1Mouse Anti-HDM Dermatophagoides pteronyssinus & Dermatophagoides farinae
Serum IgE Antibody Detection ELISA Kits
Catalog # 3037 and 3081
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify mouse anti-house dust mite (HDM) Dermatophagoides pteronyssinus or
Dermatophagoides farinae house dust mite IgE antibodies

FORMAT:

Precoated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

5 hours

STANDARD RANGE:

50 - 0.8 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum & Plasma (pre-treatment acceptable)

RECOMMENDED SAMPLE DILUTIONS:

1:10 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:HDM DP: Intra-Assay (1.7-5%)/Inter-Assay (3.2-5.3%)/Spiking Test (90-103%)
HDM DF: Intra-Assay (2.5-3.4%)/Inter-Assay (2.5-3.0%)/Spiking Test (96-106%)NOTES:

N/A© 2023 Chondrex, Inc. All Rights Reserved, 3037 & 3081 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 & Dermatophagoides farinae
Serum IgE Antibody Detection ELISA Kits
Catalog # 3037 and 3081
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: DP) and Dermatophagoides farinae (Der f: DF), more than 20 types of HDM allergens are defined
based on 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 classical ovalbumin-induced asthma
model which preferentially activates adaptive immunity.
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 toreduce allergy or asthma symptoms in patients (13). In general, mouse serum antigen-specific antibody levels for antibodies like IgA andIgG tend to be higher than IgE levels. Thus, it is difficult to detect antigen specific IgE antibody levels due to the competition for the antigenic
determinant on the antigen by other types of antibodies. This kit is designed to detect HDM specific IgE antibodies in mouse serum andworks for both IgEa (Balb/c) and IgEb (C57BL/6) allotypes equally.
To study the immune response to allergens and allergen-specific pathological effects in mouse models, Chondrex, Inc. provides mouse
ELISA kits for assaying IgE antibodies against HDM DP (Catalog # 3037) and HDM DF (Catalog # 3081) as well as othersubtypes/subclasses. Chondrex, Inc. also offers ELISA kits for assaying antibody subtypes/subclasses against many kinds of allergens aswell as total immunoglobulin subtypes/subclasses. Please visit www.chondrex.com for more information.
Note: Other antibody subtype ELISA kits against HDM as well as HDM antigen detection kits are currently under development. Please contact
Chondrex, Inc. at 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 KitMouse 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 f Catalog #
3072
3073
3074
3076
3081
Coming soon!
3075
Coming soon!

© 2023 Chondrex, Inc. All Rights Reserved, 3037 & 3081 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

Standard Anti-HDM Mouse IgE Antibody (30371)
3037: Biotinylated HDM DP (30373)
3081: Biotinylated HDM DF (30813)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30314)

1 vial

50 ng, lyophilized-20°C

1 vial100 µl-20°C

1 bottle

50 ml

-20°C

Solution D - Streptavidin Peroxidase Dilution Buffer (9055)
Solution E - Biotinylated HDM Dilution Buffer (30374)
Streptavidin Peroxidase (9029)
TMB Solution (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)

1 bottle
1 bottle
2 vials
2 vials
1 bottle
1 bottle
1 bottle

20 ml
10 ml
50 µl
0.2 ml
20 ml
10 ml
50 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Anti-Mouse IgE Antibody Coated ELISA Plate (Yellow)

1 each

96-well (8-well strips x 12)-20°C

ASSAY 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 Biotinylated HDM into wells

Incubate at room temperature for 2 hours.
Wash plate.
Add 100 ml of diluted Streptavidin Peroxidase into wells

Incubate at room temperature for 30 minutes.
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, 3037 & 3081 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

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.
NOTE 8: Serum IgE antibodies are a mixture of multiple antibodies with a variety of affinity ranges. The OD value obtained in ELISA for anantibody assay depends on the antibody concentration as well as the antibody affinity towards an antigen. In general, ELISA data using amonoclonal antibody as a standard does not reflect the absolute levels of IgE. Therefore, IgE levels determined using this kit should be
expressed as ng of IgE per ml.
NOTE 9: If the total IgE concentration in a sample is higher than 500 ng/ml, the sample must be diluted to lower the total IgE levels below
500 ng/ml because the anti-HDM IgE value obtained from this ELISA is lower than the actual value due to competition from non-anti-HDM
IgE antibodies present in the sample (Figure 2). Therefore, it is strongly recommended that total IgE levels be determined first using theMouse Total IgE Assay Kit (Catalog # 3005).

PLATE MAPPING
1234

56789101112A

5050

S1S1S9S9S17S17S25S25

S33S33B25

25

S2S2S10

S10

S18

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

H

B

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples© 2023 Chondrex, Inc. All Rights Reserved, 3037 & 3081 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

5ASSAY PROCEDURE
1.

Prepare Standard Dilutions: The recommended standard range is 0.8 - 50 ng/ml. Dissolve one vial of Standard (50 ng/vial) in 1 ml ofSample/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 50 ng/ml solution with an equal volume of Solution C 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 standard solutions.
1000 ml
250 ml

250 ml

250 ml

250 ml

250 ml

250 ml23456781000 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.

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 (1:10 or more) immunizedwith various HDM products and types (DP or DF) will vary depending on the immunization schedule and timing of serum collection. Ingeneral, no IgE antibodies against HDM are observed in normal serum at a 1:10 dilution.

3.

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.

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 Biotinylated HDM: Dilute one vial of biotinylated HDM in 10 ml Biotinylated HDM Dilution Buffer (Solution E). Add 100 µl ofbiotinylated HDM solution to each well and incubate at room temperature for 2 hours.
Strip #246810126.

Biotinylated HDM (µl)
1733506682100

Solution E (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.

© 2023 Chondrex, Inc. All Rights Reserved, 3037 & 3081 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

67.

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: 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 #2468
1012TMB (µl)
3466100132164200Chromogen Dilution Buffer (ml)
1.7
3.3
5.0
6.68.210.0

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-HDM Serum IgE 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 theantibody concentration (ng/ml) in the original test samples.

© 2023 Chondrex, Inc. All Rights Reserved, 3037 & 3081 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

7Figure 1 - Typical Standard Curves for the Anti-HDM Serum IgE Antibody ELISA Kits

DF IgEDP IgE
10.0

10.0

R² = 0.9881

1.0

OD 450 nm

OD 450 nm

R² = 0.9999

0.1

1.0

0.1

0.0

0.0
0.1

1100.1100

110100Anti-HDM DF Serum IgE Antibodies (ng/ml)

Anti-HDM DP Serum IgE Antibodies (ng/ml)

VALIDATION DATA
Table 1 - Reproducibility Data for the Mouse Anti-HDM DP Serum IgE Antibody ELISA Kit
TestIntra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

1.6 ng/ml
5.0
5.3
90%

6.3 ng/ml
2.7
5.190%

25 ng/ml
1.7
3.2
103%

Table 2 - Reproducibility Data for the Mouse Anti-HDM DF Serum IgE Antibody ELISA Kit
Test
1.6 ng/ml
6.3 ng/ml
25 ng/ml
Intra-Assay CV (%)
3.4
2.5
3.0
Inter-Assay CV (%)
2.5
2.7
3.0
Spike Test* (%)
96%
100%
106%
*Known amounts of anti-HDM IgE antibodies were added to samples and then diluted with Sample/Standard/Secondary Antibody
Dilution Buffer to assay anti-HDM IgE antibodies by ELISA.
Figure 2 - Influence of Non-Anti-HDM IgE Antibodies in Samples
120%

Recovery of Anti-HDM IgE

100%
80%
2.5 ng/ml

60%

10 ng/ml

40%
20%0%0110100

1000

10000

Total IgE (ng/ml)© 2023 Chondrex, Inc. All Rights Reserved, 3037 & 3081 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

8TROUBLESHOOTING
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: ARandomized 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, 3037 & 3081 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