Assessment of Prolamins from Different Oat Varieties Using R5-Based Sandwich ELISA

Introduction: Cereal grains contain a protein called gluten consisting of prolamins and glutelins. The prolamin fraction from wheat and related cereals possess sequences that provoke celiac disease (CD). The role of oat-derived prolamins, or avenins, in CD remains controversial. Consequently, many food regulatory entities exclude oats from their gluten classification scheme. In those jurisdictions, enforcement of gluten-free labeling laws requires the use of methods for gluten detection/quantification that selectively report prolamins from wheat, barley, and rye, but not from oats.  Currently, most such assay use the R5 monoclonal antibody (mAb).  However, it is not known whether R5-based assays or other assays perform uniformly with different oat cultivars.

Purpose:  To assess a panel of 19 certified oat cultivars for gluten content using an R5 mAb-based ELISA sandwich assay. 

Methods:  A panel of 19 oat cultivars were assessed for gluten content using a sandwich ELISA based on the R5 mAb.  This antibody binds to epitopes present in triticeae prolamins but absent from avenin from oats. Both native and denatured prolamins were tested. Nested analysis of 3 negative and 3 positive cultivars was performed using Western blot analysis with R5 mAb as well as a non-R5-based LFD assay obtained from Pi Bioscientific. The R5 mAb used in this study was provided under a license agreement from the Spanish National Research Council (CSIS).

Results: Reproducible prolamin detection was seen for only three of the 19 oat cultivars tested (designated 9, 11, and 14) using the R5 ELISA. The R5 positive oat cultivars and a subset of R5 negative oats were additionally tested using Western blotting with R5, as well as with a Skerrit-antibody based ELISA (ELISA SYSTEMS), and LFDs based on novel antibody reagents developed at IEH, all in sandwich format. 

Significance: The results obtained from testing select oat cultivars highlight features of existing gluten detection systems in relation to avenin detection which should be considered when performing gluten analysis in oats.