Lateral Flow Devices (EZ Gluten) and Gluten Analysis

Thank you to Thomas Grace of Bia Diagnostics, www.biadiagnostics.com for sharing his expertise on Lateral Flow Devices

What are lateral flow devices (LFDs)?  

LFDs are used to qualitatively or semi-quantitatively determine whether gluten is present in a food product. There are several LFDs on the market for testing food for gluten, including the Ridascreen Quick Gliadin manufactured by R-biopharm, EZ Gluten manufactured by Elisa Technologies, AgraStrip Gluten G12 Test Kit manufactured by Romer Labs, and Gluten Tox manufactured by Biomedal Diagnostics. Specifics on each of these tests are included at the end of the Q & A section.

How do LFDs work?

LFDs are usually what we think of as “dipstick” tests. Most employ sandwich type methodologies. They utilize 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 (gluten) and the conjugate can recognize its epitope (binding site), under the right conditions they will bind together. Now that they are “hooked” together as they come in contact with the line of antibodies that are fixed to the strip, these antibodies will also bind to the protein forming a sandwich complex, “sandwiching” the protein (gluten) between the two antibodies. As the conjugate complex starts to accumulate on the surface of the strip the “nano” particles start to become visible. The more protein (gluten) in the sample the more antibody-protein-conjugate binding will take place and the darker the line becomes.

It should be noted that unless the LFD has been shown to reliably detect pure wheat, barley or rye cultivars (e.g. undiluted wheat flour), it should have a hook line to avoid false negative results with high concentrations of gluten.  A hook line is simply a small amount of analyte protein on the surface of the strip that will capture any “free” antibody that has not been bound with the sample. A positive hook line shows that the strip is not over loaded and the assay is valid. The lack of any hook line alerts the user that the test is over loaded and the sample should be diluted further. 

Under what circumstances is it okay for manufacturers to use LFDs?

LFDs can be great tools for helping manufacturers in defining their Hazard Analysis Critical Control Point programs and determining if they are in compliance with their specific programs. Manufacturers can use LFDs to test raw ingredients, to check surfaces between product runs to be sure they are clean, and in some cases to test finished products. In every case, LFDs require validation to 1) demonstrate compatibility with the manufacturer’s product, 2) demonstrate the ability to detect known amounts of gluten in the product (spiked/recovery), and 3) prove similar results can be found with the R5 ELISA method which most people consider the “gold standard” for gluten testing.

How would a manufacturer go about validating an LFD?

Validation usually would consist of taking an ingredient, ingredient mix, and/or finished product negative control and running the test on the sample. If the test is negative, the manufacturer would then take the samples or sample mixes and add a 20 part per million gluten spike (a proportional mix of wheat gliadin, barley hordein, and rye secalin) into each sample (the gluten spike should be less than 10% of ingredient) and run the test again to be sure they get a positive result for each of the spiked samples.

If all samples are positive at the level expected, they would then take pure wheat flour and test it to be sure they don’t get false negative results with an over abundance of contaminant. Also, finished products should be periodically checked (every batch if produced on shared equipment or every few months if not) via third party labs using CODEX/AOACRI approved methods.

What are the limitations of using a LFD to test food for gluten?

Manufacturers utilizing LFDs must keep in mind that these tests:

  1. Are qualitative methods used to screen for specific proteins or compounds.
  2. Require two antibody binding sites to be present on the gluten protein or peptide in order for the methods to detect gluten. If only one antibody binding site is present (as may be the case with highly hydrolyzed foods and ingredients) than no sandwich complex can be formed and no test line will be visible and a false negative result will be reported.
  3. Are only meant for detecting trace amounts of protein. When more than trace amounts are present, false negative results may occur.
  4. Should never be used for finished product validation in and of themselves. Only a fully validated method such as ELISA or PCR or HPLC should be used for validating finished products.

Should consumers use LFDs to test products for gluten?

Consumers generally should not use these devices for home use because of the extreme variance in food products–each LFD should be validated for each specific product. Why? The burger they make at home will be different from the burger they get a McDonalds. The fat content will be different. The salt content will be different. The sauces will be different. All of these variables might make one burger compatible with the LFD and the other not. Something as “innocent” as a pinch of salt or a little vinegar can be all it takes for an LFD to produce a false negative or false positive result. For most consumers it is impractical or impossible to validate each product they would want to test before they actually test it.

Ridascreen Quick Gliadin: From the Manufacturer*

  • “RIDA® QUICK Gliadin can be used as a swab test for the gluten determination on surfaces in the hygiene control and for the qualitative detection of gliadin/gluten in raw material. The test should only be used for the detection of small amounts of gluten (contaminations). The gliadin/gluten determination can be carried out for numerous raw materials after a simple ethanol extraction. Further applications, e.g. for processed foods and chocolate, are available upon request.”
  • “The immunochromatographic test employs the monoclonal R5-antibody which is specific for the detection of gliadin from wheat and prolamins from rye and barley.”
  • “Results are read visually. Generally, the higher the analyte level in the sample, the stronger the red color of the test band will be.”

*From manufacturer website

EZ Gluten: From the Manufacturer*

  • “The EZ Gluten Test is an easy to use home test that will quickly detect the presence of gluten in foods. It is sensitive enough to detect gluten as low as 10 parts per million (ppm). This simple test is also small and portable enough for use in restaurants or when traveling. It can be used to test individual ingredients in foods, or to test finished and cooked products.”
  • “The EZ Gluten Test uses the anti-omega gliadin antibody developed by Skerritt and Hill for detection.”
  • “The test strip can be read visually for the presence of gluten in the sample.”

Tricia’s note: The EZ Gluten uses the omega-gliadin antibody that has very low cross-reactivity with barley (4 to 8 percent). If barley is a contaminant in the food product it will not be detected (or severely underestimated) using this test.

*From product package insert

AgraStrip Gluten G12 Test Kit: From the Manufacturer*

  • “The AgraStrip Gluten G12 Test Kit is a lateral flow immunochromatographic assay that determines a semi-quantitative level for the presence of gluten.”
  • “The Test Kit uses a new monoclonal antibody called G12 that specifically recognizes the pathogenic fragment of the gliadin protein present in gluten. This fragment is called 33-mer and triggers the auto-immune reaction in celiac patients.”
  • “The G12 antibody used in the test kit reacts with some oat varieties.”
  • Results can be read visually.

*From product package insert

Gluten Tox: From the Manufacturer*

  • “GlutenTox Home is a rapid and user-friendly test for the detection of gluten in food and drinks which is harmful for people who suffer from celiac disease.”
  • “GlutenTox Home contains a new antibody called G12. It was developed to specifically recognize the toxic fraction of gluten present in wheat, barley, rye, and oat that triggers the auto-immune response in celiac patients.”

*From manufacturer website

Tricia’s note: In the US oats not contaminated with wheat, barley, or rye are allowed in gluten-free diets. However, controversy over oats still exists.

Dietitians attending FNCE 2011: Please make plans to join us at the pre-FNCE workshop, “Celiac Disease Toolkit: Guiding Your Patients through the Gluten-Free Diet” on Saturday, September 24 from 9 am to 3:30 pm. Thomas Grace of Bia Diagnostics will be in attendance and will be discussing testing methods for gluten, including lateral flow devices.

Reminder: I have started a new website Gluten Free Watchdog (www.glutenfreewatchdog.org) to test products for gluten. Products from Arrowhead Mills, Authentic Foods, Bob’s Red Mill, General Mills, Holly’s Oatmeal, Mary’s Gone Crackers, Namaste Foods, Orgran, and Pamela’s have been tested. Products to come include those made by Lundberg Farms, Think Thin, Twin Valley Mills, and Novartis (Benefiber). Please visit the site and browse the specific products tested and share your thoughts about what you would like to see tested next.