Anti-Human Tissue Transglutaminase (anti-tTG) IgA ELISA

$470.00

The Eagle Biosciences Anti-Human Tissue Transglutaminase (anti-tTG) ELISA Assay Kit is designed and validated for the quantitative determination of IgA autoantibodies against human tissue transglutaminase in human serum.  The Anti-Human Tissue Transglutaminase (anti-tTG) ELISA Assay Kit is for research use only and should not be used in diagnostic procedures.

SKU: HTG31-K01 Categories: , ,

Anti-Human Tissue Transglutaminase (anti-tTG) IgA ELISA

For Research Use Only

Size: 1×96 wells
Sensitivity: 3 U/ml
Dynamic Range: 10 – 300 IU/ml
Incubation Time: 2 hours
Sample Type: Serum
Sample Size: 10 µL

Additional Information

Assay Principle

The Anti-Human Tissue Transglutaminase (anti-tTG) ELISA Assay Kit is an enzyme immunoassay for the quantitative or semi-quantitative determination of IgA autoantibodies to tissue transglutaminase in human serum.

Autoantibodies of the diluted samples, positive control, and calibrators react with human tissue transglutaminase immobilized on the solid phase of a microtiter plate. The Anti-Human Tissue Transglutaminase (anti-tTG) ELISA Assay Kit guarantees the specific binding of anti-tTG IgA autoantibodies of the specimen under investigation by employing highly purified, activated recombinant human tTG for coating. Following 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 anti-human-IgA-antibodies conjugated to 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 100 µl calibrators 0 – 4  (quantitative) or 100 µl of calibrator 2 (semi-quantitative), 100 µl positive control, and 100 µl diluted patient samples into the respective wells.
  3. Seal plate, incubate 60 min at room temperature.
  4. Decant, then wash each well three times using 300 µl wash solution (made of B).
  5. Add 100 µl of conjugate (D) solution to each well.
  6. Seal plate, incubate 30 min at room temperature.
  7. Decant, then wash each well three times using 300 µl wash solution (made of B).
  8. Add 100 µl of substrate (E) to each well.
  9. Incubate 15 min protected from light at room temperature.
  10. Add 100 µl of stop solution (F) to each well and mix gently.
  11. Read the OD at 450 nm versus 620 or 690 nm within 30 min after adding the stop solution.

Manual

Product Manual


Publications

Citations

  • Schuppan D, Hahn EG: IgA anti-tissue transglutaminase: setting the stage for celiac disease screening. Eur J Gastroenterol Hepatol. 2001 13 635-7.
  • Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D: Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997 3 797-801.