Gluten-Free: How Can You Prove It?

Gluten Analysis

With regulations in place, appropriate detection methods for gluten in food are needed. Several technologies that give qualitative and quantitative results, such as specific antibody-based tests (e.g., enzyme-linked immunosorbent assays or lateral flow assays), polymerase chain reaction methods, and newer concepts like mass spectrometry are available—all with varying degrees of commercialization. An analytical test system should preferably be able to detect epitopes involved in celiac disease.

The fact that gluten is a complex mixture of proteins and occurs in a wide range of unprocessed as well as processed matrices creates a huge challenge in terms of correct quantification, making it difficult to find a suitable reference material. In 1985, the Working Group on Prolamin Analysis and Toxicity was founded in Europe. Its first task was to establish a recognized gluten standard for gliadin.

By extracting gliadins from a selection of the most common wheat varieties, the group managed to get a reference material. The IRMM [Institute for Reference Material and Measurements] initially accepted the group’s gliadin standard as a certified reference material, but later withdrew its acceptance because of discussions on the selection and the number of wheat species used to generate it, as well as purity issues. However, because it is still the only reference that has some acceptance, it has been widely used for calibration of test systems.

Enzyme-linked immunosorbent assays are the recommended method for the detection of gluten in food, and a large number of test kits are available commercially. Immunological methods apply antibodies that have been raised against different prolamin fractions or specific sequences that are harmful. Different test kits do not necessarily give similar results, for several reasons. These include different specificities of the polyclonal and monoclonal antibodies used, different extraction methods, and different materials used to calibrate the assays.

Numerous monoclonal and polyclonal antibodies have been developed for gluten testing, but only some are accepted on a broader basis. In the late 1980s, the Skerritt antibody was developed. This monoclonal antibody was raised against wheat gliadin from an Australian wheat variety and recognizes high molecular weight glutenin subunits and the heat stable subfraction called ω-gliadins, which makes the Skerritt antibody suitable for gluten analysis in processed foods. Even so, because the quantitation is based on the amount of ω-gliadins, which differ among cereal species, results can differ considerably. Moreover, the Skerritt antibody only has a weak response to hordein.

Another monoclonal antibody used for the detection of gluten is the R5 antibody, developed by Professor Enrique Mendez in Spain. The R5 antibody was raised against rye secalin, but showed strong cross reactivity to wheat gliadin. It also detects proteins from soy and lupin that are not harmful prolamins, however. The change in direction from detecting prolamins to detecting immunotoxic peptides that play a role in the pathogenesis of celiac disease led to the development of a next generation of antibodies. The G12 antibody employed in the AgraQuant Gluten G12 ELISA and AgraStrip Gluten G12 Lateral Flow Test belongs to this next generation.

Next-Generation Analysis

The G12 antibody specifically recognizes the 33-mer of the gliadin protein present in gluten. This toxic fragment was identified by the University of Stanford and published in 2002 in a paper in Science. The G12 antibody, which was raised against this 33-mer peptide using knowledge gained from this publication, recognizes the hexapeptide sequence QPQLPY and similar peptides found in barley, rye, and oats.