Both the Food and Drug Administration (FDA)  and the Alcohol and Tobacco Tax and Trade Bureau (TTB) appear to be on the same page when it comes to assessing the gluten content of fermented or hydrolyzed foods. The FDA writes in its August 3, 2011 Federal Register Notice regarding gluten-free labeling that, “FDA recognizes that for some food matrices (e.g., fermented or hydrolyzed foods), there are not currently available validated methods that can be used to accurately determine if these foods contain < 20 ppm gluten.” The TTB writes in their interim policy on gluten content statements that they agree “with FDA that there are no scientifically valid methods for accurately measuring the gluten content of fermented products…”

So, why exactly is it so hard to measure the gluten content of hydrolyzed and fermented products? Here are five reasons…

  1. Hydrolyzed or fermented products (e.g., beer made using barley malt) contain gluten peptide fragments.
  2. Sandwich ELISAs, including the two that have been formally validated in multi-laboratory research studies (R5 Mendez ELISA and the Moringa Wheat Protein ELISA) can NOT accurately detect the gluten peptide fragments found in highly hydrolyzed or fermented products. Sandwich ELISAs require two epitopes or antibody binding sites. The R5 ELISA specifically requires the epitope QQPFP which stands for glutamine—glutamine—proline—phenylalanine—proline. When gluten protein is broken down into peptide fragments, two epitopes may not be available. For example, say you have the following protein where A represents any amino acid and QQPFP represents the antibody binding site:


If this protein undergoes hydrolysis, the following protein fragments may result:


Only fragment “a” would be measured by the sandwich R5 Mendez ELISA; fragments “b” and “c” would not.

  1. What is known as the competitive R5 ELISA is used to measure protein fragments where only one epitope is available BUT it has not yet been formally validated in a multi-laboratory research trial. According to R-biopharm (the manufacturer) a collaborative study is planned.
  2. Historically, the competitive R5 ELISA is not compatible with a cocktail extraction—the extraction of choice for heated foods. Prior to his passing I was exceedingly fortunate to co-author an article with the late Enrique Mendez, PhD, the developer of both the sandwich and competitive R5 ELISAs (Thompson T, Mendez E. Commercial assays to assess gluten content of gluten-free foods: Why they are not created equal. J Am Diet Assoc. 2008;108:1682-1687). We state in the paper, “One drawback of the competitive R5 ELISA is that currently it is only compatible with an ethanol extraction. Ethanol is capable of completely extracting prolamins from foods containing native proteins only. Once proteins have been heated and denatured, ethanol is no longer capable of extracting all prolamin fractions. Ideally, in heated foods, a cocktail extraction should be used. The competitive R5 ELISA along with an ethanol extraction cannot as accurately assess the gluten content of foods containing both heated and hydrolyzed gluten. The competitive R5 ELISA can reasonably assess the hydrolyzed gluten content in foods, while the hydrolyzed prolamin fragments, which have not been heated (or heated to a lesser extent), can be extracted with 60% ethanol.” NOTE: An article was recently published in the scientific literature regarding a new extraction solution for the competitive R5 ELISA. It is called UPEX and according to study authors may be used on foods that have been both hydrolyzed and heat treated. To the best of my knowledge this extraction solution is not commercially available at the present time.
  3. It may be difficult to relate the toxicity of gluten peptide fragments to intact gluten protein.

Here’s hoping that the competitive R5 ELISA is formally validated very soon AND a compatible cocktail extraction is developed (and commercially available) to go along with it.

© 2012 by Tricia Thompson, MS, RD. All rights reserved. This article may not be reprinted, reposted, or republished without the express written permission of Tricia Thompson

Beer: Why it is so hard to assess fermented and hydrolyzed products for gluten