Gluten testing

There are many tests available for detecting gluten. It is important to be aware of their intended uses. As will be clear from reading the descriptions of some of these tests, accurately testing for this protein is not easy.                                                        

ELISA TESTS

Omega-gliadin ELISA: a sandwich enzyme-linked immunosorbent assay based on monoclonal antibodies to the omega-gliadin fraction of wheat. This ELISA is intended to be used to assess the gluten content of heated and unheated food. The major drawback of this ELISA is that it greatly underestimates barley hordein content in barley-contaminated foods.

Standard R5 ELISA (R7001 Ridascreen Gliadin): a sandwich enzyme-linked immunosorbent assay based on the R5 monoclonal antibody to the potentially celiac toxic epitope (i.e., antibody-binding site) QQPFP (glutamine-glutamine-proline-phenylalanine-proline). This assay has been validated by the Prolamin Working Group of the Codex Alimentarius Commission. This ELISA is intended to be used to assess the gluten content of heated and unheated food. The major drawback of this ELISA is that it overestimates barley hordein content in barley contaminated foods.

Fast R5 ELISA (R7002 Ridascreen Fast Gliadin): a sandwich enzyme-linked immunosorbent assay based on the R5 monoclonal antibody to the potentially celiac toxic epitope (ie, antibody-binding site) QQPFP (glutamine-glutamine-proline-phenylalanine-proline). This assay is a less sensitive version of the standard sandwich R5 ELISA. This version was not validated by the Prolamin Working Group.

According to Thomas Grace, “The primary reason to use the 7001 is that it’s more sensitive. As with all methods, as you approach the LLOQ (Lower Limit of Quantitation) your margin of error proportionally goes up. Therefore as you shift your LLOQ closer to the 20ppm level you are likewise increasing your degree of uncertainty of 20ppm in your results.” 

Sandwich ELISAs can NOT accurately quantify gluten that has been highly hydrolyzed

Why? Sandwich ELISAs require two epitopes (i.e. antibody-binding sites on the gluten protein). When a protein has been hydrolyzed or broken apart into smaller protein fragments, it can lack the two necessary epitopes and, therefore, determination of gluten content might not be accurate. If a sandwich ELISA is used to assess the gluten content of a product containing hydrolyzed gluten, the reported gluten content is likely to be underestimated.

Competitive R5 ELISA (R7011 Gliadin Competitive): a competitive enzyme-linked immunosorbent assay based on the R5 monoclonal antibody to the potentially celiac toxic epitope (ie, antibody-binding site) QQPFP (glutamine-glutamine-proline-phenylalanine-proline). The competitive ELISA requires only one epitope to work. As a result, this ELISA is intended to be used on products containing hydrolyzed ingredients, such as malt, that have been broken down into smaller protein fragments. The major drawback of this ELISA is that results are expressed as gluten peptides versus gluten. Unfortunately, it is difficult to evaluate peptide concentration in terms of parts per million of gluten.

Lateral Flow

Quick Gliadin (R7003 RidaQuick Gliadin):  I am not as familiar with Lateral Flow tests so I asked Thomas Grace to explain them:

“Lateral Flow Devices (LFDs) are qualitative methods used to screen for specific proteins or compounds. Most employ sandwich type methodologies, that is, utilizing a line of fixed antibody on a surface strip and a second antibody conjugated with colored “nano” size particles (on the sample addition end of the strip). When a liquid sample extract is applied to the strip, the conjugate and the sample start to migrate across the surface of the strip together. If the sample extract has the protein or compound present and the conjugate can recognize its epitope they will bind together. As they come in contact with the line of antibodies that are fixed to the strip they will also bind to the protein forming a sandwich complex, “sandwiching” the protein between the two antibodies. As the conjugate starts to accumulate on the surface of the strip the “nano” particles start to become visible. The more antibody-protein-conjugate present the darker the line becomes.

Again, with many of these methods they require two epitope binding sites to be present on the single protein in order for the methods to detect that protein. If only one epitope (as may be the case with highly hydrolyzed foods and ingredients) is present than no sandwich complex can be formed and no test line will be visible and a false negative result will be reported.

These types of tests are only meant for detecting trace amounts of protein. When more than trace amounts are present, false negative results may occur.

Lateral flow tests can be great tools for helping manufacturers in defining their HACCP programs and determining if they are in compliance of their specific programs. Manufacturers can use them to test raw ingredients, to check surfaces to be sure they are cleaned between product runs and in some cases finished products.  In every case, they do require validation to show compatibility with the manufacturers matrixes, incurred samples and prove comparability with the gold standard methods.

Again they should never be used for finished product validation in themselves and only a fully validated method such as ELISA or PCR or HPLC should be used for validating finished products.

Note: When it comes to testing for gluten, regardless of whether a manufacturer uses wheat only in their facility, the method used should be able to detect low concentrations of barley and rye because of the high degree of cross contamination in the field, transportation, storage and processing.”

Thanks, Thom!!                                                                                                                

Important note: In my opinion, the standard sandwich R5 ELISA (Ridascreen 7001) remains the best available all-round assay for gluten determination. If the product being tested is highly hydrolyzed, the competitive R5 ELISA also should be used (Ridascreen R7011). All labeled gluten-free food should be routinely tested with one or both of these assays. State-of-the-art testing of finished gluten-free products is the most important step manufacturers should take to ensure their labeled gluten-free foods truly are gluten free.

For a more in-depth discussion of the sandwich omega-gliadin ELISA, sandwich R5 ELISA, and competitive R5 ELISA please see the article that I co-authored with the late Dr. Enrique Mendez (developer of the sandwich and competitive R5 ELISAs).  

Thompson T, Mendez E. Commercial Assays to Assess Gluten Content of Foods: Why They Are Not Created Equal. J Am Diet Assoc. 2008;108:1682-1687.

© 2010 by Tricia Thompson