Have Questions About Food Allergen Testing?

The most common methods used in the food industry include immunoassays like enzyme-linked immunosorbent assays (ELISA) and lateral flow devices (LFDs), as they detect the protein causing the allergic reaction. Each method has its own advantages and is used based on the type of allergen and food matrix  involved, as well as on the type of result needed, time-to-result required or purpose of the analysis, among others. Other methods, like PCR or liquid chromatography, are used to troubleshoot and confirm results that might be unclear (or when there is no immunoassay available) Read more about the different allergen testing methods  here.

An immunoassay is a biochemical test that measures a molecule's presence or concentration through the interaction between an antibody and an antigen (in this case, the allergen).

ELISA stands for Enzyme-Linked Immunosorbent Assay and is a common laboratory technique used for detecting and quantifying substances such as allergens. ELISA tests are sensitive and can measure allergen concentrations.

LFDs (lateral flow devices) are simple, rapid diagnostic devices used to detect target analytes, like allergens. LFDs are widely used for on-site testing due to their convenience and speed.

PCR or Polymerase Chain Reaction is a method used to amplify DNA sequences allowing for their detection and, in some cases, quantification. In allergen testing, PCR can detect DNA sequences specific to known allergens.

False positive or false negative results are test results that incorrectly indicate the presence (positive) or absence (negative) of an allergen.   Where a false positive could have an economic impact on a food producer, since this means repetition of assays, confirmation of results, and the possibility of stopped production lines, repetition of cleaning procedures, or wrongly elimination of product batches; a false negative result could end in product recalls, damage of the reputation of the producer, but could also have a direct impact in the health of the consumer Because of this, it is important to know the limitations of the methods applied, and make sure that all required validations have been carried about. 

The LOD (Limit of Detection) is the lowest amount of an allergen that can be detected and distinguished from a true blank, in a defined matrix, by a testing method, but not quantified as an exact value.

LOQ (Limit of Quantification) is the lowest amount of an allergen that can be quantitatively measured with acceptable precision and accuracy.

Cross-contamination is the unintentional transfer of allergens to a food product  . The allergens can come from multiple sources, like raw materials, other food products or additives, among others. They can be transferred in multiple stages and through multiple processes that take place not only during the production process (e.g., during weighing, mixing, packaging, etc.), but also before the introduction of raw materials and ingredients into a production plant, like their harvesting, storage or transporting. 

To choose the most adequate kit, it is necessary to first consider several aspects that will tell us the type of kit to choose: what is the actual contamination source, what is the type of sample (environmental, raw materials, finished products, liquids, pastes, etc.), if the result needed is qualitative or quantitative, the purpose of the analysis (monitoring, validations, etc.), how long can be waited for the result, how much money can be invested (also depending on the number of samples required), etc. Furthermore, after this, an evaluation of the possible kits needs to be carried about: unfortunately, they cannot be used directly “off the shelf”. Their suitability for the particular allergen/matrix combination of interest needs to be checked. Therefore, it is important to know if similar matrices have been already validated, and it is always recommended that the customer runs a validation or a matrix suitability check.

While ELISA is widely used for allergen detection, it has limitations:

• Food processing can modify target proteins, affecting the extractability of the proteins and also the formation of the antibody-protein complex. 
• Antibodies can present cross-reactivity to some foods, which limits the applicability of the methods. This can be sometimes sorted out with the use of highly-specific antibodies. 
• High specificity of an antibody to a protein of the allergen  can lead to false negatives, because the detectability of said protein could be compromised while other allergenic proteins could still be present.
• The allergen source of contamination at production plants, rarely exactly matches the form of allergen used as immunogens, or as calibrators, thus making quantitation less exact.
• There is a general lack of certified reference materials for most allergens, what means limitation in how different methods can be compared and how methods can be calibrated.
• Some matrices may present challenges (e.g., pH, high concentration of salts, content of polyphenols, etc.), that result in the need of customizing or adjusting the methods. Some of these, might not be solvable.

PCR (polymerase chain reaction) amplifies small DNA fragments to detect allergens. It's particularly useful for highly processed foods where DNA is more stable than proteins. PCR can detect allergens like celery, which are difficult to identify with antibodies because of the high cross-reactivity they normally present. However, PCR cannot differentiate between DNAs coming from different tissues, like egg or milk and the corresponding chicken meat or beef.

Mass spectrometry offers high accuracy in identifying proteins and peptides, even if they are partially degraded or modified by processing. It can detect multiple allergens simultaneously and is highly reliable. However, it requires skilled personnel, significant initial investment, and more time for results compared to immuno-based methods. Furthermore, LC-MS faces several of the same challenges that ELISA have, like the mismatch between contaminants and calibrators (in particular in regard to the digestion pattern of the protein), or the matrix effects.

No single method can be considered the best solution for all situations. ELISA and LFDs (Lateral Flow Devices) are widely used for their speed, cost-effectiveness, and directly detection of the allergic protein PCR (Polymerase Chain Reaction) is better suited for specific cases, such as foods with ingredients that cross-react. Mass spectrometry, although more advanced, is still evolving in the field of allergen testing. It is used in cases where a single method cannot deliver an unequivocal result.

Firstly, the definition of the analyte itself is normally vague and the regulations do not match the science (e.g., “tree nuts” vs. specific nuts vs. different forms of processed nuts vs. individual allergenic proteins). This lack of definition is one of the reasons for the non-comparability of different methods, together with the lack of reference materials and reference methods; cross-reactivity issues that immunoassays may present; requirements of high sensitivity given by lack of appropriate allergen level limits in foods; and the versatility of the allergen sources that makes it probable that the calibrators do not match the real allergens in the production plant; among others. Furthermore, processing foods can change the characteristics of allergenic proteins, making them more difficult to detect. Processes like heat-accelerated chemical reactions, protein aggregation, emulsion formation, and pH changes can alter allergenic proteins' structure and solubility, affecting their detectability with traditional methods. 

Some of the current approaches include: 

• Development of reference materials
• Development of guidelines for the use of multiple methods for problematic matrices (tool-box approach), 
• Better definition of allergens (e.g., specific nuts vs. "tree nuts")
• Establishment of limits of allergen content in food
• Developing improved extraction methods to increase the solubility of aggregated proteins.
• Raising new sets of antibodies that are specifically targeted at detecting processed allergens. 

Matrix effects in allergen testing refer to the influence of other components within the sample (the "matrix") on the accuracy and reliability of the allergen test results. The matrix is essentially the entire composition of the sample, excluding the allergen being tested for. Matrix effects can lead to increased variability, false positives, or false negatives in test results.

Incurred samples are those in which a known amount of the food allergen is incorporated during processing, closely mimicking the actual manufacturing conditions of the sample matrix. Spiked samples, on the other hand, involve adding a known amount of allergen to a matrix as received from the supplier or manufacturer. This distinction is important because the recovery of allergens can vary significantly between incurred and spiked samples.

According to the 2010 guidelines by the Association of Analytical Communities (AOAC), an ideal recovery rate for allergens in sample matrices ranges from 80 to 120%. This range accounts for the efficiency of both the extraction step and the ELISA procedure. However, due to challenges with certain matrices and the variability between incurred and spiked samples, recovery rates between 50 and 150% are considered acceptable, provided they are consistent.

The "hook effect" refers to a phenomenon by which overloading a testing device with a high concentration of allergen leads to a false negative result. This effect is more likely to be observed in specific testing scenarios, such as when deliberately testing with pure allergens or very high concentrations to validate the functionality of the test kits. These scenarios are not representative of routine allergen testing, as, particularly in the food industry, the concentrations of allergens in samples are usually well within the test's optimal detection range. 
In the hook effect, the quantity of allergen present surpasses the capacity of the detection antibody that is attached to the colored labeling material (often colloidal gold or colored latex). Consequently, the excess unlabeled allergen moves along the test membrane more rapidly than the heavier color-labelled allergen, saturating all the binding sites on the capture antibodies that are immobilized on the membrane surface.
As a result, when the color-labelled allergen eventually arrives at these sites, there are no available binding sites left. This causes the color-labelled allergen to continue moving to the wicking pad at the end of the test device without binding. Consequently, the color-labelled allergen cannot generate the colored test line that typically indicates a positive result. 

If allergen management is in place, you should be dealing with traces, not large amounts. If allergens are an ingredient, or if it is known that there is a high risk of contamination, then strip tests might not be the best option. From the result of the strip test alone, you cannot distinguish between a true negative and the hook effect. What you can do is diluting the sample/extract and run the assay again or test the sample with an appropriate ELISA kit (please consider that strips are designed for screening).

ATP swabs are not allergen-specific, as they detect ATP from all kinds of sources (including microorganisms) without differentiating between allergens and non-allergens. Furthermore, ATP levels vary in food, and the detection limits are not sensitive enough for allergen monitoring. The US Food and Drug Administration requires protein-specific testing, and the ATP testing does not correlate well with allergen-specific testing.

The technology used in ELISA assays and LFDs is based on antibodies that recognize epitopes associated with specific allergens. Moreover, the composition of the extraction buffer can differ among allergens, meaning there is no single kit capable of detecting all allergens.

However, what you can do is focus your allergen validation on the component with the highest allergenic load. Since allergens are proteins, the component with the highest protein level will have the most significant allergenic potential. If two or more allergens are part of a formula, you don’t have to validate the removal of all; you can concentrate your validation on the allergen present in the highest amount. However, if allergens are present in different forms (for example, one is in a paste and another in a liquid), it may be necessary to validate the removal of both allergens. Furthermore, there are some allergens known for being more “sticky” to others to certain surfaces, and therefore require extra cycles of cleaning.

The “critical” areas should be swabbed, meaning those places (like corners and hidden spots) where cleaning is more challenging, and allergens could still linger. If a visual inspection reveals that an area is not clean, then another cleaning step should be undertaken before swabbing.

Do not only swab flat and smooth areas that are easy to clean. Instead, focus more on food contact surfaces that are harder to clean, such as crevices and joints. Also, only use swabs that are provided with every AgraStrip kit or the AgraQuant Swabbing kit, because these swabs have been extensively validated and are proven to work. Using cheaper swabs may not capture allergens as effectively from equipment surfaces, and allergens might not be released from the swab. Sometimes, recycled milk cartons are used in the production of cheap swabs, which could lead to false positives for milk allergens.

In those cases, the possibility of testing rinse water is also a good option.

Steve Taylor & Joseph Baumert's article, “Best Practices in Allergen Swabbing,” in Food Safety Magazine (June/July 2013) offers further recommendations on this topic. For more information, visit: www.foodsafetymag-digital.com/foodsafetymag/20130607

The method provided in the test kits has been rigorously tested and validated. The instructions are determined to be the optimal conditions for conducting the test. The color development will continue over time and may yield an invalid result if not read at the correct time. If you wish to store the strip for your records, you should either take a picture of the result zone immediately after the required amount of time has passed or cut off the filter and wicking pad areas, storing only the result zone of the strips.

Gluten is not the same as wheat; rather, it is a protein composite found in several grains, including wheat, rye, barley, and certain varieties of oats (due to cross-contamination during processing rather than inherent gluten content). Gluten consists of two main protein groups: prolamins and glutelins. In wheat, these are specifically called gliadins and glutenins, respectively.

Gluten ELISA assays can detect this protein composite, but due to the nature of ELISA assays – particularly the antibodies used in these assays – it is not possible to distinguish whether the detected gluten originated from wheat, rye, or barley.

For instance, when comparing the two most used antibodies in gluten detection, we find that they were raised against different epitopes, yet both show cross-reactivity. The G12 antibody (incorporated in our AgraStrip(R) and AgraQuant(R) products) was raised against the 33-mer of wheat gliadin but also cross-reacts with rye and barley. Similarly, the R5 antibody was raised against rye secalin but also cross-reacts with wheat and barley.

This is due to the complex nature of gluten's composition. Prolamins (known as gliadin in wheat) are the alcohol-soluble fraction of gluten. Therefore, prolamins are extracted using alcohol-water mixtures, such as 50% (v/v) aqueous propan-1-ol or 60-70% (v/v) aqueous ethanol. Approximately half of the prolamins are present as monomers, but the other half are present in polymers that are not soluble in alcohol-water mixtures. To reduce the disulfide bonds between prolamin polymers, reducing agents such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) are used, often in conjunction with heat treatment.

If you’ve been to a grocery store lately, you’ve seen labels with: “May contain [allergenic food]” or “Produced in facilities where [allergenic food] has been processed”. But what’s behind a “may contain” label? PALs, such as those described above, are used to indicate the potential presence of allergens due to cross-contact. They should be based on a thorough risk assessment and not used as a substitute for Good Manufacturing Practices . Unfortunately, this is not always the way they are used in the present. Read more about the precautionary allergen labeling here.

Accurate labeling, based on scientific data, helps to inform consumers honestly about their options and establishes brand trust. Labels should not be misleading, ambiguous, or confusing, and should avoid stating every possible allergen as this can be perceived as overly protective and not useful.  

The proposed changes include:

• Removal of certain allergens (soybean, brazil nuts, macadamia nuts, pine nuts, and oats) from the global priority list.
• Inclusion of sesame as a global priority allergen.
• Active monitoring of emerging allergens (like kiwi, insect protein) for potential inclusion.
• Establishment of reference doses for allergens to inform allergen management and labeling decisions.
• Development of a regulatory framework for Precautionary Allergen Labeling (PAL).

The requirement for soybean labeling will depend on regional regulations, as it's been moved to a secondary priority list. However, sesame will likely need to be included in allergen labeling in countries that are members of the World Trade Organization (WTO), as it's added to the global priority list.

As of now 2024, implementing PAL is not mandatory. A proposed regulatory framework for PAL in the CODEX Alimentarius is under development, aiming for a more standardized and risk assessment-based approach. The implementation of PAL will depend on the completion of this framework and subsequent regulations. Among others, this framework contemplates the use of a symbol on the food products labels, that indicates that a risk-based allergen management plan has been implemented and used before determining the need of a PAL for those products. 

No, the proposed guidelines recommend against using PAL if allergen levels do not exceed the action levels. This approach is to prevent the overuse of PAL and ensure it's based on a proper allergen risk assessment. 

No, the threshold levels are intended for deciding the necessity of PAL and should not be used as criteria for “free-from” labeling. Such claims require adherence to specific standards ensuring the safety and validity of the claim.

Food allergen management involves all documented practices and measures that aim to identify, minimize, control, or eliminate the presence of allergens in all levels of a company involved in the food supply chain. It's crucial for ensuring consumer safety, maintaining trust in your brand, and complying with mandatory allergen content declarations in food products.

Companies should focus on clear identification and avoidance of cross-contact. This includes:

• Work with providers that have allergen management plans in place and relevant food safety certification where needed.
• Request certificate of analysis of the incoming materials.
• Sampling materials upon reception to verify allergen status.
• Keeping allergenic materials sealed and clearly marked.
• Storing materials in isolated or clearly demarcated areas.
• Store allergenic materials at floor level to avoid cross-contamination caused by possible leakage or spilling.
• Implementing measures appropriate to the nature of the materials (liquid, powder, granulate, etc.).
• Handle allergenic material with dedicated clothing and utensils.

Recommended practices include:

• Include the Allergens Management as part of the HAPPC system in the manufacturing process, paying special attention on the risk points of contamination. 
• Using dedicated premises or production lines for goods with defined allergen profiles.
• Implementing effective segregation and validated cleaning programs. This also includes temporal segregation (scheduling).
• Ensuring that all ingredients processed match those listed in the recipe.
• Ensuring that the products match their packaging and label. 
• Regularly verifying the effectiveness of the allergen management plan.

If a product change introduces new allergens, re-evaluate the allergen risk according to the management plan. Communicate these changes clearly to consumers, for example, with labels such as “now contains…” or “new recipe” and ensure old packaging materials are removed and destroyed to avoid confusion.

Cleaning is crucial in allergen management. Validate and regularly test cleaning methods for effectiveness, using specific analytical methods for allergens. Employ single-purpose cleaning materials, adapt layouts to facilitate cleaning, and prefer wet cleaning methods over dry ones to minimize the risk of cross-contamination.

Cleaning validation is crucial in allergen management as it ensures that cleaning procedures effectively reduce allergens to acceptable levels. This is a key component of any allergen control program, helping to prevent cross-contamination and ensuring consumer safety.

Key steps include:

1. Determining the objective of cleaning, focusing on allergen removal.
2. Ensuring organizational support and a multi-departmental team approach.
3. Reviewing the hygienic design of equipment and the floor layout  to identify hard-to-clean areas.
4. Reviewing current cleaning programs and applicable regulations.
5. Determining the worst-case scenario for soiling.
6. Choosing appropriate analytical tests for validation, such as ELISA and LFDs.

Verification, by definition, is the process of ensuring that your validation remains applicable. It involves ongoing surveillance of your validation and should occur at a scheduled frequency over a set time period. Verification is often a scaled-down form of monitoring compared to your validation, aiming to ensure that nothing within your allergen management program has changed over time.

Verification can be performed through environmental testing of swabs and rinse waters, and it can also involve testing of finished products specifically. For verification, the use of lateral flow device test kits is recommended. A qualitative presence/absence test is sufficient for continuous monitoring of your allergen program to ensure that no allergens have entered your systems.

If anything within your process changes over time - be it a vendor, a sanitizing agent, or something within the facility - it is important to re-evaluate your validation to ensure that your verification scheme remains effective and compliant.

A cleaning validation program must be tailored to the specific needs of a production environment. This involves understanding the products, process lines, and the efficacy of current and previous cleaning programs. The chosen validation method should be robust enough to meet both internal requirements and external accrediting body standards.