Human Anti-Gliadin IgG ELISA Kit

Sample

Serum

Species Reactivity

Human

Detection Method

iELISA

Intended Use

This microplate based EIA (enzyme immunoassay) kit is intended for the quantitative determination of human anti-gliadin IgG level in serum.

Contents of Kit

Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
1. Gliadin Coated Microplate
One microplate with 12 x eight strips (96 wells total) coated with highly purified gliadin antigen. The plate is framed and sealed in a foil Ziploc bag with a desiccant. This reagent should be stored at 2 – 8°C and is stable until the expiration date on the kit box.
2. Anti-hIgG Tracer Antibody
One vial containing 0.6 mL concentrated horseradish peroxidase (HRP)-conjugated anti-human IgG tracer antibody in a stabilized protein matrix. This reagent must be diluted with Tracer Antibody Diluent before use. This reagent should be stored at 2 – 8°C and is stable until the expiration date on the kit box.
3. Tracer Antibody Diluent
One vial containing 12 mL ready-to-use buffer. It should be only used for antibody dilution according to the assay procedures. This reagent should be stored at 2 – 8°C and is stable until the expiration date on the kit box.
4. ELISA Wash Concentrate
One bottle contains 30 mL of 30 fold concentrate. Before use the contents must be diluted with 870 mL of distilled water and mixed well. Upon dilution this yields a working wash solution containing a surfactant in phosphate-buffered saline with a non-azidepreservative. The diluted wash solution should be stored at room temperature and is stable until the expiration date on the kit box.
5. ELISA HRP Substrate
One bottle contains 12 mL of tetramethylbenzidine (TMB) with hydrogen peroxide. This reagent should be stored at 2 – 8°C and is stable until the expiration date on the kit box.
6. ELISA Stop Solution
One bottle contains 12 mL of 0.5 M sulfuric acid. This reagent should be stored at 2 – 8°C or room temperature and is stable until the expiration date on the kit box.
7. Gliadin IgG Calibrators
Five vials each contain human anti-Gliadin IgG in a liquid bovine serum albumin-based matrix with a non-azide preservative. Refer to vials for exact concentration for each calibrator. After the first use, the calibrators should be stored at -20°C or below for long term storage.
8. Gliadin IgG Controls
Two vials each contain human anti-Gliadin IgG in a liquid bovine serum albumin-based matrix with a non-azide preservative. Refer to vials for exact concentration range for each control. After the first use, the calibrators should be stored at -20°C or below for long term storage.
9. Patient Sample Diluent
One bottle contains 60 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.

Storage

This test kit must be stored at 2 – 8°C upon receipt. For the expiration date of the kit refer to the label on the kit box. All components are stable until this expiration date.
Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.

Precision

Intra-assay precision:

Inter-assay precision:

Sensitivity

The sensitivity of this gliadin IgG EIA as determined by the 95% confidence limit on 20 duplicate determination of zero calibrator is about 1 U/mL.

General Description

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.

Standard Curve

A typical absorbance data and the resulting calibrator curve from human gliadin IgG ELISA are represented. This curve should not be used in lieu of calibrator curve run with each assay.