1

Mouse Anti-Gliadin Antibody Subtype/Subclass ELISA Kits
Catalog # 3051, 3052, 3053, 3054, and 3055
For Research Use Only - Not Human or Therapeutic Use

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

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Indirect ELISAASSAY TIME:

3.5 hours

STANDARD RANGE:

3051 (IgG): 10 - 0.16 ng/ml

3052 (IgG1): 50 - 0.8 ng/ml

3053 (IgG2a) : 10 - 0.16 ng/ml
3054 (IgG2b) : 10 - 0.16 ng/ml
3055 (IgM): 50 - 0.8 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Serum & Plasma

RECOMMENDED SAMPLE DILUTIONS:

1:1000 (at least)

CHROMOGEN:

TMB (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

3051: Intra-Assay (2.2-7.7%)/Inter-Assay (5.5-8.6%)/Spiking Test (99-109%)
3052: Intra-Assay (3.0-8.4%)/Inter-Assay (6.8-8.7%)/Spiking Test (104-109%)
3053: Intra-Assay (3.5-4.8%)/Inter-Assay (1.8-4.5%)/Spiking Test (96-106%)
3054: Intra-Assay (1.8-4.1%)/Inter-Assay (4.0-8.3%)/Spiking Test (105-108%)3055: Intra-Assay (2.5-3.4%)/Inter-Assay (2.3-4.9%)/Spiking Test (97-101%)NOTES:N/A© 2021 Chondrex, Inc. All Rights Reserved, Gliadin 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

2Mouse Anti-Gliadin Antibody Subtype/Subclass ELISA Kits
Catalog # 3051, 3052, 3053, 3054, and 3055
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

AmountStorage1 vial

Lyophilized-20°C

IgG (30511) – 10 ng
IgG1 (30521) – 50 ng
IgG2a (30531) – 10 ng
Standard
IgG2b (30541) – 10 ng
IgM (30551) – 50 ng
IgG (30113)
IgG1(30133)
Secondary Antibody
IgG2a (30153)(peroxidase-conjugated
IgG2b (30163)
polyclonal antibodies)
IgM (30173)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30106)
TMB Solution (90023)

2 vials

50 µl

-20°C

2 bottles
2 vials

50 ml
0.2 ml

-20°C
-20°C

Chromogen Dilution Buffer (90022)
Stop Solution - 2N Sulfuric Acid (9016)
Wash Buffer, 20X (9005)
Gliadin Coated ELISA Plate (Blue)

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

© 2021 Chondrex, Inc. All Rights Reserved, Gliadin 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

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 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: 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-Gliadin IgG Antibody ELISA Kit (Cat # 3051).

ASSAY OUTLINE
Add 100 ml of diluted standards or samples in wells
Incubate at room temperature for 2 hours.
Wash plates.

Add 100 ml of diluted Secondary Antibody
Incubate at room temperature for 1 hour.
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© 2021 Chondrex, Inc. All Rights Reserved, Gliadin 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-Gliadin IgG, G2a and G2b Antibody ELISA Kits

Example of the Mouse Anti-Gliadin IgG1 and M Antibody ELISA Kits
123456789101112A50

50

S1

S1

S9S9

S17

S17

S25

S25

S33

S33

S34

B

2525

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

S35

S35

C12.5

12.5

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

S28

S36

S36

D

6.3

6.3

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

S29

S29

S37

S37

E

3.1

3.1

S5

S5

S13

S13

S21

S21

S29

S29

S37

S37

S22

S30

S30

S38

S38

F

1.6

1.6

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

S23

S23

S31

S31

S39

S39

G

0.8

0.8

S7S7S15

S15

S23

S23

S31

S31

S39

S39

S24

S24

S32

S32

S40

S40

HB

B

S8S8

S16

S16

S24

S24

S32

S32

S40

S40

123456789101112A

1010

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

55

S2

S2

S10

S10

S18

S18

S26

S26

S34

C

2.52.5

S3

S3

S11

S11

S19

S19

S27

S27

D

1.25

1.25

S4

S4

S12

S12

S20

S20

S28

E

0.63

0.63

S5

S5

S13

S13

S21

S21

F

0.31

0.31

S6

S6

S14

S14

S22

G0.16

0.16

S7

S7

S15

S15

HB

BS8S8

S16

S16Samples

Standards

SamplesStandards

ASSAY PROCEDURE
1.

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 ml2345678250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 mlIgG/ IgG2a/ IgG2b: 10 ng/ml

5 ng/ml

2.5 ng/ml

1.25 ng/ml

0.63 ng/ml

0.31 ng/ml

0.16 ng/ml

Blank

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

11000 ml

IgG1/IgM:

2.

250 mlPrepare Sample Dilutions: The dilution of serum from mice immunized with gliadin varies (1:1000 or more) depending on theimmunization schedule and timing of serum collection. In general, no antibodies against OVA are observed in normal serum at a 1:1000
dilution.
NOTE: Because regular mouse chows may contain gluten, unimmunized, naive mice may have anti-gliadin antibodies in serum.

© 2021 Chondrex, Inc. All Rights Reserved, Gliadin 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

53.

Add Standards and Samples: Add 100 ml 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 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 #
2
4
6
8
1012

2nd Antibody (µl)
81725
334250Solution 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 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
100132
164
200

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

8.

Stop: Stop the reaction with 50 µl of 2N Sulfuric Acid (Stop Solution) to each well.

9.

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
an example of a standard curve for anti-Gliadin Ig antibodies.

© 2021 Chondrex, Inc. All Rights Reserved, Gliadin 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

64.

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-Gliadin Antibody ELISA Kits
10.010.010.0
R² = 0.9522

0.1

1.00.00.00.1

1101.00.10.1

0.00.1Anti-Gliadin IgG Antibodies (ng/ml)

1101000.1

110

Anti-Gliadin IgG2a Antibodies (ng/ml)

Anti-Gliadin IgG1 Antibodies (ng/ml)
10.0

10.0

R² = 0.9834

R² = 0.9937

1.0OD 450 nm

OD 450 nm

R² = 0.994

OD 450 nm

1.0

OD 450 nm

OD 450 nm

R² = 0.9919

1.00.10.1

0.00.00.1

110Anti-Gliadin IgG2b Antibodies (ng/ml)

0.1110100Anti-Gliadin IgM Antibodies (ng/ml)

VALIDATION DATA
Table 1 - Reproducibility Data for the Mouse Anti-Gliadin IgG Antibody ELISA Kit
Test
0.31 ng/ml
1.25 ng/ml
Intra-Assay CV (%)
2.2
7.7
Inter-Assay CV (%)
8.6
7.1
Spike Test* (%)
101%99%5 ng/ml
5.0
5.5
109%

Table 2 - Reproducibility Data for the Mouse Anti-Gliadin IgG1 Antibody ELISA Kit
Test
1.6 ng/ml
6.3 ng/ml
Intra-Assay CV (%)
8.2
8.4
Inter-Assay CV (%)
7.7
8.7Spike Test* (%)
106%
104%

25 ng/ml
3.0
6.8
109%

Table 3 - Reproducibility Data for the Mouse Anti-Gliadin IgG2a Antibody ELISA Kit© 2021 Chondrex, Inc. All Rights Reserved, Gliadin 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

7TestIntra-Assay CV (%)
Inter-Assay CV (%)
Spike Test* (%)

0.31 ng/ml
4.8
2.996%

1.25 ng/ml
3.5
4.5
106%

5 ng/ml
4.0
1.8106%

Table 4 - Reproducibility Data for the Mouse Anti-Gliadin IgG2b Antibody ELISA Kit
Test
0.31 ng/ml
1.25 ng/ml
Intra-Assay CV (%)
3.8
4.1
Inter-Assay CV (%)
6.0
4.0
Spike Test* (%)
105%
105%

5 ng/ml
1.8
8.3
108%

Table 5 - Reproducibility Data for the Mouse Anti-Gliadin IgM Antibody ELISA Kit
Test
1.6 ng/ml
6.3 ng/ml
25 ng/ml
Intra-Assay CV (%)
2.5
3.2
3.4
Inter-Assay CV (%)
4.9
4.8
2.3
Spike Test* (%)
98%
97%
101%
*Known amounts of anti-gliadin antibodies were added to samples and then diluted with Sample/Standard/Secondary Antibody Dilution
Buffer to assay anti-gliadin antibodies by ELISA.

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

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, Gliadin 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