1

Mouse Anti-Crude Peanut Extract Antibody Subtype/Subclass ELISA Kits
Catalog # 3056, 3057, 3058, 3059, 3060, 3061, and 3062
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify mouse anti-crude peanut extract (CPE) antibodies

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

4.5 hours

STANDARD RANGE:

3056 (IgG): 25 - 0.4 ng/ml

3057 (IgG1): 50 - 0.8 ng/ml

3058 (IgG2a) : 25 - 0.4 ng/ml
3059 (IgG2b) : 5 - 0.08 ng/ml
3060 (IgG3): 50 - 0.8 ng/ml

3061 (IgA): 50 - 0.8 ng/ml

3062 (IgM): 50 - 0.8 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum & Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:100 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

3056: Intra-Assay (3.3-7.4%)/Inter-Assay (4.0-10.4%)/Spiking Test (102-108%)3057: Intra-Assay (1.9-7.2%)/Inter-Assay (0.8-3.7%)/Spiking Test (93-103%)
3058: Intra-Assay (3.6-4.5%)/Inter-Assay (5.4-9.0%)/Spiking Test (91-110%)3059: Intra-Assay (4.6-5.8%)/Inter-Assay (4.9-9.8%)/Spiking Test (104-109%)
3060: Intra-Assay (3.1-4.1%)/Inter-Assay (2.3-6.6%)/Spiking Test (104-110%)3061: Intra-Assay (4.4-9.4%)/Inter-Assay (3.3-5.4%)/Spiking Test (91-105%)
3062: Intra-Assay (3.2-5.8%)/Inter-Assay (3.7-9.4%)/Spiking Test (94-107%)

NOTES:

N/A© 2022 Chondrex, Inc. All Rights Reserved, CPE Isotypes 1.0
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-Crude Peanut Extract Antibody Subtype/Subclass ELISA Kits
Catalog # 3056, 3057, 3058, 3059, 3060, 3061, and 3062
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).
Mouse peanut allergy models have been used to study the pathogenesis of the peanut allergy and to help develop new treatments. Themouse models 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 forstudying the efficacy of protective effects against the development of allergic reactions (3–9).
To evaluate the humoral immune response against CPE in mouse allergy models, Chondrex, Inc. provides ELISA kits for assaying mouseanti-CPE subtype and subclass antibodies including IgA, IgM, IgG, IgG1, IgG2a, IgG2b, and IgG3 antibodies. Chondrex, Inc. also offers
ELISA kits for assaying anti-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.

KIT COMPONENTS
ItemIgG (30561) – 25 ng
IgG1 (30571) – 50 ng
IgG2a (30581) – 25 ng
StandardIgG2b (30591) – 5 ng
IgG3 (30601) – 25 ng
IgA (30611) – 25 ng
IgM (30621) – 50 ng
IgG (30113)
Secondary Antibody
IgG1 (30133)(peroxidase-conjugated
IgG2a (30153)
polyclonal antibodies)
IgG2b (30163)
IgG3 (30393)
IgA (30183)
IgM (30173)
Solution B - Blocking Buffer (30105)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30106)
TMB Solution (90023)
Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
Crude Peanut Extract Coated ELISA Plate (Green)

Quantity

AmountStorage

1 vialLyophilized-20°C

2 vials

50 µl

-20°C

1 bottle
1 bottle
2 vials

10 ml
50 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

© 2022 Chondrex, Inc. All Rights Reserved, CPE Isotypes 1.016928 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

3NOTES 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 for
disposal.
NOTE 8: Depending on the isotypes, subtypes, and targeting epitopes of antibodies, the binding affinity of individual antibodies varies
significantly. Therefore, the total IgG antibody concentration calculated as the sum of individual IgG subtypes might not perfectly match thetotal IgG concentration as determined by the Mouse Anti-CPE IgG Antibody ELISA Kit (Cat # 3056).

ASSAY OUTLINE© 2022 Chondrex, Inc. All Rights Reserved, CPE Isotypes 1.0
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

PLATE MAPPING
Example of the Mouse Anti-CPE IgG1, IgG3, IgA, and IgM Antibody ELISA Kits
123456789101112A

50

50

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

25

25

S2

S2

S10

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

F1.6

1.6

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G0.80.8

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39HBB

S8S8

S16

S16

S24

S24

S32

S32

S40

S40

StandardsSamples

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. Dilute the standard stock
accordingly to get the first stock solution. Then serially dilute it with Solution C. For example, mix 250 µl of the first stock solution withan equal volume of Solution C to make the second stock solution, and then repeat it five more times. The original standard stock solution
may 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 ml

250 ml

250 ml

250 ml

IgG2b:

5 ng/ml

2.5 ng/ml

1.3 ng/ml

0.63 ng/ml

0.31 ng/ml

0.16 ng/ml

0.08 ng/ml

Blank

IgG/ IgG2a:25 ng/ml

12.5 ng/ml

6.3 ng/ml

3.1 ng/ml

1.6 ng/ml

0.8 ng/ml

0.4 ng/ml

Blank

IgG1/IgG3/IgA/IgM:

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

© 2022 Chondrex, Inc. All Rights Reserved, CPE Isotypes 1.016928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

53.

Prepare Sample Dilutions: The dilution of serum from mice immunized with CPE varies (1:100 or more) depending on the immunization
schedule and timing of serum collection. In general, no antibodies against CPE are observed in normal serum at a 1:100 dilution.

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.
Strip #
2468
1012

2nd Antibody (µl)
81725
334250Solution 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 #24681012

TMB (µl)
3466
100132164200Chromogen 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.

© 2022 Chondrex, Inc. All Rights Reserved, CPE Isotypes 1.0
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

6CALCULATING 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
sample standard curves for anti-CPE Ig 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 original test samples.

Figure 1 - Typical Standard Curves for the Anti-CPE Antibody ELISA Kits
10.0

10.00.1

0.0

1.0

1.00.10.1

0.00.11101000.00.1

Anti-CPE IgG Antibodies (ng/ml)

1101000.1

Anti-CPE IgG1 Antibodies (ng/ml)

0.0

1.0

0.1

0.00.1

1

10100

Anti-CPE IgG2b Antibodies (ng/ml)

100

R² = 0.994

OD 450 nm

OD 450 nm

0.1

1010.0R² = 0.944

R² = 0.9913

1.01

Anti-CPE IgG2a Antibodies (ng/ml)

10.010.0

OD 450 nm

R² = 0.9814

OD 450 nm

1.0

OD 450 nm

OD 450 nm

10.0

R² = 0.9703

R² = 0.9901

1.00.1

0.00.11101000.1

Anti-CPE IgG3 Antibodies (ng/ml)

110100Anti-CPE IgA Antibodies (ng/ml)

10.0

OD 450 nm

R² = 0.9977

1.00.1

0.00.1110100Anti-Gliadin IgM Antibodies (ng/ml)© 2022 Chondrex, Inc. All Rights Reserved, CPE Isotypes 1.016928 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

VALIDATION DATATable 1 - Reproducibility Data for the Mouse Anti-CPE IgG Antibody ELISA Kit
Test
0.5 ng/ml
2.5 ng/ml
Intra-Assay CV (%)
4.3
7.4
Inter-Assay CV (%)
9.3
4.0
Spike Test* (%)
102%
108%

10 ng/ml
3.3
10.4
107%

Table 2 - Reproducibility Data for the Mouse Anti-CPE IgG1 Antibody ELISA Kit
Test
1.6 ng/ml
6.3 ng/ml
Intra-Assay CV (%)
7.1
7.2
Inter-Assay CV (%)
3.7
3.3
Spike Test* (%)
95%
93%

25 ng/ml
1.90.8103%

Table 3 - Reproducibility Data for the Mouse Anti-CPE IgG2a Antibody ELISA Kit
Test
1.0 ng/ml
6.0 ng/ml
Intra-Assay CV (%)
3.6
4.2
Inter-Assay CV (%)
9.0
5.4
Spike Test* (%)
95%
91%

20 ng/ml
4.5
7.9110%

Table 4 - Reproducibility Data for the Mouse Anti-CPE IgG2b Antibody ELISA Kit
Test
0.1 ng/ml
0.63 ng/ml
Intra-Assay CV (%)
5.8
4.6
Inter-Assay CV (%)
9.8
5.7
Spike Test* (%)
106%
104%

2.5 ng/ml
5.6
4.9109%

Table 5 - Reproducibility Data for the Mouse Anti-CPE IgG3 Antibody ELISA Kit
Test
1.5 ng/ml
8 ng/ml
Intra-Assay CV (%)
3.3
4.1
Inter-Assay CV (%)
6.6
2.3
Spike Test* (%)
110%
105%

35 ng/ml
3.1
4.5104%

Table 6 - Reproducibility Data for the Mouse Anti-CPE IgA Antibody ELISA Kit
Test
1.6 ng/ml
Intra-Assay CV (%)
7.1
Inter-Assay CV (%)
3.4
Spike Test* (%)
105%

25 ng/ml
4.4
3.391%

6.3 ng/ml
9.4
5.496%

Table 7 - Reproducibility Data for the Mouse Anti-CPE IgM Antibody ELISA Kit
Test
1.3 ng/ml
8 ng/ml
20 ng/ml
Intra-Assay CV (%)
3.2
5.4
5.8
Inter-Assay CV (%)
4.83.7
9.4
Spike Test* (%)
107%
101%
94%
*Known amounts of anti-CPE antibodies were added to samples and then diluted with Sample/Standard/Secondary Antibody Dilution
Buffer to assay anti-CPE antibodies by ELISA.

© 2022 Chondrex, Inc. All Rights Reserved, CPE Isotypes 1.0
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. 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. 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).

© 2022 Chondrex, Inc. All Rights Reserved, CPE Isotypes 1.0
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