Anti-Gliadin IgG ELISA Kit

$340.00

The Eagle Biosciences Gliadin IgG ELISA Assay Kit is used for the quantitative determination of IgG antibodies against deamidated gliadin in human serum. The Gliadin IgG ELISA Assay Kit is for research use only and should not be used in diagnostic procedures.

SKU: GDG31-K01 Categories: , ,

Anti-Gliadin IgG ELISA Kit

For Research Use Only

Size: 1×96 wells
Dynamic Range: 10-300 U/mL
Incubation Time: 2 hours
Sample Type: Serum
Sample Size: 10 µL

Additional Information

Assay Principle

The Gliadin IgG ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of IgG antibodies to deamidated gliadin.   The antibodies of the calibrators, positive control, and diluted samples react with fragments of deamidated gliadin immobilized on the solid phase of microtiter plates. After an incubation period of 60 min at room temperature (18-25°C), unbound serum components are removed by a wash step.

The bound autoantibodies react specifically with the anti-human-IgG-antibodies conjugated to the horseradish peroxidase (HRP) within the incubation period of 30 min at room temperature. Excessive conjugate is separated from the solid-phase immune complexes by the following wash step.  HRP converts the colorless substrate solution of 3,3’,5,5’-tetramethyl­benzidine (TMB) added into a blue product. This enzyme reaction is stopped by dispensing an acidic solution (H2SO4) into the wells after 15 min at room temperature turning the solution from blue to yellow.

The optical density (OD) of the solution at 450 nm is directly proportional to the amount of specific antibodies bound. The standard curve is established by plotting the concentrations of the antibodies of the calibrators (x-axis) and their corresponding OD values (y-axis) measured. The concentration of antibodies of the specimen is directly read off the standard curve. Evaluating the test by a semi-quantitative method is also possible.

Assay Procedure

  1. Bring all reagents to room temperature (18-25°C) before use. Mix gently, avoid foam.
  2. Dispense into the respective wells: 100 µl calibrators 0 – 4  (quantitative) or 100 µl of calibrator 1 (semi-quantitative),100 µl positive control, 100 µl diluted samples
  3. Seal plate, incubate 60 min at room temperature.
    Decant, then wash each well three times using 300 µl wash solution (made of B).
  4. Add 100 µl of conjugate (D) solution to each well.
  5. Seal plate, incubate 30 min at room temperature.
  6. Decant, then wash each well three times using 300 µl wash solution (made of B).
  7. Add 100 µl of substrate (E) to each well.
  8. Incubate 15 min protected from light at room temperature.
  9. Add 100 µl of stop solution (F) to each well and mix gently.
  10. Read the OD at 450 nm versus 620 or 690 nm within 30 min after adding the stop solution.

Manual

Product Manual


Publications

References

    • Lerner A, Kumar V, Iancu TC: Immunological diagnosis of childhood celiac disease: comparison between antigliadin, antireticulin and antiendomysial antibodies. Clin Exp Immunol 1994; 95:78-82
    • Schuppan D: Current concepts of celiac disease pathogenesis. Gastroenterology. 2000;119: 234-42.
    • Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D: Identification of tissue transglutaminase as the autoantigen of celiac disease. Nature Med 1977, 3:797-80

Recent Citations

  • Biggs, CM;Kostjukovits, S;Dobbs, K;Laakso, S;Klemetti, P;Valta, H;Taskinen, M;Mäkitie, O;Notarangelo, LD;, (2017). Diverse Autoantibody Reactivity in Cartilage-Hair Hypoplasia. J. Clin. Immunol.