Correcting the Record: Gluten Free Watchdog’s Testing of Trader Joe’s Gluten-Free Almost Everything Bagels

Background

For those of you who have not been following the Moms Across America testing of gluten-free labeled products, and in particular Trader Joe’s Gluten-Free Almost Everything Bagels, here are links to the test results which have been moved to the public side of the website, along with a more concise summary:

Test results: https://www.glutenfreewatchdog.org/product/trader-joes-gf-almost-everything-bagels-2024/1348

Summary: https://www.glutenfreewatchdog.org/news/test-result-from-moms-across-america-on-trader-joes-gluten-free-almost-everything-bagels-possibly-false-positive/

Current situation

There has recently been a mischaracterization of Gluten Free Watchdog posted by another website covering this testing issue. The following statement was posted on celiac.com and attributed to John Fagan, Ph.D., Chief Science Officer, Health Research Institute. This is the lab that did the testing for Moms Across America.

“I was surprised at the Gluten Watchdog’s article. And the fact that the product contained no chocolate raises questions about their explanation. The obvious explanation for the discrepancy between the Gluten Watchdog’s results and ours is not even considered in their article, namely that they did not test the same sample that we tested. They didn’t ask for that sample but we told them that we would be quite happy to set up an opportunity for them to test that sample. I suspect they would have confirmed our result. Also, during our phone call with Watchdog, they did not suggest the modification of the method where extra protein is added.”

About this statement, Scott Adams of celiac.com writes:

“I received this reply from John Fagan, Ph.D. or Health Research Institute regarding the Romer G12 ELISA tests that were run by his lab for the MMA study, and it includes his response to the Gluten-Free Watchdog article. His reply makes me even more suspicious about the article Gluten-Free Watchdog, as he even offered to send them a sample from the same batch that they tested, which they refused (they tested a different batch to somehow try to prove that the tests done by Health Research Institute may have been done incorrectly).”

Bottom line

Gluten Free Watchdog sent two email requests to John Fagan asking to test retained samples. We did not receive a reply. Gluten Free Watchdog never received an offer from Health Research Institute to test any of their retained samples, including the bagels. During the testing done by Bia Diagnostics, and in consultation with Romer Labs (manufacturer of the G12 ELISA) and R-Biopharm (manufacturer of the R5 ELISA) we discovered that the bagel results differed depending upon whether extra protein was included in the testing process. Both Romer and R-Biopharm suggested ingredients in the bagels that might be causing non-specific binding that may have led to false positive results when testing the bagels with the G12 ELISA without extra protein. These suggested ingredients were tested at Bia Diagnostics. Testing brown rice flour and sorghum flour without added protein resulted in non-specific binding. Please read on for more information.

Correcting the Record

• Adrian Rogers*, formerly of Romer Labs (the manufacturer of the G12 ELISA used by HRI to test food for gluten) and I spoke with John Fagan on June 24^th. At the time Gluten Free Watchdog had not yet commissioned any testing on the products tested by Moms Across America.  
  • For those of you unfamiliar with Adrian, he is the Development Manager at BioCheckUK and is a specialist in immunoassay development with a focus on gluten and food allergen detection. He helped develop the AgraQuant gluten G12 ELISA from Romer Labs. (Information added 8/19)
• After our call, I sent an email to John Fagan copying Adrian Rogers asking to test their retained samples for the products that were certified gluten-free and had tested above 20 parts per million of gluten. It read:

“Hi John,

Thank you so much for speaking with us today. Would you consider sending some of your retained sample for 3 of the 4 products (not the bagels) that tested above 20 ppm to Bia Diagnostics for testing using the R5 ELISA? It would be good to know whether the issue we are seeing is due to sampling or antibody detection. 

Thank you.

Kind regards,

Tricia

• We did not receive a reply.
• In a Gluten Free Watchdog post to Facebook on June 27, the following statement was included:

“Going forward, it would be best to test retained sample from the HRI lab with both the R5 ELISA and the G12 ELISA. Short of that, it might be helpful to test new samples with both assays. To that end, Gluten Free Watchdog will be sending Simple Mills almond flour crackers, Simple Mills brownie mix, and Made Good double chocolate cookies to the lab for testing using both the R5 ELISA and the G12 ELISA.”

• The products mentioned above were tested by Bia Diagnostics on July 1^st.
• On July 2^nd, the following email was sent to John Fagan copying Adrian Rogers. Again, I asked to test retained samples:

“Hi John,

Hope this email finds you well.

I sent Bia Diagnostics 3 boxes of product certified gluten-free by GFCO that tested above 20 ppm of gluten based on your testing:

Simple Mills Almond Flour Crackers Fine Ground Sea Salt

Simple Mills Almond Flour Brownie Mix

Made Good Soft Baked Double Chocolate Mini Cookies

[Testing data redacted]

Would you be willing to send some retained sample from the above three products to Bia for testing using the R5 and G12 so we can try to figure out what might be going on?

Thank you.

Tricia”

• We did not receive a reply.
• After not receiving responses to my two requests to test retained samples, I did not send any additional emails to John Fagan.
• Gluten Free Watchdog never received an offer from Health Research Institute to test any of their retained samples, including the bagels.
• During our June 27^th conversation with John Fagan, we did not suggest any modification to the testing protocol. We had not yet done our own testing. We had not yet figured out that non-specific binding may be causing false positive results.
• Gluten Free Watchdog received consumer requests to test the Trader Joe’s Gluten-Free Almost Everything Bagels commissioned for testing by Moms Across America. Three bags of bagels with three different best by dates were tested by Bia Diagnostics on July 9 and July 10 using both the G12 ELISA and the R5 ELISA.
• Results are available at https://www.glutenfreewatchdog.org/product/trader-joes-gf-almost-everything-bagels-2024/1348. Please look at the results or the information below will not make sense.

Summary of the bagel testing

Gluten Free Watchdog commissioned two rounds of testing. In the first round, a milk additive was included when testing the bagels using the R5 ELISA. This was done because the instructions call for the addition of milk additive when testing tannin and polyphenol containing food samples. A milk additive was not included when testing the bagels using the G12 ELISA. According to Bia Diagnostics, the kit instructions for the G12 call for fish gelatin (extra protein) when testing chocolate containing samples. The bagels do not contain chocolate. In round two of testing, we decided to switch the protocol. We did not use milk additive when testing using the R5. We did use milk additive when testing using the G12. We then had to put on our thinking caps to figure out what might be causing the discrepancy in results between the R5 and G12 when following the kit instructions for both assays. This involved reaching out to both R-Biopharm (manufacturer of the R5 ELISA) and Romer Labs (manufacturer of the G12 ELISA) for their thoughts regarding non-specific binding when testing the bagels using the G12 and what ingredient(s) in the bagels might be causing it.

For those of you interested in the precise wording of the R5 and G12 kit instructions, screen shots have been included below (added 8/20).

For the Agraquant Gluten 12 ELISA:

For the Ridascreen Gliadin R5 ELISA:

Non Specific Binding

• What is non-specific binding? Enzyme-linked immunosorbent assays (ELISAs) like the R5 and G12 are based on what is called an antigen-antibody reaction. If gluten is present in the food samples that are being tested, it will bind to the antibodies in the R5 and G12. Sometimes what is called non-specific binding occurs—meaning something other than gluten binds to the antibodies. This may cause false positive results. To help prevent non-specific binding, extra protein in the form of milk protein or fish gelatin may be included in the testing process.
• Based on the advice of Romer Labs and R-Biopharm, labeled gluten-free poppyseeds, quinoa, sorghum flour, and brown rice flour (all ingredients in the bagels) were sent to the lab for testing using the G12 ELISA. Samples were tested in duplicate with and without a milk additive. The poppyseeds and quinoa tested below the lower limit of quantification (< 4 parts per million of gluten) with and without a milk additive. The brown rice flour and sorghum flour tested < 4 ppm with milk additive BUT tested with quantifiable gluten when milk additive was not used. Based on these findings, it appears that there may be non-specific binding when brown rice flour and sorghum flour are tested with the G12 without extra protein.
• Bottom line: It appears that the brown rice flour and sorghum flour ingredients (and possibly others) may have caused non-specific binding when testing the bagels without added protein. This may have caused false positive results.

Folks this is how science works. If something doesn’t make sense, it must be investigated to try to figure out why. And that’s what we did.