
Wash plate.
Add 100 ml of diluted standards and samples into wells

Incubate at room temperature for 2 hours.
Wash plate.
Add 100 ml of diluted secondary antibody solution into wellsIncubate at room temperature for 1 hour.
Wash plate.
Add 100 ml of TMB solution into wells

Incubate at room temperature for 25 minutes.

Add 50 ml of Stop Solution into wells

Read plates at 450 nm/630 nm© 2023 Chondrex, Inc. All Rights Reserved, DP2 Isotypes 1.1
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

PLATE MAPPING
Example of the Mouse Anti-Der p2 IgG Antibody ELISA Kit
123456789101112A12.5

12.5

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

6.3

6.3

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C

3.1

3.1

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D

1.6

1.6

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

0.8

0.8

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

0.4

0.4

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

0.2

0.2

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

H

B

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

StandardsSamples

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 fordisposal.

© 2023 Chondrex, Inc. All Rights Reserved, DP2 Isotypes 1.1
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

5ASSAY PROCEDURE
1.

Add Blocking Buffer: Add 100 µl of the Sample/Standard Dilution Buffer (Solution B) to each well and incubate at room temperaturefor 1 hour.

2.

Prepare Standard Dilutions: Please see the figure below for each assay’s recommended standard range. Dissolve one vial of Standard
in 1 ml of Sample/Standard Dilution Buffer (Solution B) and keep it as a standard stock. Then serially dilute it with Solution B. For
example, mix 250 µl of the stock solution with an equal volume of Solution B to make the second stock solution, and then repeat it five
more times. The remaining stock solution can be stored at -20⁰C for use in a future assay. Chondrex, Inc. recommends making fresh
serial dilutions for each assay.
1000 ml
250 ml

250 ml

250 ml

250 ml

250 ml

250 ml23456781000 ml

250 ml

250 ml

250 ml

250 ml250 ml

250 ml

250 mlIgG/IgG2a/IgG2b:12.5 ng/ml

6.3 ng/ml

3.1 ng/ml

1.6 ng/ml

0.8 ng/ml

0.4 ng/ml

0.2 ng/ml

Blank

IgM:

100 ng/ml

50 ng/ml

25 ng/ml

12.5 ng/ml

6.3 ng/ml

3.1 ng/ml

1.6 ng/ml

Blank

1

3.

Prepare Sample Dilutions: An important point to note is that the composition of HDM can exhibit variations depending on the vendor,batch, and manufacturing process. These variations can result in differing levels of antigens in the final HDM product, which when usedto immunize mice, can impact the serum antibody levels against those antigens. The dilution of mouse serum (1:100 or more) immunizedwith various HDM products and types (DP or DF) will vary depending on the immunization schedule and timing of serum collection. In
general, no antibodies against Der p2 are observed in normal serum at a 1:100 dilution. If serum samples require a lower dilution than
1:100, please contact support@chondrex.com.

4.

Wash: Dilute 50 ml of 20X wash buffer 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.

5.

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

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 theplate by inverting it and blotting on a paper towel to remove excess liquid. Do not allow the plate to dry out.

7.

Add Secondary Antibody: Dilute one vial of Secondary Antibody in 10 ml Secondary Antibody Dilution Buffer (Solution C). Add 100µl of secondary antibody solution to each well and incubate at room temperature for 1 hour.

© 2023 Chondrex, Inc. All Rights Reserved, DP2 Isotypes 1.1
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

Strip #2468
1012

2nd Antibody (µl)
81725334250

Solution C (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 theplate 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: Use new tubes when preparing TMB solution. Just prior to use, prepare TMB solution with Chromogen Dilution
Buffer as shown in the following table. Add 100 µl of TMB solution to each well 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.

CALCULATING RESULTS
1.

Average the duplicate OD values for the standards, blanks (B), and test samples.

2.

Subtract the averaged blank OD values from the averaged OD values of the standards and test samples.

3.

Plot the OD values of the standards against the ng/ml of antibody standard. Using a log/log plot will linearize the data. Figure 1 shows
examples of standard curves for anti-Der p2 antibodies.

4.

The antibody concentration in test samples can be calculated using regression analysis. Multiply it by the sample dilution factor to obtain
the antibody concentration in original test samples.

© 2023 Chondrex, Inc. All Rights Reserved, DP2 Isotypes 1.1
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

7

Figure 1 - Typical Standard Curves for the Anti-Der p2 Antibody ELISA Kits
IgG2aIgG
10.00

10.00

R² = 0.9961

1.00

OD 450 nm

OD 450 nm

R² = 0.998

1.00

0.10

0.10

0.01
0.1

110

0.1

100

1100

IgM

IgG2b10.00

10.00

R² = 0.991

R² = 0.9923

1.00OD 450 nm

OD 450 nm

10

Anti-DP2 IgG2a Concentration (ng/ml)

Anti-DP2 IgG Concentration (ng/ml)

1.00

0.10

0.10

0.01

0.01
0.1

110100110100Anti-DP2 IgM Concentration (ng/ml)

Anti-DP2 IgG2b Concentration (ng/ml)

VALIDATION DATA
Table 1 - Reproducibility Data for the Mouse Anti-Der p2 IgG Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

0.4 ng/ml
4.9
6.8
104%

1.6 ng/ml
4.6
7.794%

6.3 ng/ml
1.3
2.894%

Table 2 - Reproducibility Data for the Mouse Anti-Der p2 IgG2a Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

0.4 ng/ml
4.1
10.1
100%

1.6 ng/ml
1.2
2.496%

6.3 ng/ml
3.6
8.499%

Table 3 - Reproducibility Data for the Mouse Anti-Der p2 IgG2b Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

0.4 ng/ml
5.1
5.595%

1.6 ng/ml
4.7
5.892%

6.3 ng/ml
5.0
5.793%© 2023 Chondrex, Inc. All Rights Reserved, DP2 Isotypes 1.1
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

8Table 4 - Reproducibility Data for the Mouse Anti-Der p2 IgM Antibody ELISA Kit
Test
3.1 ng/ml
12.5 ng/ml
50 ng/ml
Intra-Assay CV (%)
1.1
0.9
2.9
Inter-Assay CV (%)
6.3
6.1
3.3
Spike Test* (%)
110%
106%
109%
*Known amounts of anti-Der p2 antibodies were added to samples and then diluted with Sample/Standard Dilution Buffer to assay
anti-Der p2 antibodies by ELISA.

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

REFERENCES
1. M. Masoli, D. Fabian, S. Holt, R. Beasley, G. I. f. A. G. Program, The global burden of asthma: executive summary of the GINA
Dissemination Committee report. Allergy 59, 469-478 (2004).
2. K. Weiss, S. Sullivan, C. Lyttle, Trends in the cost of illness for asthma in the United States, 1985-1994. J Allergy Clin Immunol 106,493-9 (2000).
3. L. G. Gregory, C. M. Lloyd, Orchestrating house dust mite-associated allergy in the lung. Trends Immunol 32, 402-411 (2011).
4. V. D. Gandhi, C. Davidson, M. Asaduzzaman, D. Nahirney, H. Vliagoftis, House dust mite interactions with airway epithelium: role inallergic airway inflammation. Curr Allergy Asthma Rep 13, 262-270 (2013).
5. W. R. Thomas, W. Smith, House-dust-mite allergens. Allergy 53, 821-832 (1998).
6. A. Jacquet, The role of innate immunity activation in house dust mite allergy. Trends Mol Med 17, 604-611 (2011).
7. A. Custovic, S. C. Taggart, H. C. Francis, M. D. Chapman, A. Woodcock, Exposure to house dust mite allergens and the clinical activityof asthma. J Allergy Clin Immunol 98, 64-72 (1996).
8. I.-J. Bau, G.-J. Wang, A Highly Sensitive Electrochemical Impedimetric Nanobiosensor for Dust Mite Antigen Der p2 Detection. ASME
2011 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference, 3–9 (2012).
9. S. C. Yin, E. C. Liao, C. L. Chiu, C. Y. Chang, J. J. Tsai, Der p2 Internalization by Epithelium Synergistically Augments Toll-like ReceptorMediated Proinflammatory Signaling. Allergy Asthma Immunol. Res. 7, 393–403 (2015).
10. V. B. Joshi, A. Adamcakova-Dodd, X. Jing, A. Wongrakpanich, K. N. Gibson-Corley, P. S. Thorne, A. K. Salem, Development of a poly(lactic-co-glycolic acid) particle vaccine to protect against house dust mite induced allergy. AAPS J. 16, 975–985 (2014).
11. G. Li, Z. Liu, N. Zhong, B. Liao, Y. Xiong, Therapeutic effects of DNA vaccine on allergen-induced allergic airway inflammation in mousemodel. Cell. Mol. Immunol. 3, 379–384 (2006).

© 2023 Chondrex, Inc. All Rights Reserved, DP2 Isotypes 1.1
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