Anti-Gliadin IgG ELISA Kit

Catalog No. ABIN1305148

Product Details

Target: Anti-Gliadin IgG

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 1-200 U/mL

Minimum Detection Limit: 1 U/mL

Application: ELISA

Purpose: This microplate based EIA (enzyme immunoassay) kit is intended forthe quantitative determination of human anti-gliadin IgG level inserum. The assay is a useful tool in the aid of diagnosis of celiacdisease.

Brand: ED™

Sample Type: Serum

Analytical Method: Quantitative

Components: 1. Gliadin Coated Microplate
Two bottles each contains 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

Application Details

Sample Volume: 10 μL

Assay Time: 3 - 4 h

Plate: Pre-coated

Protocol: This EIA is designed, developed and produced for the quantitativemeasurement of human anti-gliadin IgG level in test sample. Theassay utilizes the microplate based enzyme immunoassay techniqueby coating highly purified gliadin antigen onto the wall of microtiterwell.Assay calibrators, controls and human serum samples containinganti-gliadin IgG are added to microtiter wells of a microplate that wascoated with a highly purified gliadin antigen on its wall. After the firstincubation period, the unbound protein matrix is removed in thesubsequent washing step. A horseradish peroxidase conjugatedrabbit anti-human IgG subclass specific antibody (tracer antibody) isadded to each well. After an incubation period an immunocomplex ofgliadin human anti-gliadin IgG HRP-conjugated tracer antibodyis formed if there is human anti-gliadin antibody present in the testsample. The unbound tracer antibody is removed in the subsequentwashing step. HRP-conjugated tracer antibody bound to the well isthen incubated with a substrate solution in a timed reaction and thenmeasured in a spectrophotometric microplate reader. The enzymaticactivity of the tracer antibody bound to the human IgG on the wall ofthe microtiter well is directly proportional to the amount of humananti-gliadin antibody level in the sample. A calibrator curve isgenerated by plotting the absorbance versus the respective humananti-gliadin antibody concentration for each calibrator on point-topointor 4-parameter fit. The concentration of human anti-gliadinantibody in test samples is determined directly from this calibratorcurve.

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 IgG 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:

Patient sample needs to be diluted 1:101 with assay buffer before being measured.
(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 IgG calibrators (30042-30046), controls (30047-30048), 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-hIgG 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 IgG 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.

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

Handling

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

Target Details

Target: Anti-Gliadin IgG

Target Type: Antibody