NOTICE: This blog is no longer being updated, so medical information may no longer be accurate.

Detecting Gluten in Food: The differences between Lateral Flow and ELISA testing

Last time Heidi was kind enough to have me guest post, we talked a bit about the ways that different antibodies are used to check foods for gluten — and I ruined Where’s Waldo for a few people.

Today, I want to talk a bit about the way different tests use those antibodies to check foods for gluten. Rest assured, no cartoons were harmed in the making of this post—but it’s a long one, I’ll warn you.

Antibody-Based Gluten Tests: Lateral Flow Devices and ELISA

Generally speaking, antibody-based gluten tests fall into two categories: lateral flow devices and ELISA tests. Both types of test have a place in modern gluten detection—many companies use both in their quality control practices. But, have you ever wondered what these words actually mean?

Well, let’s start with what the tests have in common when it comes to gluten:

  • an antibody: could be G12, R5, Skerritt, etc.
  • a sample: could be solid or liquid, but you’ve got to test something for gluten!
  • an extraction process: the gluten has to be separated from other sample components, so that the antibody can easily find and interact with it
  • an extraction solution: almost always, the extraction process involves shaking the sample in a special solution
  • something that changes color: teeny-tiny beads in an LFD, and an enzyme-activated substrate in an ELISA

Now, what’s different?

  • ha! There’s no way I can get this part to fit in bullet points.  You’re funny.  Let’s keep going, shall we?

Lateral Flow Devices

With lateral flow devices (hereafter LFDs), the extracted sample (in solution) is applied to the end of a test strip of some sort.  The test strip is essentially a piece of paper, with the antibody and colored beads preloaded at strategic parts. The solution, which is full of maybe-gluten, flows laterally along the strip—laterally!—until it reaches the antibody.

If the antibody finds something to latch onto, the colored beads get activated and a positive test line appears.  This is the same process as with a home pregnancy test, which are also lateral flow devices—except of course those antibodies are looking for pregnancy hormones instead of a gluten fragment.

Heidi demonstrated this in her post on Gluten Free Boxed Cereals when she tested several brands of cereal and some vital wheat gluten flour with the GlutenTox Home Test Kit:

Just like with a pregnancy test, the answer is qualitative: either the test finds the specific fragment of gluten it’s looking for, or it doesn’t.  A lateral flow test can only tell you yes or no.

There are, of course, plenty of variables and even a few exceptions.  But in general, the above is what you’re looking at when you look at an LFD.  Among their advantages: LFDs are comparatively inexpensive to produce, easier to use, can be read by the naked eye, don’t necessarily require lab equipment, and provide accurate results fairly quickly.


Enzyme-Linked ImmunoSorbent Assays, or ELISAs, are more complex.  They take longer, they require highly specific lab equipment, temperature controls, and they’re more costly to produce and run.  Outside of gluten, one of the most commonly-discussed uses for ELISA testing is checking blood for antibodies to HIV.

Within the world of gluten detection, ELISAs are important because they allow greater degrees of fine-tuning and specification. Unlike qualitative LFDs, ELISAs are quantitative: results are not yes/no, but a specific quantity (usually mg/Kg or ppm when we’re talking gluten).

Like LFDs, ELISAs also involve introducing the sample to an antibody and seeing if there’s a match.  Instead of the antibody going on a piece of paper, though, it generally winds up affixed to the bottom of a PVC tray.  The extraction process is more involved, as is the process of trying to get the antibody and the potential antigen to bind together—at strategic times, at specific temperatures, different solutions are applied to the tray and then washed off.  If the antibody finds what it’s looking for, an enzyme that linked to it will do its job and change the color of the enzyme’s substrate, indicating the presence of gluten.

Also unlike most LFDs, ELISA tests are not read by the naked eye.  Instead, a reading machine compares the intensity of the color of each sample to the colors of a positive and a negative control. By analyzing the color, the machine can determine precisely how much gluten is in the sample. Pretty neat, right?

The two most common types of ELISA test when testing for gluten are Sandwich and Competitive ELISAs. To explain the differences very quickly:

Sandwich ELISAs

Sandwich ELISAs try to trap any gluten in the sample between two layers of related antibody. This is good, but misses out on any molecules that have been processed, fermented, or hydrolyzed enough to break into pieces that only have one epitope. Depending on the sample and the antibody, these small pieces could still be toxic.

Competitive ELISAs

Competitive ELISAs, on the other hand, make any gluten in the sample compete with a known antigen for landing spots on the antibody. The nice thing about a competitive ELISA is that it can find even fragments of potentially toxic molecules, because it only requires a single epitope to catch something.


If you’d prefer a more technical explanation, I recommend the following links:

Sinauer Associates and Sumanas, Inc. have great step-by-step illustrations of both types of ELISA test as well as lateral flow pregnancy tests.

Thermo Fisher Scientific have good technical explanations for the different components of an ELISA test (NB: they sell many ELISA tests but none for gluten).

The National Institutes of Health’s center for Translational Therapeutics also has a good overview with simple illustrations of both Sandwich and Competitive ELISAs, as well as another kind called antigen-down.

As a quick summary:

  • Both ELISA and Lateral Flow tests play important roles in preventing cross-contamination of gluten-free foods.
  • Lateral Flow tests are generally quicker and less expensive than ELISAs, but provide less information and have fewer variables to tinker with.
  • There are different types of ELISA test; a reputable lab will recommend one or the other (or multiple!) depending on the sample.
  • ELISA and Lateral Flow are simply categories. Each can be created with different antibodies, extraction solutions, etc. So, simply knowing that a food was ELISA-tested does not tell us much about whether or not it is safe to eat.

Phew! That wasn’t so bad now, was it?  If you made it all the way down here, go get yourself a cookie or some sushi or a gold star sticker: you earned it!


  1. I have an unidentified autoimmune disease that I call the Margo disease since not one of my 8 doctors in the Penn Health system can shed any insight into it. I am 32 and experience strange (and often incapacitating) symptoms. I took the Elisa act test in may 2011 (through personal research – not on the advice of any of my doctors). Eliminating my food “allergens” has greatly improved my life. I put allergens in quotes because my many doctors discount my personal findings. I believe they are true allergens. Food plays a crucial role in how we feel and how our bodies function. The Elisa act test was worth every penny. It’s results improved my life.

  2. Great explanation! Though not perfect, it is great that these types of tests are available and being used.

  3. Shirley @ gfe says

    I am grateful for this explanation and resource links. A second and third reading did help. 😉 Your explanation is very good, Emily … I think I just have a mental block on the testing details. So, ironically, it seems that the false negatives that occur in celiac testing for diagnosis seem to carry over to testing of food for gluten? 🙁 So is the takeaway that Competitive ELISA tests are the very best way to go, but are not affordable for the average consumer (hence, the need for a program like Gluten Free Watchdog where the cost is spread among all subscribers)? Are there any times when a competitive ELISA will provide a false negative?


    • Thanks Shirley! I’m trying to find a clear way to answer questions — it’s not a particularly clear subject matter!
      Regarding tests for celiac disease and how the results might overlap; I’m hesitant to comment. I just don’t know enough about the people-testing (as opposed to food-testing) to feel confident in an answer. In terms of a ‘best’ test, there isn’t, unfortunately, a clear-cut winner (even absent price). Different situations call for one or the other (or multiple). That being said:- I can’t think of a situation in which a Competitive ELISA would find *less* gluten than other tests (assuming the other variables were all the same).- I know that a big part of the process of bringing GlutenTox Home to market was proving that its yes/no answers would match the quantitative results of ELISA tests. I think we discussed some of the studies on the G12 LFDs, which have both Sandwich and Competitive comparisons (if not just let me know.; the short version is: everything matches up).There’s also an underlying question: What does positive/negative mean? More specifically: what is the particular test actually looking for? Does a negative result actually indicate that the food will not cause harm to someone with celiac disease (or vice versa)?Researchers are increasingly able to pinpoint the parts of gluten-containing grains that trigger celiac reactions; one of the reasons I’m so happy to be distributing GlutenTox is that the G12 antibody was developed after researchers identified the 33mer peptide as highly toxic — so the antibody was designed to respond to a part of this peptide specifically. The same is not true of the other commonly available antibodies, many of which were developed before the 33mer was found.It’s tempting to treat gluten as a single, solid, yes/no item — but of course that’s not true, and gluten has lots of components that interact differently with people’s bodies. So when we say something tested positive or negative for gluten, it’s a bit of a simplified answer.

      I have a feeling that this might have raised as many questions as it answers, but let me know!

  4. Great post – great explanation!

  5. Finally I actually understand this stuff! I heard about the differences in testing but really had no idea about the specifics. I do not think this is something the average “gluten-free public” is aware of. Thank you. 


  1. […] for Gluten: Not So Simple There are two categories of tests currently in use: the enzyme-linked immunosorbent assay (ELISA) test and the lateral flow tests. […]