Samples are extracted based on matrix. Standards and samples are added to the wells, and present gliadin will bind to specific capture antibodies. The result is an antibody-antigen-complex. Components not bound by the antibodies are then removed in a washing step. Antibody conjugated to peroxidase is added and this antibody-conjugate is bound to the Ab-Ag complex. Any unbound enzyme conjugate is then removed in a washing step. Enzyme substrate and chromogen are added to the wells and incubated. Bound enzyme conjugate converts the colorless chromagen into a blue product. The addition of the stop reagent leads to a color change from blue to yellow. The measurement is made photometrically at 450 nm. The absorbance is proportional to the gliadin concentration of the sample. This result is then multiplied by 2 in order to obtain the gluten concentration (gliadin usually represents 50% of the proteins present in gluten).
Raw and processed foods
Matrices that are incapable of dissolving in an 80% alcohol solution.
Concentrated food additives, colors, and flavors may cause interference in this analysis.
Hydrolyzed and fermented proteins may not be detected using ELISA methods for allergen testing. Due to the breakdown of the proteins into small peptides or amino acids, they may become undetectable by this assay, but still could be allergenic and cause an allergic reaction.
Microwell Strip Reader
This method is intended for the quantitative analysis of prolamins (plant storage proteins having high content of the amino acid proline) originating from wheat (gliadin), rye (secalin), and barley (hordein).