1Mouse Anti-Gliadin IgE Antibody ELISA Kit
Catalog # 3050
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify mouse anti-gliadin IgE antibodies

FORMAT:

Precoated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

4 hours

STANDARD RANGE:

1000 - 16 pg/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:

Intra-Assay (5.6–6.1%)/Inter-Assay (3.4-8.1%)/Spiking Test (105-109%)

NOTES:

N/A© 2021 Chondrex, Inc. All Rights Reserved, 3050 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-Gliadin IgE Antibody ELISA Kit
Catalog # 3050
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Wheat is the most widely consumed food grain in the world. Wheat proteins are categorized into four fractions based on their solubility insolvents: water (albumins), water containing salt (globulins), alcohol (gliadins), and alkali or acid solution (glutelin) (1). One of these proteins,
gliadin consists of alpha, beta, gamma, and omega types which are between 28 – 55kDa, and has repeated sequences which show highantigenicity. Gliadins play a critical role in activating both the innate and adaptive immune response, which results in the immune-mediatedinjury of the intestine such as high intestinal permeability and lamina propria inflammatory cell infiltration. Therefore, gliadins can trigger
Celiac disease (CD) which is an autoimmune disorder in genetically susceptible individuals. Many CD patients even have antibodies which
recognize major epitopes (QQFPQQQ, QQIPQQQ, and QQLPQQQ) in omega gliadin (2).
Mouse CD models have been widely used to study the pathogenesis of gliadin and its immune responses. The mice who receive gliadin
have significantly higher serum gliadin specific IgE and IgG1 antibodies than controls (3–6). To evaluate the humoral immunity against gliadin
in the mouse CD models, Chondrex, Inc. provides ELISA kits for assaying mouse anti-gliadin subtype and subclass antibodies, including
IgE, IgM, IgG, IgG1, IgG2a, and IgG2b antibodies. Chondrex, Inc. also offers ELISA kits for assaying anti-ovalbumin and house dust mite
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
Item

Quantity

AmountStorage

Standard Mouse Anti-Gliadin IgE Antibody (30501)
Biotinylated Gliadin (30503)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30106)

1 vial1 vial
1 bottle

1000 pg, lyophilized
100 µl
50 ml

-20°C
-20°C
-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)

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

20 ml
50 µl
0.2 ml
20 ml
10 ml
50 ml

-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© 2021 Chondrex, Inc. All Rights Reserved, 3050 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

3

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 100 µl of a stock solution in 10 ml of buffer for 12 strips, then for 6 strips, dilute 50 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: 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 beexpressed as pg of IgE per ml.
NOTE 9: If the total IgE concentration in a sample is higher than 10 ng/ml, the sample must be diluted to lower the total IgE levels below 10ng/ml because the anti-gliadin IgE value obtained from this ELISA may be lower than the actual value due to competition from non-antigliadin IgE antibodies in sample. Therefore, it is strongly recommended that total IgE levels be determined first using the Mouse Total IgEAssay Kit (Catalog # 3005).

PLATE MAPPING
123456789101112

A

1000

1000

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

500

500

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C250

250

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D125125

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E6363

S5S5S13

S13

S21

S21

S29

S29

S37

S37F3131S6S6S14

S14

S22

S22

S30

S30

S38

S38G1616S7S7S15

S15

S23

S23S31S31

S39S39HB

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

StandardsSamples© 2021 Chondrex, Inc. All Rights Reserved, 3050 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

4ASSAY OUTLINE
Add 100 ml of diluted standards or samples in wellsIncubate at room temperature for 1.5 hours.
Wash plates.
Add 100 ml of diluted Biotinylated Gliadin

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

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: The recommended standard range is 16 - 1000 pg/ml. Dissolve one vial of Standard (1000 pg/vial) 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 1000 pg/ml solution with an equal volume of Solution C to make a 500 pg/ml solution, andthen repeat it five more times for 250, 125, 63, 31, and 16 pg/ml standard solutions.
1000 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 ml1000 pg/ml

500 pg/ml

250 pg/ml

125 pg/ml

63 pg/ml

31 pg/ml

16 pg/ml

Blank

12.

250 mlPrepare Sample Dilutions: The serum dilution of mice immunized with gliadin will vary (1:10 or more) depending on the immunization
schedule and timing of serum collection. In general, no IgE antibodies against gliadin are observed in normal serum at a 1:10 dilution.

© 2021 Chondrex, Inc. All Rights Reserved, 3050 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

53.

Add Standards and Samples: Add 100 µl of standards, Solution C (blank), and samples to wells in duplicate. Incubate at room
temperature for 1.5 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 Gliadin: Dilute one vial of biotinylated gliadin in 10 ml Sample/Standard/Secondary Antibody Dilution Buffer (SolutionC). Add 100 µl of biotinylated gliadin solution to each well and incubate at room temperature for 1.5 hours.
Strip #24681012Biotinylated Gliadin (µl)
1733506682
100Solution C (ml)
1.7
3.3
5.0
6.6
8.2
10.0

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 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 #24681012

Streptavidin 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 #24681012TMB (µl)
3466100132
164
200

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

© 2021 Chondrex, Inc. All Rights Reserved, 3050 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

6

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 pg/ml of antibody standard. Using a log/log plot will linearize the data. Figure 1 shows
an example of a standard curve for anti-gliadin IgE antibodies.

4.

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

Figure 1 - A Typical Standard Curve for the Anti-Gliadin IgE Antibody ELISA Kit
10.0

OD 450 nm

R² = 0.9886

1.00.10.0
101001000

Anti-Gliadin IgE Antibodies (pg/ml)

VALIDATION DATA
Table 1 - Reproducibility Data for the Mouse Anti-Gliadin IgE Antibody ELISA Kit
Test
31 pg/ml
125 pg/ml
500 pg/ml
Intra-Assay CV (%)
5.6
6.0
6.1
Inter-Assay CV (%)
3.4
3.5
8.1
Spike Test* (%)
105%
106%
109%
*Known amounts of anti-gliadin IgE antibodies were added to samples and then diluted with Sample/Standard/Secondary Antibody
Dilution Buffer to assay anti-gliadin IgE antibodies by ELISA.

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

© 2021 Chondrex, Inc. All Rights Reserved, 3050 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

7

REFERENCES
1. N. Inomata, Wheat allergy. Curr. Opin. Allergy Clin. Immunol. 9, 238–243 (2009).
2. F. Battais, T. Mothes, D. A. Moneret-Vautrin, F. Pineau, G. Kanny, Y. Popineau, M. Bodinier, S. Denery-Papini, Identification of IgEbinding epitopes on gliadins for patients with food allergy to wheat. Allergy. 60, 815–821 (2005).
3. R. Abe, N. Matsukaze, Y. Yamaguchi, M. Akao, H. Kumagai, H. Kumagai, Wheat gliadin deamidated by cation-exchange resins inducesoral tolerance in a mouse model of wheat allergy. Journal of Food Bioactives. 2, 119–128 (2018).
4. R. Abe, S. Shimizu, K. Yasuda, M. Sugai, Y. Okada, K. Chiba, M. Akao, H. Kumagai, H. Kumagai, Evaluation of reduced allergenicity ofdeamidated gliadin in a mouse model of wheat-gliadin allergy using an antibody prepared by a peptide containing three epitopes. J.
Agric. Food Chem. 62, 2845–2852 (2014).
5. M. Bodinier, M. Leroy, S. Ah-Leung, F. Blanc, O. Tranquet, S. Denery-Papini, J.-M. Wal, K. Adel-Patient, Sensitization and elicitation ofan allergic reaction to wheat gliadins in mice. J. Agric. Food Chem. 57, 1219–1225 (2009).
6. P. Gourbeyre, S. Denery-Papini, C. Larré, J.-C. Gaudin, C. Brossard, M. Bodinier, Wheat gliadins modified by deamidation are more
efficient than native gliadins in inducing a Th2 response in Balb/c mice experimentally sensitized to wheat allergens. Mol. Nutr. Food
Res. 56, 336–344 (2012).

© 2021 Chondrex, Inc. All Rights Reserved, 3050 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