X6: A Novel Antibody for Potential Use in Gluten Quantification
Authors: Shatalova, Aleksandrina; Shatalov, Ivan; Lebedin, Yuri
Gliadin is a fraction of gluten, known to trigger celiac disease in susceptible people. To date, the life-long gluten-free diet is used for the prevention of this disease. Hence, methods for gluten control in foods are of significant importance. Being one of the most-used methods used for this purpose, ELISA should use high-affinity antibodies to gliadin peptides involved into celiac process. This study investigates the characteristics of a novel anti-gliadin antibody X6. We found the QXQPFPXP site to be a recognized epitope that provides specific binding of the antibody to cereal prolamins involved in celiac disease manifestation. A specificity study using immunoblotting shows the recognition of wheat, barley and rye proteins-as well as alpha-gliadin homologs from non-edible cereals (Dasypyrum villosum). Reactivity to avenin was less pronounced, as this protein does not contain the PFP motif most critical for antibody recognition. The proteins ofZea maysandSetaria italicawere not recognized by X6. X6-based ELISA highly correlated with R5 and G12, which are Codex Alimentarius standards in the quantitative assessment of gluten content (Pearson's R = 0.86 and 0.87, respectively). Qualitative assessment revealed no significant differences between R5 and G12 and X6.
Characterization of Celiac Disease-Related Epitopes and Gluten Fractions, and Identification of Associated Loci in Durum Wheat
Authors: Taranto, Francesca; D'Agostino, Nunzio; Catellani, Marcello; Laviano, Luca; Ronga, Domenico; Milc, Justyna; Prandi, Barbara; Boukid, Fatma; Sforza, Stefano; Graziano, Sara; Gulli, Mariolina; Visioli, Giovanna; Marmiroli, Nelson; Badeck, Franz-W.; Minervini, Anna Paola; Pecorella, Ivano; Pecchioni, Nicola; De Vita, Pasquale; Francia, Enrico
While durum wheat is a major food source in Mediterranean countries, storage (i.e., gluten) proteins are however responsible for celiac disease (CD), a serious autoimmune disease that occurs in genetically predisposed subjects. Different gluten epitopes-defined as "immunogenic" (IP) and "toxic" (TP) peptides-are involved in the pathology and their content in wheat grain depends on environmental and genetic factors. Detection of IP and TP is not trivial, and no work has been conducted so far to identify the genomic regions associated with their accumulation in wheat. In the present study, a genome-wide association study was performed on a durum wheat collection to identify marker-trait associations (MTAs) between 5730 high quality SNPs and the accumulation of CD-related peptides and gluten protein composition measured in two consecutive cropping seasons (2015/2016 and 2016/2017). High-molecular-weight glutenin subunits (HMW-GS) were more stable between the two years, and differences in total gluten proteins were mainly due to low-molecular-weight glutenin subunits (LMW-GS) and accumulation of gliadins. In the first instance, association tests were conducted on yellow pigment content (YP), a highly inheritable trait with a well-known genetic basis, and several significant MTAs were found corresponding to loci already known for being related to YP. These findings showed that MTAs found for the rest of the measured traits were reliable. In total, 28 significant MTAs were found for gluten composition, while 14 were found to be associated with IP and TP. Noteworthy, neither significant (-log10p> 4.7) nor suggestive (-log10p> 3.3) MTAs for the accumulation of CD-triggering epitopes were found onGli-A1/Glu-A3andGli-B1/Glu-B3loci, thus suggesting regulatory rather than structural gene effect. A PBF transcription factor on chromosome 5B, known to be involved in the regulation of the expression of CD-related peptides, was identified among the positional candidate genes in the LD-decay range around significant SNPs. Results obtained in the present study provide useful insights and resources for the long-term objective of selecting low-toxic durum wheat varieties while maintaining satisfactory gluten quality.