Have Questions About Food Allergen Testing?

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.

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.

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

No single method can be considered the gold standard for all situations. ELISA and LFDs (Lateral Flow Devices) are widely used for their speed and cost-effectiveness. PCR (Polymerase Chain Reaction) is better suited for specific cases, such as highly processed foods. 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.

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.

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 dilute the sample/extract and run the assay again or test the sample with an appropriate ELISA kit.

ATP swabs are not allergen-specific, as they detect ATP from all kinds of sources, including microorganisms. 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.

General protein kits are very unspecific, detecting all proteins without differentiating between allergens and non-allergens. Their sensitivity is not sufficient for allergen monitoring and 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.

The “critical” areas should be swabbed, meaning those places (like corners and hidden spots) where cleaning is more challehoonging, 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.

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

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.

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.

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 identifying, minimizing, controlling, or eliminating 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.

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.

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.