Wheat cultivar and species influence variability of gluten ELISA analyses based on polyclonal and monoclonal antibodies R5 and G12

Reliable analytical methods are needed to ascertain that gluten-free products contain less than 20 mg/kg of gluten and are safe for celiac disease patients. Enzyme-linked immunosorbent assays (ELISAs) based on monoclonal (mAb) or polyclonal (pAb) antibodies are most commonly used, but the effects of environmental and genetic variability of gluten on the results are largely unknown. To gain more fundamental insights, the influence of various gluten compositions due to ten different cultivars of common wheat and two cultivars each of spelt, durum wheat, emmer and einkorn on ELISA responses was studied using the R5 and G12 mAbs and one pAb. Reversed-phase high-performance liquid chromatography served as an independent reference method to determine gliadin, glutenin and gluten concentrations. Analyses of flour blends spiked to defined gluten contents and concentration-absorbance curve arrays revealed that the pAb ELISA was less affected by variability of gluten than the R5 and G12 ELISAs. Clear differences in mAb responses to hexaploid, tetraploid and, especially, diploid wheat species were observed and the pAb ELISA was the only kit to detect gluten from einkorn. The findings highlight the importance of understanding the complexity of gluten as an analyte to improve reference materials and antibodies for gluten analysis.