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The Gluten ELISA Assay was designed to quantitate low levels of gluten in food ingredients as well as in prepared and processed foods and beverages. The kit is provided with a standard curve based on the levels of gluten found in cooked breads made with varying levels of all-purpose wheat flour, but the kit can also detect rye and barley. However, when read on a wheat standard curve, equivalent concentrations of barley protein give results that are approximately 25% of wheat results. For this reason, a separate set of barley standards is available for accurately quantitating barley (Catalog No. 510821) in samples suspected to contain barley but not wheat, rye or other related grains.
The Gluten ELISA Assay uses a monoclonal antibody (401.21) which recognizes both the gliadin and glutenin fractions of gluten. This antibody is fixed to the wells of the 96-well plate, and binds to available gluten when samples or standards are added to the well. Any unbound material is removed in the first wash step. The Gluten Conjugate, which is a solution containing the same 401.21 antibody bound to the horse radish peroxidase (HRP) enzyme, is then added and allowed to bind any gluten present in the wells. The second wash step removes any unbound gluten conjugate. Finally, a color substrate (TMB) is added, which causes a blue color change in proportion to the amount of HRP present in the well. The Stop Solution stops the TMB reaction and changes the blue color to yellow, and the intensity of this yellow color is then read on a plate reader.
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