1

Mouse Anti-HDM Dermatophagoides pteronyssinus Antibody
Subtype/Subclass ELISA Kits
Catalog # 3030, 3034, 3035, 3036, 3038, 3039, and 3046
For Research Use Only - Not Human or Therapeutic Use
PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kits to quantify mouse anti-house dust mite (HDM) Dermatophagoides pteronyssinus
antibodies

FORMAT:

Precoated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

3030 (IgG): 100 - 1.6 ng/ml

3034 (IgG1): 100 - 1.6 ng/ml

3035 (IgG2b) : 10 - 0.16 ng/ml
3036 (IgM): 1000 - 16 ng/ml

3038 (IgG2a) : 50 - 0.8 ng/ml
3039 (IgG3): 50 - 0.8 ng/ml

3046 (IgA): 500 - 8 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:

3030: Intra-Assay (5.5-9.6%)/Inter-Assay (2.9-7.0%)/Spiking Test (100-109%)3034: Intra-Assay (1.7-5.4%)/Inter-Assay (3.6-12.6%)/Spiking Test (90-106%)
3035: Intra-Assay (5.8-10.1%)/Inter-Assay (3.1-9.8%)/Spiking Test (103-111%)
3036: Intra-Assay (3.1-8.3%)/Inter-Assay (0.9-4.8%)/Spiking Test (102-105%)
3038: Intra-Assay (2.7-6.3%)/Inter-Assay (7.4-11.5%)/Spiking Test (87-96%)
3039: Intra-Assay (1.5-7.2%)/Inter-Assay (1.5-9.3%)/Spiking Test (100-109%)3046: Intra-Assay (2.4-7.7%)/Inter-Assay (7.8-10%)/Spiking Test (96-100%)

NOTES:

These kits may recognize anti-HDM Dermatophagoides farinae antibodies due to thehomology between the two species of house dust mite.

© 2023 Chondrex, Inc. All Rights Reserved, HDM DP Isotypes 3.0
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

Mouse Anti-HDM Dermatophagoides pteronyssinus Antibody
Subtype/Subclass ELISA Kits
Catalog # 3030, 3034, 3035, 3036, 3038, 3039, and 3046
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). 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 (2, 3). 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 researched allergens (4-6).
Previously, asthma was considered an inflammatory airway disease mediated by the adaptive immune system, particularly type 2 helper Tcells (7). However, recent studies indicate that the innate immune system is also involved in triggering an inflammatory response in both
asthma patients and animal models (8-10). Airway remodeling and inflammatory changes significantly vary depending on the types ofallergens (11). To meet such needs, a mouse HDM-induced asthma model is a useful tool to dissect the pathological roles of the adaptive
and innate immune systems activated by the different HDM elements. This is an advantage over the classic ovalbumin-induced asthma
model which activates adaptive immunity preferentially. Recently, it was reported that HDM-specific sublingual immunotherapy (SLIT) is
more efficacious at preventing the development of allergic inflammatory reactions than subcutaneous immunotherapy in mouse models (12).
This SLIT protocol has been approved as a treatment to reduce allergy or asthma symptoms in patients (13).
To study the immune response to allergens and allergen-specific pathological effects in mouse models, Chondrex, Inc. provides mouse antiHDM antibody ELISA kits listed below, including anti-HDM IgE ELISA kits. Chondrex, Inc. also offers ELISA kits for assaying anti-OVA
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, HDM DP Isotypes 3.0
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

3

KIT COMPONENTS
Item

Quantity

AmountStorage

1 vialLyophilized-20°C

IgG (30301) - 100 ng
IgG1 (30341) - 100 ng
IgG2b (30351) - 10 ng
Standard
IgM (30361) - 1000 ng
IgG2a (30381) - 50 ng
IgG3 (30391) - 50 ng
IgA (30461) - 500 ng
IgG (30113)
IgG1 (203011)
Secondary Antibody
IgG2b (30353)(peroxidase-conjugated
IgM (30363)
polyclonal antibodies) IgG2a (203012)
IgG3 (30393)
IgA (30463)
Solution B - Blocking Buffer (30313)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30314)
TMB Solution (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)

2 vials

50 µl

-20°C

1 bottle
1 bottle
2 vials
1 bottle
1 bottle

10 ml
50 ml
0.2 ml
20 ml
10 ml

-20°C
-20°C
-20°C
-20°C
-20°C

Wash Buffer, 20X (9005)
HDM Extract from Dermatophagoides pteronyssinus coated ELISA Plate (Brown)

1 bottle
1 each50 ml
96-well (8-well strips x 12)

-20°C
-20°C

PLATE MAPPING
Example of the Mouse Anti-HDM Der p IgG Antibody ELISA Kit
1

2

3

4

5

6

7

8

9

10

11

12

A

100

100

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

50

50

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C

25

25

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D

12.5

12.5

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

6.3

6.3

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

3.1

3.1

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

1.6

1.6

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© 2023 Chondrex, Inc. All Rights Reserved, HDM DP Isotypes 3.0
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

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 wells

Incubate 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

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 the
protocol 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.

© 2023 Chondrex, Inc. All Rights Reserved, HDM DP Isotypes 3.0
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

5

ASSAY PROCEDURE
1.

Add Blocking Buffer: Add 100 µl of the Blocking Buffer (Solution B) to each well and incubate at room temperature for 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/Secondary Antibody Dilution Buffer (Solution C) and keep it as a standard stock. Then serially dilute it with
Solution C. For example, mix 250 µl of the stock solution with an equal volume of Solution C to make the second stock solution, andthen 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 ml

2

3

4

5

6

7

8

1000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 mlIgG/IgG1:

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

IgG2a/IgG3:

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

IgG2b:

10 ng/ml

5 ng/ml

2.5 ng/ml

1.25 ng/ml

0.63 ng/ml

0.31 ng/ml

0.16 ng/ml

Blank

IgM:

1000 ng/ml

500 ng/ml

250 ng/ml

125 ng/ml

63 ng/ml

31 ng/ml

16 ng/ml

Blank

IgA:

500 ng/ml

250 ng/ml

125 ng/ml

63 ng/ml

31 ng/ml

16 ng/ml

8 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 used
to 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 HDM 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 C (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 Sample/Standard/Secondary Antibody Dilution Buffer(Solution C). Add 100 µl of secondary antibody solution to each well and incubate at room temperature for 1 hour.

© 2023 Chondrex, Inc. All Rights Reserved, HDM DP Isotypes 3.0
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

Strip #
2
4
6
8
10
12

2nd Antibody (µl)
8
17
25
33
42
50

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 #
2
4
6
8
10
12

TMB (µl)
34
66
100
132
164
200

Chromogen 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
an example of a standard curve for anti-HDM Der p IgG antibodies.

4.

The ng/ml of antibody in test samples can be calculated using regression analysis. Multiply it by the sample dilution factor to obtain the
antibody concentration (ng/ml) in original test samples.

© 2023 Chondrex, Inc. All Rights Reserved, HDM DP Isotypes 3.0
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

7

Figure 1 - Typical Standard Curves for the Anti-HDM Der p Antibody ELISA Kits
IgG

IgG1

10.0

0.1

0.1

0.0

0.0
1

10

100

100

10.0

IgG2a
10.0

100.0

1000.0Anti-Der p HDM IgM Concentration (ng/ml)

IgG3
R² = 0.9987

1.0

0.1

0.0
0.1

1.0

10.0

Anti-Der p HDM IgG2a Concentration (ng/ml)

100.0

0.1

1.0

10.0

100.0

Anti-Der p HDM IgG3 Concentration (ng/ml)

IgA

10.00

OD 450 nm

10

1.0

0.1
10.0

1

Anti-Der p HDM IgG2b Concentration (ng/ml)

R² = 0.9953

0.1

0.0

0.1

OD 450 nm

IgM

OD 450 nm

OD 450 nm

10

Anti-Der p HDM IgG1 Concentration (ng/ml)

R² = 0.9996

1.0

1.0

0.1
1

Anti-Der p HDM IgG Concentration (ng/ml)

10.0

R² = 0.9955

1.0

OD 450 nm

1.0

IgG2b
10.0

R² = 0.9955

R² = 0.9955

OD 450 nm

OD 450 nm

10.0

R² = 0.9964

1.00

0.10

0.01
1

10

100

1000

Anti-Der p HDM IgA Concentration (ng/ml)© 2023 Chondrex, Inc. All Rights Reserved, HDM DP Isotypes 3.0
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

8

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

3.2 ng/ml
5.8
7.0
101%

12.5 ng/ml
9.6
4.0
109%

50 ng/ml
5.5
2.9
100%

Table 2 - Reproducibility Data for the Mouse Anti-HDM Der p IgG1 Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

3.2 ng/ml
5.4
12.6
90%

12.5 ng/ml
4.4
3.6
106%

50 ng/ml
1.7
5.5
106%

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

0.32 ng/ml
5.8
3.9
103%

1.3 ng/ml
6.0
9.8
104%

5 ng/ml
10.1
3.1
111%

Table 4 - Reproducibility Data for the Mouse Anti-HDM Der p IgM Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

32 ng/ml
8.3
3.8
103%

125 ng/ml
4.0
4.8
105%

500 ng/ml
3.1
0.9
102%

Table 5 - Reproducibility Data for the Mouse Anti-HDM Der p IgG2a Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

1.6 ng/ml
6.3
7.4
87%

6.3 ng/ml
4.4
10.8
92%

25 ng/ml
2.7
11.5
96%

Table 6 - Reproducibility Data for the Mouse Anti-HDM Der p IgG3 Antibody ELISA Kit
Test
Intra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

1.6 ng/ml
1.5
7.4
107%

6.3 ng/ml
7.2
9.3
109%

25 ng/ml
4.7
1.5
100%

Table 7 - Reproducibility Data for the Mouse Anti-HDM Der p IgA Antibody ELISA Kit
Test
16 ng/ml
63 ng/ml
250 ng/ml
Intra-Assay CV (%)
3.3
7.7
2.4
Inter-Assay CV (%)
10.0
9.9
7.8
Spike Test* (%)
99%
96%
100%
*Known amounts of anti-HDM Der p antibodies were added to samples and then diluted with Sample/Standard/Secondary Antibody
Dilution Buffer to assay anti-HDM Der p antibodies by ELISA.

© 2023 Chondrex, Inc. All Rights Reserved, HDM DP Isotypes 3.0
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

9

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. L. G. Gregory, C. M. Lloyd, Orchestrating house dust mite-associated allergy in the lung. Trends Immunol 32, 402-411 (2011).
3. 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).
4. W. R. Thomas, W. Smith, House-dust-mite allergens. Allergy 53, 821-832 (1998).
5. 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).
6. A. Jacquet, The role of innate immunity activation in house dust mite allergy. Trends Mol Med 17, 604-611 (2011).
7. L. Cohn, J. A. Elias, G. L. Chupp, Asthma: mechanisms of disease persistence and progression. Annu Rev Immunol 22, 789-815 (2004).
8. J. R. Murdoch, C. M. Lloyd, Chronic inflammation and asthma. Mutat Res 690, 24-39 (2010).
9. J. C. Virchow et al., Efficacy of a House Dust Mite Sublingual Allergen Immunotherapy Tablet in Adults With Allergic Asthma: A
Randomized Clinical Trial. JAMA 315, 1715-1725 (2016).
10. S. Hagner et al., House Dust Mite-Specific Sublingual Immunotherapy Prevents the Development of Allergic Inflammation in a Mouse
Model of Experimental Asthma. Int Arch Allergy Immunol 170, 22-34 (2016).
11. C. S. Stevenson, M. A. Birrell, Moving towards a new generation of animal models for asthma and COPD with improved clinicalrelevance. Pharmacol Ther 130, 93-105 (2011).
12. P. Moingeon et al., Immune mechanisms of allergen-specific sublingual immunotherapy. Allergy 61, 151-165 (2006).
13. Z. Aryan, E. Compalati, E. Comapalati, G. W. Canonica, N. Rezaei, Allergen-specific immunotherapy in asthmatic children: from thebasis to clinical applications. Expert Rev Vaccines 12, 639-659 (2013).

© 2023 Chondrex, Inc. All Rights Reserved, HDM DP Isotypes 3.0
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