Recognition of Gliadin and Glutenin Fractions in Four Commercial Gluten Assays

Abstract

Gluten sensitivity affects nearly 1 of the population in the United States and Europe. To help these consumers avoid the health issues that result from gluten consumption, the U.S. Food and Drug Administration is attempting to establish a definition and testing protocol for gluten-free foods. Establishing this protocol depends on accurate tests that can detect and quantitate gluten. There are multiple immunoassays available for the quantitation of gluten, and most are based on one of two antibodies. These antibodies, known as the Skerritt and R5 antibodies, were examined through the use of four commercial test kits for their ability to detect the two main components of gluten, known as gliadin and glutenin, in wheat. Commercial tests based on the Skerritt and R5 antibodies demonstrated differing affinities for gliadin and glutenin, with the Skerritt-based tests recognizing glutenins more strongly, and the R5 tests recognizing gliadins more strongly. Analysis of 40 processed food samples of unknown gluten content revealed differences in gluten detection and quantitation between the Skerritt-based and R5-based assays. These discrepancies in test results may be the result of the antibody affinity differences between the Skerritt- and R5-based tests, the solubility differences between gliadins and glutenins, or a combination of these and other factors.

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

“…We believe this is valuable information to aid the understanding of mAb specificities and the selection of a set of appropriate gluten markers. The results obtained also suggest, in accordance with previous research (Allred and Ritter 2010;van Eckert et al 2010;Sharma 2012), that assay antibodies could interact with glutenin proteins, mainly HMW-GS. That certain antibodies also react with the glutelin fraction has already been shown by Rallabhandi et al (2015) and Lexhaller et al (2016).…”

“…Sharma (2012) reported that the 401.21 mAb could be reacting to LMW-GS because these proteins could be derived from modified components of the w-gliadin fraction. However, our experiments and previous reports strongly suggest it is more likely that the 401.21 mAb reacts to HMW-GS fractions (Allred and Ritter 2010;van Eckert et al 2010) as well as the w-gliadin proteins (Skerritt and Hill 1990) but not LMW-GS.…”

Analyzing Gluten Content in Various Food Products Using Different Types of ELISA Test Kits

“…However, ELISA is very expensive, and the reproducibility of the results varies depending on the ELISA test kit type. Differences have been noted in the affinity for gliadin and glutenin of the R5 and the 401/21 antibodies across test kits [ 27 ]. Differences have also been observed between values obtained using R5 sandwich and competitive methods for gluten-containing cereals [ 22 ].…”

Detection and Quantitation of Gluten in Fermented-Hydrolyzed Foods by Antibody-Based Methods: Challenges, Progress, and a Potential Path Forward

“…A polyclonal antibody against gluten proteins is also available from the Morinaga Institutes of Biological Sciences, Inc., (MIoBS). The Skerritt antibody was raised against wheat gliadin and has been shown to recognize the HMW glutenins (16–18). The R5 antibody was raised against rye secalin and strongly binds to the QQPFP, QQQFP, LQPFP, and QLPFP epitopes in α-/β-, ω-, and γ-gliadins (19, 20).…”