1Mouse Anti- Crude Peanut Extract (CPE) IgE Antibody ELISA Kit
Mouse Anti- Ara h3 (Peanut Allergen) IgE Antibody ELISA Kit
Catalog # 3063 and 3071
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kits to quantify mouse anti-crude peanut extract (CPE) or anti-Ara h3 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:

3063: Intra-Assay (1.1-8.8%)/Inter-Assay (1.2-7.4%)/Spiking Test (92-94%)
3071: Intra-Assay (1.3-4.5%)/Inter-Assay (1.0-4.1%)/Spiking Test (93-106%)NOTES:

N/A© 2023 Chondrex, Inc. All Rights Reserved, 3063 & 3071 1.1
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2

Mouse Anti-Crude Peanut Extract (CPE) IgE Antibody ELISA Kit
Mouse Anti-Ara h3 (Peanut Allergen) IgE Antibody ELISA Kit
Catalog # 3063 and 3071
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Immediate hypersensitivity reactions to peanuts, an IgE-mediated food allergy, have been a major public health concern for many years,
particularly in westernized countries where peanut allergies can persist into adulthood. For allergic patients, avoidance currently remains theonly viable option (1).
Eleven potentially important peanut allergens have been identified. Ara h1, Ara h2, Ara h3, and Ara h6 have been designated the major
peanut allergens. Ara h2 and Ara h6, two highly related 2S albumins, especially contribute to the development of allergic reactions (2). Mousepeanut allergy models have been used to study the pathogenesis of the peanut allergy and to help develop new treatments. The mousemodels can be induced by administration of crude peanut extract (CPE) or each purified Ara allergen and evaluated for the humoral immune
responses such as serum anti-IgE and IgG antibodies against the allergen, T-cell mediated immune response associated cytokines levels,
as well as body temperature and clinical signs of anaphylaxis. These factor changes observed in the disease models are useful for studying
the efficacy of protective effects against the development of allergic reactions (3–9). Additionally, the choice of mouse strain may beimportant, as reactivity to peanut allergens may vary depending on the genetic background. For example, specific IgE responses were mainly
directed against Ara h3 in C57BL/6 and BALB/c mice, while Ara h2 was the main target in C3H mice. (10, 11)
To evaluate the humoral immune response against CPE in mouse allergy models, Chondrex, Inc. provides ELISA kits for assaying mouseanti-CPE and anti-CPE Ara h3 IgE antibodies (Catalog # 3063 and 3071). Chondrex, Inc. also offers ELISA kits for assaying anti-CPE,
ovalbumin, house dust mite, and gliadin antibody subtypes/subclasses as well as total immunoglobulin subtypes/subclasses. For more
information, please visit www.chondrex.com or contact support@chondrex.com.

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

CPE Catalog #305630573059306230633058
3060
3061

Ara h2 Catalog #3077
Coming Soon!
3078
3079
Coming soon!
Coming soon!
Coming soon!
3080

Ara h3 Catalog #
3082
3083
Coming soon!
Coming soon!
3071
Coming soon!
Coming soon!
Coming soon!

© 2023 Chondrex, Inc. All Rights Reserved, 3063 & 3071 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

Quantity

AmountStorage

Standard Mouse Anti-CPE IgE Antibody (30631)
Biotinylated CPE (30633)
Biotinylated Ara h3 (30713)
Solution B - Sample/Standard/Detection Antibody Dilution Buffer (67015)

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)
Streptavidin Peroxidase (9029)
TMB Solution (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
Anti-Mouse IgE Antibody Coated ELISA Plate (Yellow)

2 bottles
2 vials
2 vials
1 bottle
1 bottle
1 bottle
1 each20 ml
50 µl
0.2 ml
20 ml
10 ml
50 ml
96-well (8-well strips x 12)

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

ASSAY OUTLINE
Add 100 ml of blocking buffer in wells
Incubate at room temperature for 1 hour.
Wash plates.
Add 100 ml of diluted standards or samples in wells
Incubate at room temperature for 1.5 hours.
Wash plates.
Add 100 ml of diluted Biotinylated CPE (3063) or Biotinylated Ara h3 (3071)
Incubate at room temperature for 1.5 hours.
Wash plates.
Add 100 ml of diluted Streptavidin Peroxidase
Incubate at room temperature for 30 minutes.
Wash plates.
Add 100 ml of TMB solution

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

Read plates at 450 nm/630 nm© 2023 Chondrex, Inc. All Rights Reserved, 3063 & 3071 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
1

2

3

4

5

6

7

8910

1112

A

50

50

S1

S1

S9S9S17

S17

S25

S25

S33

S33

B

25

25

S2S2

S10

S10

S18

S18

S26

S26

S34

S34C12.5

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

S39H

B

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples

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 an
antibody 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 beexpressed as ng of IgE per ml.

© 2023 Chondrex, Inc. All Rights Reserved, 3063 & 3071 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 Blocking Buffer (Solution B) to each well and incubate at room temperature for 1 hour.

2.

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 B) and keep it as a standard stock. Then serially dilute it with Solution
B. For example, mix 250 µl of the 50 ng/ml solution with an equal volume of Solution B 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

3.

Prepare Sample Dilutions: An important point to note is that the composition of CPE mixtures can exhibit variations depending on thevendor, batch, and manufacturing process. These variations can result in differing levels of antigens in the final CPE product, which
when used to immunize mice, can impact the serum antibody levels against those antigens. The dilution of serum from mice immunizedwith CPE antigens varies (1:100 or more) depending on the immunization schedule and timing of serum collection. In general, noantibodies against CPE are observed in normal serum at a 1:100 dilution. If serum samples require a lower dilution than 1:100, pleasecontact 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 1.5 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 Biotinylated CPE OR Biotinylated Ara h3: Prepare biotinylated CPE OR Biotinylated Ara h3 with Streptavidin Peroxidase Dilution
Buffer (Solution D) as shown in the following table. Do not combine biotinylated CPE and biotinylated Ara h3. Add 100 µl of biotinylated
solution to each well and incubate at room temperature for 1.5 hours.
Strip #24681012Biotinylated Tracer (µl)
1733506682100

Solution D (ml)
1.7
3.3
5.0
6.6
8.2
10.0

© 2023 Chondrex, Inc. All Rights Reserved, 3063 & 3071 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

68.

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 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

10. 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.
11. 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
4681012TMB (µl)
3466100132
164
200

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

12. Stop: Stop the reaction with 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 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-CPE IgE and anti-CPE Ara h3 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, 3063 & 3071 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

7Figure 1 - Typical Standard Curves for the Anti-CPE and Anti-CPE Ara h3 IgE Antibody ELISA Kits
10.00

10.00

R² = 0.9975

R² = 0.9996

1.00

OD 450 nm

OD 450 nm

1.00

0.10

0.01

0.10

0.01

0.00

0.00

0.1

110
Anti-CPE Antibodies (ng/ml)

100

0.1

110
Anti-CPE Ara h3 Antibodies (ng/ml)

100

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

1.25 ng/ml
8.8
7.494%

5 ng/ml
6.0
4.1
93%

25 ng/ml
1.1
1.292%

Table 2 - Reproducibility Data for the Mouse Anti-Ara h3 IgE Antibody ELISA Kit
Test
2.1 ng/ml
8.3 ng/ml
33.2 ng/ml
Intra-Assay CV (%)
4.5
1.3
2.0
Inter-Assay CV (%)
1.0
2.2
4.1
Spike Test* (%)
106%
93%
94%
*Known amounts of anti-CPE or anti-CPE Ara h3 IgE antibodies were added to samples and then diluted with Sample/Standard/Secondary
Antibody Dilution Buffer to assay anti-CPE or anti-CPE Ara h3 IgE antibodies by ELISA.

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

© 2023 Chondrex, Inc. All Rights Reserved, 3063 & 3071 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

8

REFERENCES
1.

A. W. Burks, Peanut allergy. Lancet. 371, 1538–1546 (2008).

2.

Y. Zhuang, S. C. Dreskin, Redefining the major peanut allergens. Immunol. Res. 55, 125–134 (2013).

3.

J. J. Dolence, Induction of Peanut Allergy Through Inhalation of Peanut in Mice. Methods Mol. Biol. 2223, 19–35 (2021).

4.

X. M. Li, D. Serebrisky, S. Y. Lee, C. K. Huang, L. Bardina, B. H. Schofield, J. S. Stanley, A. W. Burks, G. A. Bannon, H. A. Sampson,
A murine model of peanut anaphylaxis: T- and B-cell responses to a major peanut allergen mimic human responses. J. Allergy Clin.
Immunol. 106, 150–158 (2000).

5.

M.-J. Bae, H. S. Shin, E.-K. Kim, J. Kim, D.-H. Shon, Oral administration of chitin and chitosan prevents peanut-induced anaphylaxis ina murine food allergy model. Int. J. Biol. Macromol. 61, 164–168 (2013).

6.

M. Kulis, X. Chen, J. Lew, Q. Wang, O. P. Patel, Y. Zhuang, K. S. Murray, M. W. Duncan, H. S. Porterfield, A. W Burks, S. C. Dreskin,
The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize
peanut-allergic mice. Clin. Exp. Allergy. 42, 326–336 (2012).

7.

L. M. Chang, Y. Song, X.-M. Li, H. A. Sampson, M. Masilamani, Dietary Elimination of Soybean Components Enhances Allergic Immune
Response to Peanuts in BALB/c Mice. Int. Arch. Allergy Immunol. 166, 304–310 (2015).

8.

C. Zhou, T. Ludmila, N. Sun, C. Wang, Q. Pu, K. Huang, H. Che, BALB/c mice can be used to evaluate allergenicity of different foodprotein extracts. Food Agric. Immunol. 27, 589–603 (2016).

9.

C. Bowman, M. K. Selgrade, Differences in allergenic potential of food extracts following oral exposure in mice reflect differences indigestibility: potential approaches to safety assessment. Toxicol. Sci. 102, 100–109 (2008).

10. M. Paolucci, V. Homère, Y. Waeckerle-Men, N. Wuillemin, D. Bieli, N. Pengo, T. Sonati, T. M. Kündig, P. Johansen, Strain matters inmouse models of peanut-allergic anaphylaxis: Systemic IgE-dependent and Ara h 2-dominant sensitization in C3H mice. Clin. Exp.
Allergy. 53(3), 550-560 (2023).
11. G. S. Ladics, L. M. J. Knippels, A. H. Penninks, G. A. Bannon, R. E. Goodman, C. Herouet-Guicheney, Review of animal models
designed to predict the potential allergenicity of novel proteins in genetically modified crops. Regul. Toxicol. Pharmacol. 56, 212–224(2010).

© 2023 Chondrex, Inc. All Rights Reserved, 3063 & 3071 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