Anti-Gliadin IgA Antibody ELISA Kit

Details for Product No. ABIN1305147, Supplier: Log in to see
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Detection Method
Method Type
Competition ELISA
Detection Range
1-200 U/mL
Minimum Detection Limit
1 U/mL
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Supplier Product No.
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Purpose This microplate based EIA (enzyme immunoassay) kit is intended for the quantitative determination of human anti-gliadin IgA level in serum. The assay is a useful tool in the aid of diagnosis of celiac disease.
Brand ED™
Sample Type Serum
Analytical Method Quantitative
Detection Method Colorimetric
Specificity The microplates are coated with highly purified alpha gliadin. No cross-reactivity to other autoantibodies has been observed.
Components 1. Gliadin Coated Microplate Two bottles each contain 30 mL phosphate buffer with protein stabilizers and preservative. The reagent is ready to use. This reagent should be stored at 2-8 °C and is stable until the expiration date on the kit box.
Material not included 1. Precision single channel pipettes capable of delivering 10 µL, 50 µL, 100 µL, and 1000 µL, etc.
2. Repeating dispenser suitable for delivering 100 µL.
3. Disposable pipette tips suitable for above volume dispensing.
4. Disposable 12 x 75 mm or 13 x 100 glass tubes.
5. Disposable plastic 1000 mL bottle with caps.
6. Aluminum foil.
7. Deionized or distilled water.
8. Plastic microtiter well cover or polyethylene film.
9. ELISA multichannel wash bottle or automatic (semi-automatic) washing system.
10. Spectrophotometric microplate reader capable of reading absorbance at 450 nm.
Target Type Antibody
Background Celiac disease or gluten-sensitive enteropathy is characterized by atrophy of the small intestinal villi leading to a so-called flat mucosa occurring in both adults and children. It is caused by a pathological intolerance to gliadin, resulting in inflammation and atrophy of the mucosa of the small intestine. Clinical manifestations include malabsorption with symptoms of diarrhea, steatorrhea and nutritional and vitamin deficiencies. Secondary immunologic illnesses, such as atopic dermatitis, dermatitis herpetiformis, alopecia and aphthous ulcers may be the primary presentation. As celiac disease is caused by the uptake of gluten, consequently a gluten-free diet cures the disease completely and thus has to be maintained for lifetime. Renewed consumption of gliadin leads to a recurrence of the disease symptoms. The disease is HLA-associated (>95 % of patients have DQ2 encoded by DQA1*0501 and DQB1*0201) and manifests at any age. A high incidence range up to 1:300 was found in European countries and approximately 1:250 in the United States. Clinical diagnosis of celiac disease is made by small intestinal biopsy and supported by serological markers. Human antibodies against gliadin and tissue Transglutaminase (tTG) are major serological markers. Circulating IgG and IgA antibodies to gliadin are found in the serum of most but not all celiac disease patients. Both IgG and IgA antibodies are detected in sera of patients with gluten-sensitive enteropathy. It was reported that IgA antibodies are less sensitive but more specific markers of the disease and their measurement is useful in following disease activity and monitoring maintenance of a gluten-free diet. IgG antibodies appear to be more sensitive but less specific markers of disease than IgA. It is recommended that both antibodies should be measured due to the high incidence of IgA deficiency among celiac patients, which may mask the disease. Antibody testing is also important in detecting individuals who are at risk for having celiac disease but have no symptoms, in individuals with atypical symptoms or extra-intestinal manifestations of celiac disease and in individuals with presumed celiac disease who fail to respond to a gluten-free diet. Patients with positive antibody tests must undergo small intestine biopsy to confirm the diagnosis and assess the degree of mucosal involvement. Antibodies to gliadin may be the only serological marker in neonates, as anti-tTG and EMA auto-antibodies are not present at this age. Consequently anti-gliadin antibodies are the earliest serological marker for pediatricians when diagnosing celiac disease.
Sample Volume 10 μL
Assay Time 4 h
Plate Pre-coated
Protocol This EIA is designed, developed and produced for the quantitative measurement of human anti-gliadin IgA level in test sample. The assay utilizes the microplate-based enzyme immunoassay technique by coating highly purified gliadin antigen onto the wall of microtiter well. Assay calibrators, controls and human serum samples containing anti-gliadin IgA are added to microtiter wells of a microplate that was coated with a highly purified gliadin antigen on its wall. After the first incubation period, the unbound protein matrix is removed in the subsequent washing step. A horseradish peroxidase-conjugated rabbit anti-human IgA subclass specific antibody (tracer antibody) is added to each well. After an incubation period an immunocomplex of gliadin human anti-gliadin IgA HRP-conjugated tracer antibody is formed if there is human anti-gliadin antibody present in the test sample. The unbound tracer antibody is removed in the subsequent washing step. HRP-conjugated tracer antibody bound to the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the tracer antibody bound to the human IgA on the wall of the microtiter well is directly proportional to the amount of human anti-gliadin antibody level in the sample. A calibrator curve is generated by plotting the absorbance versus the respective human anti-gliadin antibody concentration for each calibrator on point-to-point or 4-parameter fit. The concentration of human anti-gliadin antibody in test samples is determined directly from this calibrator curve.
Reagent Preparation

(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details

Sample Collection Only 10 µL of human serum is required for gliadin IgA measurement in duplicate. No special preparation of individual is necessary prior to specimen collection. Whole blood should be collected and must be allowed to clot for minimum 30 minutes at room temperature before the serum is separated by centrifugation (850 ? 1500xg for 10 minutes). The serum should be separated from the clot within three hours of blood collection and transferred to a clean test tube. Serum samples should be stored at 2 ? 8 C up to 48 hours and at -20 °C or below for long term storage until measurement.
Sample Preparation

(1) Label a test tube (12x75 mm).
(2) Add 1 mL of assay buffer to each tube. Pipet 10 μL of patient serum sample to the tube.

Assay Procedure

(1) Place a sufficient number of gliadin coated microwell strips in a holder to run gliadin IgA calibrators , controls , and unknown samples in duplicate.
(2) Test Configuration
(3) Add 100 µL of calibrators, controls and diluted patient serum samples into the designated microwell.
(4) Cover the plate with one plate sealer.
(5) Incubate plate at room temperature for 1 hour.
(6) Prepare working anti-hIgA Tracer Antibody Working Solution by 1:21 fold dilution of the tracer antibody with the Tracer Antibody Diluent . For each strip, it is required to mix 1 mL of Tracer Antibody Diluent with 50 µL of Gliadin IgA Tracer Antibody in a clean test tube.
(7) Remove the plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL to 400 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(8) Add 100 µL of above diluted tracer antibody working solution to each of the wells.
(9) Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
(10) Incubate plate at room temperature for 30 minutes.
(11) Remove the plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL to 400 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(12) Add 100 µL of ELISA HRP Substrate into each of the wells. (13) Cover the plate with a new plate sealer and also with aluminum foil to avoid exposure to light. (14) Incubate plate at room temperature for 20 minutes (15) Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently. (16) Read the absorbance at 450 nm within 10 minutes in a microplate reader.

Calculation of Results
  1. Calculate the average absorbance for each pair of duplicate test results.
    2. Subtract the average absorbance of the calibrator 1 (0 U/mL) from the average absorbance of all other readings to obtain corrected absorbance.
    3. The calibrator curve is generated by the corrected absorbance of all calibrator levels on the ordinate against the calibrator concentration on the abscissa using point-to-point or log-log paper. Appropriate computer assisted data reduction programs may also be used for the calculation of results. The gliadin IgA concentrations for the controls and samples are read directly from the calibrator curve using their respective corrected absorbance. If log-log graph paper or computer assisted data reduction program utilizing logarithmic transformation are used, sample having corrected absorbance between the 10 U/mL calibrator and the next highest calibrator should be calculated by the formula: Corrected absorbance (unknown) Value of unknown = x Value of the 2nd STD Corrected Absorbance (2nd STD)
Assay Precision The intra-assay precision is validated by measuring two samples in a single assay with 20 replicate determinations. The inter-assay precision is validated by measuring two samples in duplicate in 12 individual assays
Restrictions For Research Use only
Precaution of Use The reagents must be used in research laboratory and is for research use only. The source material for reagents containing bovine serum was derived in the contiguous 48 United States. It was obtained only from healthy donor animals maintained under veterinary supervision and found free of contagious diseases. Wear gloves while performing this assay and handle these reagents as if they are potentially infectious. Avoid contact with reagents containing TMB, hydrogen peroxide, or sulfuric acid. TMB may cause irritation to skin and mucous membranes and cause an allergic skin reaction. TMB is a suspected carcinogen. Sulfuric acid may cause severe irritation on contact with skin. Do not get in eyes, on skin, or on clothing. Do not ingest or inhale fumes. On contact, flush with copious amounts of water for at least 15 minutes. Use Good Laboratory Practices.
Storage 4 °C
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