Investigation of the effects of sample preparation on gluten quantitation in rye and barley flours

Proper gluten quantitation is essential for providing safe gluten-free food for patients living with celiac disease (CD). However, gluten quantitation faces several challenges: the lack of a reference method and certified reference materials, the variability of methods and the effects of genetic and environmental factors on gluten. Among all these challenges our research group focuses on gluten reference material development. Gluten content is determined by enzyme linked immunosorbent assay (ELISA) methods to obtain comparable data for the selection of cultivars used in our reference material development efforts. As ELISA methods are developed for determining low gluten concentrations, application for these special research purposes requires a 10,000-fold dilution. The formerly performed process was a post-extraction liquid dilution that proved to be sufficient for wheat samples. However, gluten contents of rye and barley samples were found to be overestimated by ELISA methods. One of the suggested reasons is the structural and solubility changes of gluten proteins during the dilution process. Therefore, our present study focuses on the comparison of the original dilution method and a revised version using solid-phase dilution in a gluten-free matrix.