Lateral Flow Devices (Nima, Gluten Tox Home, EZ Gluten) to test food for gluten: Manufacturer & Consumer Use

Lateral Flow Devices (Nima, Gluten Tox Home, EZ Gluten) to test food for gluten: Manufacturer & Consumer Use

This article was originally published in 2012 on glutenfreedietitian.com. Due to the marketing of the new consumer lateral flow device (LFD) NIMA there is a renewed interest in LFDs. An updated version of the article is republished here. If you are a manufacturer or consumer who currently uses or is thinking of using a LFD please take the time to read this article.

 Thank you to Thomas Grace of Bia Diagnostics, www.biadiagnostics.com and Adrian Rogers of Romer Labs https://www.romerlabs.com/ for sharing their expertise on Lateral Flow Devices

Background Information on LFDs

What are lateral flow devices (LFDs)?

LFDs are used to qualitatively (is gluten present or not) or semi-quantitatively* (generally, what amount of gluten is present) to determine whether gluten is present in a food product, a surface, or rinse solution. There are several LFDs on the market for testing food for gluten, including products intended for consumer use. These include Gluten Tox Home, EZ Gluten, and Nima.

* Technically the strips themselves can only be qualitative. If you adjust the extraction dilution you can adjust the LOD (limit of detection) of the strip. Currently, there are several possible ways to make the LFD semi-quantitative but each has to be extensively validated for each specific application and matrix (food) which can be very expensive and cannot be applied to a new matrix or application without first being re-validated.

How do LFDs work?

LFDs are usually what we think of as “dipstick” tests. They are very similar in design to a home pregnancy test. The tests themselves use antibodies that are very specific for the detection of gluten molecules. They utilize a line of fixed antibody on a surface strip and a second antibody, which is attached to tiny colored particles (on the sample addition end of the strip). When an extracted liquid sample is added to the strip, the particle labeled antibodies and the sample start to migrate across the surface of the strip together. If the sample extract has the protein or compound of interest present (e.g., gluten) and the particle labeled antibodies can recognize its epitope (binding site), under the right conditions they will bind together. Now that they are bound 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. This forms a sandwich complex, “sandwiching” the protein (gluten) between the two antibodies. As the whole complex starts to accumulate on the surface of the strip the tiny 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. Manufacturers should be contacted for guidance or any questions. Extensive validation should have been done on the kit and they should be able to provide information on the upper limit of detection.

See video for visual examples of a negative result, positive result, and hook effect. This video is courtesy of Romer Labs and Adrian Rogers. Generic Strip (2)

Manufacturer Use of LFDs

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

Thomas Grace: LFDs can be great tools for helping manufacturers in defining their Hazard Analysis Critical Control Point (HACCP) 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 a fully validated ELISA (enzyme linked immuno-sorbent assay).

Adrian Rogers: LFDs are commonly used as a screening device, a quick check that that the manufacturer’s HACCP program is working correctly. Any unexpected results should be followed up with further testing using a secondary method such as a quantitative ELISA. Ab

How would a manufacturer go about validating an LFD?

Thomas Grace: 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 into each sample. To be 100% sure that the method can detect all forms of gluten one should therefore use wheat gliadin, barley hordein, and rye secalins for the spikes because some methods might utilize an antibody that does not easily recognize hordeins. Keep in mind that the gluten spike should be less than 10% of ingredient. The sample, the spiked sample and gluten spike itself should all be tested using a certified ELISA method to confirm the results. Also, if a matrix or an ingredient of the matrix contains any fermented or hydrolyzed products the LFD might not detect (a sandwich ELISA may not either).

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.

Adrian Rogers: Adding to the above, it is also equally important to identify and test the source of potential contamination if possible. If the manufacturer uses a gluten containing product on site that has been processed by heat, pressure or fermentation it may increase the limit of detection of the device or not be detected at all.

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

Thomas Grace: 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. As opposed to screening, these methods should never be used for finished product validation in and of themselves, but only in conjunction with a fully certified and validated method such as an ELISA.

Adrian Rogers: As mentioned above, LFDs can be used to screen finished products as long as an in depth validation has been undertaken for a specific product from a specific manufacturer. The manufacturer should work closely with the method developer to achieve this.

Consumer Use of LFDs

Should consumers use LFDs to test products for gluten?

Thomas Grace: Currently I am not aware of any method (ELISA, LFD, PCR, MS, etc.) that can reliably be used for all possible food matrices without being validated beforehand. While it can be difficult for a fully certified food laboratory to validate a method for a specific application, for most consumers it would be impossible. Consumers generally do not have the equipment or capacity to validate these devices for their specific application also because of the extreme variance in food products. Why? The burger they make at home will be different from the burger they get at 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.

Adrian Rogers: I agree with Thom. When food manufactures use these devices they have a set sample plan and will homogenize a relatively large amount of product and sub sample from that. It would be the equivalent of putting your whole meal in a blender and taking a pea sized amount from that. I would feel uncomfortable giving out advice to individual consumers but they must test a representative sample of the meal they are about to consume.

I would also say that the consumer must get hold of as much information about the test they are using as possible, it’s strengths and more importantly it’s weaknesses. Everyone who uses the test should get hold of the validation report provided by the manufacturer that summarizes if the kit is fit for purpose and whether it meets the predetermined performance criteria for which it was designed.

Gluten Free Watchdog Note: There is a lot of interest in the portable gluten testing device called Nima. The Nima website states, “2016 will bring full system validation conducted by a third party. This third party will summarize their results, which will compare the Nima device to results from a leading antibody. The results will be published on the Nima blog and distributed in emails to all our subscribers on our email lists.” To date, such a validation report is not available for the Nima making it very difficult to fully evaluate this device. In the opinion of Gluten Free Watchdog, putting the Nima in the hands of paying consumers before the availability of a validation report may be putting the cart before the horse.

Thank you, Thom and Adrian!

Additional Information

For more information about Nima, including Gluten Free Watchdog’s test findings and current opinion on good and not so good uses for this device see https://www.glutenfreewatchdog.org/news/category/nima-sensor/

 

 

 

 

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