1Gliadin Detection ELISA Kit
Catalog # 6035
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:ELISA kit to quantify gliadin

FORMAT:

Precoated 96-well ELISA Plate with removeable strips

ASSAY TYPE:

Sandwich ELISAASSAY TIME:

4 hours

STANDARD RANGE:

20 – 0.31 ng/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Liquid samples and biological fluids (pre-treatment acceptable)

RECOMMENDED SAMPLE DILUTIONS:

1:5 (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:

Gliadin in samples must be solubilized before starting assays (please see Sample Preparationbelow)© 2022 Chondrex, Inc. All Rights Reserved, 6035 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

Gliadin Detection ELISA Kit
Catalog # 6035
For Research Use Only - Not Human or Therapeutic UseINTRODUCTION
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).
Gluten-related disorders can only be treated by life-long wheat exclusion diets (3, 4). These diets must consist only of “gluten-free” products,
which are defined as containing up to 20 mg gluten per kg of raw or cooked food, (5–9). Therefore, precise gluten monitoring systems must
be used to keep gluten levels below the acceptable threshold that defines “gluten free” limits in food (10). One such system, sandwich ELISAs
are widely used in the food industry, however, flaws exist that can affect assay reliability such as allergen size limitations, sample processing(denatured protein structures due to heating and hydrolyzing processes) and non-validated extraction protocols (gliadin is difficult to solubilize
and extract). Standardized assay systems demonstrating better accuracy and sensitivities are thus needed in the food industry.
To evaluate gliadin levels in samples, Chondrex, Inc. offers a Gliadin Detection Kit (Catalog # 6035) which employs two monoclonal
antibodies recognizing a repeating epitope among gliadins and demonstrates high assay sensitivity. Please visit www.chondrex.com for
more information.

KIT COMPONENTS
Item

Quantity

AmountStorageStandard Gliadin (60351)

1 vial20 ng, lyophilized-20°C

Anti-Gliadin Detection Antibody (60353)
Solution C - Sample/Standard/Secondary Antibody Dilution Buffer (30106)
Solution E - Detection Antibody Dilution Buffer (60356)
Solution D - Streptavidin Peroxidase Dilution Buffer (9055)
Streptavidin Peroxidase (9029)
TMB Solution (90023)

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

100 µl
50 ml
10 ml
20 ml
50 µl
0.2 ml

-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

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

1 bottle
1 bottle
1 bottle
1 each20 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, 6035 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

3ASSAY 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 Anti-Gliadin Detection Antibody

Incubate at room temperature for 1 hour.
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

PLATE MAPPING
123456789101112A

20

20

S1

S1

S9

S9

S17

S17

S25

S25

S33

S33

B

1010

S2

S2

S10

S10

S18

S18

S26

S26

S34

S34

C

55

S3

S3

S11

S11

S19

S19

S27

S27

S35

S35

D2.52.5

S4

S4

S12

S12

S20

S20

S28

S28

S36

S36

E

1.25

1.25

S5S5S13

S13

S21

S21

S29

S29

S37

S37F

0.630.63

S6S6S14

S14

S22

S22

S30

S30

S38

S38

G0.310.31

S7

S7S15

S15

S23

S23

S31

S31S39

S39

HBB

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples© 2022 Chondrex, Inc. All Rights Reserved, 6035 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

4

SAMPLE PREPARATION (11, 12)
REQUIRED MATERIALS (NOT INCLUDED)
1. Dithiothreitol (DTT) (Alternatively 2-Mercaptoethanol (2-ME) can be used)
2. 1-isopropanol (IPA) (Alternatively Isopropa-2-nol or ethyl alcohol can be used)
PROCEDURE
1.

Suspend 5-50 mg samples in 1 ml of 40% (v/v) IPA containing 1% (w/v) DTT (75mM) in water by vortex mixing.

2.

Incubate the suspension at 50 °C for 60 minutes under continuous agitation.

3.

Centrifuge for 10 minutes at 20,800 x g and collect supernatant.

4.

Dilute the supernatant at 1:5 or higher with Solution C for subsequent analysis.

NOTE: The expected highest possible gliadin concentration after extraction is 500 µg/ml due to its lower solubility. Depending on the sample
types, use an appropriate sample amount (mg) to make the initial suspension. For more information about gliadin sample preparation, pleasecontact support@chondrex.com.

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.

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: The recommended standard range is 0.31 - 20 ng/ml. Dissolve one vial of Standard (20 ng/vial) in 1 mlof 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 20 ng/ml solution with an equal volume of Solution C to make a 10 ng/ml solution, and then repeat it
five more times for 5. 2.5, 1.25, 0.63, and 0.31 ng/ml standard solutions.

© 2022 Chondrex, Inc. All Rights Reserved, 6035 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

51000 ml250 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 ml20 ng/ml

10 ng/ml

5 ng/ml

2.5 ng/ml

1.25 ng/ml

0.63 ng/ml

0.31 ng/ml

Blank

1

2.

Prepare Sample Dilutions: Dilute samples at least 1:5 with Solution C depending on the estimated gliadin level in the samples. Twoor three different sample dilutions are recommended if the gliadin levels in the samples are unknown.

3.

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.

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 Gliadin Detection Antibody: Dilute one vial of Anti-Gliadin Detection Antibody in 10 ml Detection Antibody Dilution Buffer (SolutionE). Add 100 µl of detection antibody solution to each well and incubate at room temperature for 1 hour.
Strip #24681012Gliadin Detection (µl)
1733506682100

Solution E (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 #2468
1012Streptavidin Peroxidase (µl)
81725
33
4250

Solution D (ml)
1.7
3.3
5.0
6.6
8.2
10.0

© 2022 Chondrex, Inc. All Rights Reserved, 6035 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

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 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)
3466100132164200Chromogen 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.

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 where the standard range is 0.31 to 20 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 Gliadin Detection ELISA Kit
10.0

OD 450 nm

R² = 0.993

1.00.1

0.00.1110100Gliadin (ng/ml)© 2022 Chondrex, Inc. All Rights Reserved, 6035 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

VALIDATION DATA
Table 1 - Reproducibility Data for the Gliadin Detection ELISA Kit
Test
0.6 ng/ml
2.5 ng/ml
10 ng/ml
Intra-Assay CV (%)
1.3
3.2
1.1
Inter-Assay CV (%)
4.18.94.1
Spike Test* (%)
100%
101%
101%
*Known amounts of gliadin were added to samples and then diluted with Sample/Standard/Secondary Antibody Dilution Buffer toassay gliadin 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.

C. Osorio, N. Wen, R. Gemini, R. Zemetra, D. von Wettstein, S. Rustgi, Targeted modification of wheat grain protein to reduce thecontent of celiac causing epitopes. Funct. Integr. Genomics. 12, 417–438 (2012).

4.

J. R. Biesiekierski, What is gluten? J. Gastroenterol. Hepatol. 32 Suppl 1, 78–81 (2017).

5.

K. A. Bascuñán, M. C. Vespa, M. Araya, Celiac disease: understanding the gluten-free diet. Eur. J. Nutr. 56, 449–459 (2017).

6.

S. J. Shepherd, P. R. Gibson, Nutritional inadequacies of the gluten-free diet in both recently-diagnosed and long-term patients withcoeliac disease. J. Hum. Nutr. Diet. 26, 349–358 (2013).

7.

A. Løvik, G. Skodje, J. Bratlie, M. Brottveit, K. E. A. Lundin, Diet adherence and gluten exposure in coeliac disease and self-reported
non-coeliac gluten sensitivity. Clin. Nutr. 36, 275–280 (2017).

8.

B. Cabanillas, Gluten-related disorders: Celiac disease, wheat allergy, and nonceliac gluten sensitivity. Crit. Rev. Food Sci. Nutr. 60,2606–2621 (2020).

9.

G. Domínguez-Ortega, O. Borrelli, R. Meyer, R. Dziubak, C. De Koker, H. Godwin, C. Fleming, N. Thapar, M. Elawad, F. Kiparissi, A.
T. Fox, N. Shah, Extraintestinal Manifestations in Children With Gastrointestinal Food Allergy. J. Pediatr. Gastroenterol. Nutr. 59, 210(2014).

10. U. Volta, G. Caio, F. Tovoli, R. De Giorgio, Non-celiac gluten sensitivity: questions still to be answered despite increasing awareness.
Cell. Mol. Immunol. 10, 383–392 (2013).
11. P. Kanerva, T. Sontag-Strohm, O. Brinck, H. Salovaara, A Improved extraction of prolamins for gluten detection in processed foods.
Agricultural and Food Science. 20 (2011), doi:10.2137/145960611797471525.
12. H. C. van den Broeck, A. H. P. America, M. J. M. Smulders, D. Bosch, R. J. Hamer, L. J. W. J. Gilissen, I. M. van der Meer, A
modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheat. J. Chromatogr.
B Analyt. Technol. Biomed. Life Sci. 877, 975‚Äì982 (2009).

© 2022 Chondrex, Inc. All Rights Reserved, 6035 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