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Microwells are precoated with recombinant human tissue transglutaminase (tTG) antigen, the antigen has been expressed in Baculovirus cells and the expression construct used a cDNA coding for the long spliced isoform of human tTG.
Calibrators, controls, and diluted patient samples are added to the wells and autoantibodies recognizing the tTG antigen bind during the first incubation. After washing the wells to remove all unbound proteins, purified peroxidase-labeled rabbit antihuman IgA (alpha chain specific) conjugate is added. The conjugate binds to the captured human autoantibody and the excess unbound conjugate is removed by a further wash step.
Bound conjugate is visualized with 3,3'5,5' tetramethylbenzidine substrate, which gives a blue reaction product, the intensity of which is proportional to the concentration of the autoantibody in the sample. Phosphoric acid is added to each well to stop the reaction. This produces a yellow end point color, which is read at 450 nm. Testing is performed on the Agility instrument by Dynex.(Package insert: QUANTA Lite R h-tTG IgA. Inova Diagnostics, Inc. San Diego, CA, 92131)
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