What the FDA has to say about testing for gluten

This post is part of a series covering the FDA’s Final Rule on Gluten-Free Labeling of Fermented or Hydrolyzed Foods. 

The rule is available at

https://www.federalregister.gov/documents/2020/08/13/2020-17088/food-labeling-gluten-free-labeling-of-fermented-or-hydrolyzed-foods

Bottom line

Currently (and according to FDA), it is next to impossible to accurately test food for gluten when it has been partially broken down into smaller protein fragments during certain processing procedures, such as fermentation and hydrolysis.

Examples of foods & ingredients that may contain gluten that has been partially broken down

• Soy sauce (hydrolyzed wheat protein)
• Malt vinegar (barley malt)
• Malt and malt ingredients (barley malt)
• Products containing microbes (bacteria, yeast) developed on fermentation growth media that contain a gluten source
• Products containing enzymes (harvested from microbes grown on fermentation growth media containing a gluten source)
• Beer (barley malt)

What the FDA says about assays used to assess gluten levels of fermented & hydrolyzed foods

• FDA states that they know of “no scientifically valid analytical method effective in detecting and quantifying with precision the gluten protein content in fermented or hydrolyzed foods in terms of equivalent amounts of intact gluten proteins.”
  • This includes the competitive R5 ELISA.
• According to FDA, the R5 Competitive ELISA method is not suitable for the detection and quantification of gluten in any fermented or hydrolyzed food.
• FDA stresses that the inability to detect any gluten using the R5 Competitive ELISA is not an indication of complete elimination or even a reduction of gluten.
  • The R5 antibody does not recognize all immunogenic amino acid sequences.
    • Gluten could be present in a form that is not detectable by the competitive R5 ELISA.

Note: At Gluten Free Watchdog, we test foods that could possibly contain fermented or hydrolyzed gluten with the competitive R5 ELISA. If gluten is detected, this is cause for concern. If gluten fragments are not detected, it is not possible to know if this result is a false negative.

Brief primer on testing

• An enzyme-linked immunosorbent assay (ELISA) is commonly used to assess food for gluten.
  • This assay involves an antigen-antibody reaction.
  • Antibodies in the assay will react with antigens (e.g., gluten) in the food sample.
• Two types of ELISAs are currently used.
  • Sandwich ELISAs require two epitopes or antibody binding sites on a continuous sequence of amino acids.
  • Competitive ELISAs require only one epitope or antibody binding site on a continuous sequence of amino acids.
• The sandwich R5 ELISA is used worldwide to test food for gluten.
  • Based on the R5 antibody
  • Recognizes the 5 amino acid sequence QQPFP* found repeatedly in gluten protein (*glutamine-glutamine-proline-phenylalanine-proline)
  • Used to detect and quantify intact gluten proteins from wheat, barley, and rye
  • Codex Alimentarius Type 1 Method
• The competitive R5 ELISA is a modification of the sandwich R5 ELISA.
  • Used to test food for the presence of hydrolyzed/fermented gluten
    • Gluten that has been broken down into smaller gluten peptide fragments
  • PROBLEM: The competitive R5 ELISA is not a universally approved method of analysis by all regulatory agencies, including FDA due to all of the reasons stated previously.

Question: Why can’t the sandwich R5 ELISA be used to accurately assess food for the presence of hydrolyzed gluten?

Answer: Hydrolysis may result in gluten peptide fragments that do not contain the two QQPFP epitopes necessary for the sandwich R5 ELISA to work.

• Example: A gluten protein containing the repeating epitope QQPFP with “a” representing other amino acids might appear as:
  • QQPFPaaaaaaaQQPFPaaaaaaaaaaaaQQPFPaaaaaaaQQPFPaaaaaaaQQPFPaaaaaaaQQPFP
  • If this protein is hydrolyzed it may break apart into protein fragments:
    • Fragment 1: QQPFPaaaaaaaQQPFPaa
      • Two epitopes are present so the sandwich R5 ELISA CAN detect
    • Fragment 2: aaaaaaaaaaQQPFPaa
      • One epitope is present so the sandwich R5 ELISA can NOT detect
    • Fragment 3: aaaaaQQPFPaaaa
      • One epitope is present so the sandwich R5 ELISA can NOT detect
    • Fragment 4: aaaQQPFPaaaaaaaQQPFP
      • Two epitopes are present so the sandwich R5 ELISA CAN detect

Second bottom line

If a soy sauce containing hydrolyzed wheat protein tests negative for gluten, this doesn’t mean the product is free of gluten protein.

If a malt vinegar tests negative for gluten, this doesn’t mean the product is free of gluten protein.

If a malt beverage (e.g., beer) tests negative for gluten, this doesn’t mean the product is free of gluten protein.

All the test results may mean, is that the assay used to test the soy sauce, malt vinegar, or beer isn’t capable of detecting the gluten protein fragments present in the product.

If you have any questions, please leave a comment. Your comments will help inform updates to this post. Thank you.