1Mouse Total IgE (IgEa and IgEb) Detection ELISA Kit
Catalog # 3005
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify total mouse IgE antibodies

FORMAT:

96-well ELISA plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

100 ng/ml to 1.6 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum & Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:100 or greater

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

Intra-Assay (0.3-3%)/Inter-Assay (1.9-3%)/Spiking Test (95-98%)

NOTES:© 2022 Chondrex, Inc. All Rights Reserved, 3005 6.0
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 Total IgE (IgEa and IgEb) Detection ELISA Kit
Catalog # 3005
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Type I hypersensitivity, which is characterized by an allergic reaction immediately following contact with innocuous antigens, is a typicalclinical feature of allergic diseases, such as asthma, eczema, hay fever, and urticaria. This hypersensitivity is mediated by IgE antibodies,
the so-called “atopic reagin” and the clinical features of type I hypersensitivity are described as “atopy”. IgE antibodies bind to high affinity
IgE receptors (FcεRI) on mast cells and basophils and drastically up-regulate FcεRI expression through stabilization and accumulation ofFcεRI (1), enhancing hypersensitivity responses to allergens. The specific allergen bound to IgE antibodies on the cell surface cross-links
FcεRI (2-3), which leads to the stimulation and degranulation of mast cells. This is associated with the release of a variety of proinflammatory
mediators and cytokines such as histamine, proteolytic enzymes, heparin, and chemotactic factors, which cause the symptoms associatedwith type I hypersensitivity.
In order to study the pathogenesis of allergic diseases, mice are the most practical experimental animals, due to the variety of inbred strains
and transgenic and gene knockout mice that are available. Serum IgE antibody levels are often elevated in allergic diseases and parasitic
infections, although serum IgE level alone does not reflect the allergic state and the clinical symptoms of the patient. However, it is apparent
that an elevated serum IgE level aids in the diagnosis of these diseases in humans.
The Mouse Total IgE Detection ELISA Kit (Catalog # 3005) is designed to determine total IgE levels in mouse sera. Both capture anddetection monoclonal antibodies used in this kit do not cross-react with any other mouse immunoglobulin subclasses and subtypes (IgA,IgM, IgG, IgG1, IgG2a, IgG2b, IgG2c, and IgG3) and both react equally with both IgEa (Balb/c) and IgEb (C57BL/6) allotypes, so it is notnecessary to run two separate assays using two independent IgEa and IgEb standards.

KIT COMPONENTS
Item

Quantity

AmountStorage

IgE Standard (30051)
Capture Antibody (30052)
Detection Antibody (30053)
Solution A - Coating Buffer (9052)

1 vial1 vial1 vial
1 bottle

100 ng/vial, lyophilized0.1 ml
Lyophilized
10 ml

-20°C
-20°C
-20°C-20°C

Solution B - Sample/Standard Dilution Buffer (30055)
Solution C - Detection Antibody Dilution Buffer (30056)
Solution D - Streptavidin Peroxidase Dilution Buffer (9055)
Streptavidin Peroxidase (9029)
TMB Solution (90023)
Chromogen Dilution Buffer (90022)

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

50 ml
10 ml
20 ml
50 µl/vial
0.2 ml
20 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
ELISA Plate

1 bottle
1 bottle
1 each

10 ml
50 ml

-20°C
-20°C
-20°C

96-well (8-well strips x 12)© 2022 Chondrex, Inc. All Rights Reserved, 3005 6.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

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support@chondrex.com

3ASSAY OUTLINE
Add 100 µl of diluted Capture Antibody to all wells
Incubate at 4 degrees Celsius overnight.
Wash plate.
Add 100 µl of diluted Standards and Samples
Incubate at room temperature for 2 hours.
Wash plate.
Add 100 µl of diluted Detection Antibody solution
Incubate at room temperature for 1 hour.
Wash plate.
Add 100 µl of diluted Streptavidin Peroxidase solution
Incubate at room temperature for 1 hour.
Wash plate.

Add 100 µl of TMB solution

Incubate at room temperature for 25 minutes.
Add 50 µl of Stop Solution

Read plates at 450 nm/630 nm

PLATE MAPPING
123456789101112A

100100S1S1S9S9S17

S17

S25

S25

S33

S33

B

5050

S2S2

S10

S10S18

S18

S26

S26

S34

S34C

25

25

S3S3S11S11

S19

S19S27S27

S35

S35

D12.5

12.5

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

6.36.3

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

F

3.13.1

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© 2022 Chondrex, Inc. All Rights Reserved, 3005 6.0
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: Total IgE levels in normal mouse sera is assumed to be in the 50-100 ng/ml range, whereas it will increase to several µg/ml 2weeks after immunizing with an antigen in aluminum hydroxide gel adjuvant. Total IgE levels will further increase to 10-20 µg/ml after repeat
challenges with the aerosolized antigen.

ASSAY PROCEDURE
1.

Add Capture Antibody: Dilute one vial of Capture Antibody with 10 ml of Coating Buffer (Solution A). Alternatively, dilute according tothe table below. Add 100 µl of capture antibody solution to each well and incubate at 4°C overnight. Any leftover Capture Antibody
Stock Solution may be stored at -20°C for future assays.
Strip #2
46810122.

Capture Antibody (µl)
1733
5066
82
100Solution A (ml)
1.7
3.3
5.0
6.6
8.2
10.0

Prepare Standard Dilutions: The recommended standard range is 1.6-100 ng/ml. Dissolve one vial of Standard (100 ng/vial) in 1 mlof Sample/Standard Dilution Buffer (Solution B) to make a 100 ng/ml IgE standard stock solution. Then, serially dilute it with Solution B.
For example, mix 250 µl of the standard (100 ng/ml) with an equal volume of Solution B to make a 50 ng/ml solution, and then repeat
it five more times for 25, 12.5, 6.3, 3.1, and 1.6 ng/ml standards. The remaining 100 ng/ml standard stock can be stored at -20°C for
use in a second assay. Chondrex, Inc. recommends making fresh serial dilutions for each assay.

© 2022 Chondrex, Inc. All Rights Reserved, 3005 6.0
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

51000 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 ml100 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: The suggested dilution for normal serum is 1:10 - 1:50, whereas serum from mouse immunized with
antigens varies from 1:100 to 1:1000 depending upon the immunization schedule and timing of serum collection.

4.

Dilute Wash Buffer: 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 appropriate wells in duplicate. Incubate atroom 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 Detection Antibody: Dissolve one vial of Detection Antibody in 10 ml Detection Antibody Dilution Buffer (Solution C). Alternatively,
dissolve one vial of Detection Antibody in 50 µl of Solution C and follow the dilutions in the following table. Add 100 µl of detection
antibody solution to each well and incubate at room temperature for 1 hour.
Strip #24681012Detection 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 Streptavidin Peroxidase: Dilute one vial of Streptavidin Peroxidase in 10 ml of Streptavidin Dilution Buffer (Solution D). Add 100µl of streptavidin peroxidase solution to each well and incubate at room temperature for 1 hour.
Strip #24681012Streptavidin Peroxidase (µl)
81725334250

Solution D (ml)
1.7
3.3
5.0
6.6
8.2
10.0

© 2022 Chondrex, Inc. All Rights Reserved, 3005 6.0
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

610. 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.
11. Add TMB: Use new tubes when preparing TMB. Dilute one vial of TMB with 10 ml of Chromogen Dilution Buffer just prior to use. Add100 µl of TMB solution to each well immediately after washing the plate and incubate for 25 minutes at room temperature.
Strip #2
4681012TMB (µl)
3466
100
132
164
200

Chromogen Dilution Buffer (ml)
1.7
3.3
5.0
6.6
8.2
10.0

12. Stop: Add 50 µl of 2N sulfuric acid (Stop Solution) to each well.
13. 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 blank (B), standards, 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 standards against the concentration of standard antibody (ng/ml). Using a log/log plot will linearize the data. Figure
1 shows a representative experiment where the standard range is 1.6 - 100 ng/ml.

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.

Figure 1 - A Typical Standard Curve for the Mouse Total IgE Detection ELISA Kit
10.0

R² = 0.9994

OD 450

1.0

0.1

0.0
1.0

10.0

100.0

Mouse IgE Concentration (ng/ml)© 2022 Chondrex, Inc. All Rights Reserved, 3005 6.0
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

ASSAY VALIDATION
Table 1 - Reproducibility Data for the Mouse Total IgE Detection ELISA Kit
Test
12.5 ng/ml
50 ng/ml
100 ng/ml
Intra-Assay CV (%)
0.3
2.0
3.0
Inter-Assay CV (%)
3.0
2.1
1.9
Test
3.1 ng/ml
12.5 ng/ml
50 ng/ml
Spike Test* (%)
96%
95%
98%
* Known amounts of mouse IgE antibodies were added to samples and then diluted with Sample/Standard 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.

S. Kubo, et al., Drastic up-regulation of FceRI on mast cells is induced by IgE binding through stabilization and accumulation of FceRI
on the cell surfaces. J. Immunol. 167: 3427-34 (2001).

2.

S. Kaul, Replacement of murine sera by allergen-specific monoclonal IgE antibodies: A new approach for the characterization of allergen
extracts. Dev. Biol. (Basel) 111: 109-115 (2002).

3.

A. M. Collins, et al., Antigen valency as a determinant of the responsiveness of IgE-sensitised rat basophil leukemia cells. Int. Arch.
Allergy Immunol. 107: 547-556 (1995).

4.

T. Morokata, et al., Differential susceptibility of C57BL/6 and DBA/2 mice to ovalbumin-induced pulmonary eosinophil regulated by
Th1/Th2 type cytokines. Immunol. Letter 70: 127-134 (1999).

5.

A. Oshiba, et al., Passive transfer of immediate-hypersensitivity and airway hyperresponsiveness by allergen-specific immunoglobulin
IgE and IgG1 in mice. J. Clin. Invest. 97: 1398-1408 (1996).

6.

E. Hamelmann, et al., Role of IgE in the development of allergic airway inflammation and airway hyperresponsiveness - a murine model.
Allergy 54: 297-305 Review (1999).

7.

C. Taube, et al., Mast cells, FceRI, and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen
exposure in the absence of adjuvant. J. Immunol. 172: 6398-6406 (2004).

© 2022 Chondrex, Inc. All Rights Reserved, 3005 6.0
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