Label‐Free Proteomic Analysis of Wheat Gluten Proteins and Their Immunoreactivity to ELISA Antibodies

Abstract

Cereal Chem. 94(5):820-826Enzyme-linked immunosorbent assay (ELISA) methods are currently the most widely used for gluten quantification. However, the lack of comparable measurements among commercial kits has caused much concern. Here, we studied the immunoreactivity of five commercial ELISA kits to wheat gluten fractionated by reversed-phase high-performance liquid chromatography and identified the proteins and peptides in the resulting fractions by mass spectrometry to understand the extent to which these may be contributing to the lack of comparability. The investigated monoclonal antibodies clearly demonstrated divergent responses to the fractioned wheat gluten proteins and sometimes to their initial intended targets. To make comparable gluten measurements a reality, the analytical measurement community requires a set of agreed peptide markers, known conversion factors from these markers to total gluten content, and appropriately characterized (certified) reference materials representative of gluten. † Corresponding

Evaluation of the discriminatory potential of antibodies created from synthetic peptides derived from wheat, barley, rye and oat gluten

“…Besides, the standard used to calibrate the immunoassay may also impact the results. Most authors agree on the need for better test calibration and better standards [ 11 – 19 ]. However, the use of the most appropriate reference material is still extensively debated [ 11 – 19 ].…”

“…Most authors agree on the need for better test calibration and better standards [ 11 – 19 ]. However, the use of the most appropriate reference material is still extensively debated [ 11 – 19 ]. The use of a reference material for each grain, namely wheat, barley, rye and oats, as a calibration standard makes it possible to correct or at least reduce this variability [ 9 , 11 , 20 ].…”

Mapping Coeliac Toxic Motifs in the Prolamin Seed Storage Proteins of Barley, Rye, and Oats Using a Curated Sequence Database

“…The high sequence homology between prolamins in cereal species can cause partial reactivity of the antibodies to wheat, barley, rye and oats, and the potential reactivity with contaminating wild grass species. Moreover, incomplete extraction of proteins and the use of incorrect conversion factors can further compound these issues (30)(31)(32)(33).…”

Modern Approaches in the Identification and Quantification of Immunogenic Peptides in Cereals by LC-MS/MS

“…As a consequence, the quantification can be compromised since the result is converted to gluten amount by multiplying the prolamin content by two, assuming the prolamin/glutelin ratio to be constant (Thompson and Méndez, 2008; Wieser and Koehler, 2009). ELISA methods currently cannot distinguish between the different gluten-containing cereals and are affected by the cross-reactivity of antibodies (Wieser and Koehler, 2009; Diaz-Amigo and Popping, 2013; Martínez-Esteso et al, 2017).…”

“…In this context, proteomic approaches appear to be more sensitive and reliable techniques than the currently used assays to identify gluten proteins, which present high amino acid sequence similarity and are difficult to distinguish. Especially when applying modern in tandem tools, proteomics can undoubtedly provide additional information to ELISA results, such as the confirmation of specific proteins by unraveling the peptide sequences (Martínez-Esteso et al, 2017).…”