Allergen control has become increasingly important for food manufacturers, due in large part to consumer demand for safe foods and federal regulations. There are many techniques in a responsible hazard plan for allergen-free products: reputable sourcing, established cleaning procedures, final transportation of the product, etc.
A mis-step with an undeclared allergen can be devastating to manufacturers and consumers. What is the best way verify that the steps you are taking adequately cover your critical control points?
Allergen test kits are an important data point for any food manufacturer working with allergen-free products or focused on being FALCPA-compliant. Test kits are especially important for production facilities that are using shared lines: the problem of cross contact in mixed line facilities is something that must be watched carefully.
There are several types of tests that are available to manufacturers, and more are in development. Which type of allergen test kits are right for your facility? For this discussion, we’ll be focusing on antibody methods (ELISA and LFD tests), and DNA-based methods (PCR).
Antibody-based methods: Lateral Flow Devices and ELISA
Generally speaking, antibody-based allergen tests fall into two categories: LFDs (lateral flow devices) and ELISA (enzyme-linked immunosorbent assay) tests. Both types of test have a place in hazard control—many companies use both in their quality control plans. But, have you ever wondered what these words actually mean?
Well, let’s start with what the tests have in common:
- an antibody: In gluten testing, for example, that could be G12, R5, Skerritt, etc.
- a sample to test, which could be solid or liquid, and it could come from any point in processing
- an extraction process: the allergen 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 agitating the sample in a special solution
- something that changes color: teeny-tiny beads in an LFD, and an enzyme-activated substrate in an ELISA
While there are some things the tests have in common, their differences are much more complex.
Lateral Flow Devices
With 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 points. The sample, which has been subjected to an extraction process, 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 an allergen protein fragment.
The measurement you get with a LFD is qualitative: either the test finds the specific fragment of allergen it’s looking for, or it doesn’t. A lateral flow test can only tell you yes or no. In the case of GlutenTox Pro, Sticks PLUS and Home, the sensitivity can be adjusted to a few different thresholds, allowing for semi-quantitative possibilities.
There are, of course, plenty of variables and even a few exceptions. Some lateral flow kits require lab equipment and careful temperature controls, and others do not. Most LFDs can be modified for use on environmental surfaces. 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, easy 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 and have more steps, they require highly specific lab equipment and temperature controls, and they’re more costly to produce and run. ELISA tests are used in many areas in diagnostic testing outside of just food manufacture.
Within the world of allergen 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 allergens).
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 various ‘standards’ with known amounts of the substance in question. By analyzing the color, the machine can determine precisely how much allergen is in the sample. Pretty neat, right? However, this step also explains why ELISAs can be so costly to run: instead of simply running one test on one sample (like an LFD), a technician working with ELISA must also run 4-10 control tests to compare the sample against. This means that testing 10 or 20 samples at one time (alongside 4-10 controls) is far more cost effective than 1-2 samples.
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 try to trap any allergen 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, on the other hand, make any allergen 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. However, these tests are even more delicate than Sandwich ELISAs.
DNA-based methods: Polymerase Chain Reaction (PCR)
PCR tests are very sensitive, and detect allergens on a molecular level. Once a sample is prepared, PCR uses a machine to amplify a segment of DNA, like from a sample of food, to produce lots and lots of copies. This means that you can start from a very small sample size. If DNA from an allergenic food is present in the sample, the test will find it and report the presence of the allergen.
This is particularly useful in looking for allergens that are low-protein, like celery. This technique can be used in raw or cooked products and is not affected by the heating process. DNA molecules remain intact after exposure to cooking temperatures. The technique can also be employed in highly processed foods, and in products with high levels of polyphenols, tannins or antioxidants.
Some foods won’t work with PCR, however. Since PCR looks for DNA, foods that don’t contain those molecules won’t be detected. This would include Milk, Egg Whites, and Oils. ELISA or LFD tests are a better option for these types of allergens.
PCR is also not able to answer quantitative questions: it will report a presence or absence of allergen.
As a quick summary:
- LFD tests are generally quicker and less expensive than ELISAs or PCR, but provide less information and have fewer variables to tinker with.
- There are different types of ELISA tests; a reputable lab will recommend one or the other (or multiple!) depending on the sample.
- ELISA and LFD 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.
- In contrast:
Unlike immunochemical methods, PCR looks for DNA, not for protein.
- You’ll need lab equipment and experience in a lab setting to conduct a PCR or ELISA test.
- PCR is useful for very low-protein foods, or for foods that have had their protein structures altered.
It is more expensive and complex than antibody based testing.
Sinauer Associates and Sumanas, Inc. have great step-by-step illustrations of both types of ELISA test as well as lateral flow pregnancy tests.
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.
Read this very informative Wikipedia article for a general overview of ELISA test method
Science Learning Hub has an overview article answering the question What is PCR?