The testing process appears to be simple at first glance with just a few steps:
- Sampling
- Submission
- Transport
- Registration
- Preparation
- Analysis
- Reporting.
As with many processes that appear simple, complexity arises in the detail. The most important stage is the preparation prior to sampling. Before deciding to proceed with any allergen testing you must consider the following question: ‘What do I want from my results?’
Know what you want
If you start the sampling process without a clear understanding of what you want to achieve, and you do not put the right ingredients in at the beginning, you will not get the result you want at the end. A common pitfall is starting with the simplified, ‘I want a certificate that shows no allergens have been detected in my product’ as the reason for testing. It may be the case that a ‘not detected’ result did not mean what you thought, leaving you frustrated that an unsafe food product from your facility was released to the public, undetected by your testing programme.
In this case, it is better to step back and ask: ‘Why am I testing?’. A more robust answer might be: ‘To monitor/control the risks associated with allergens within my food production process; ensuring my food is safe and affordable for all potential consumers’. This answer encompasses the original answer and adds the purpose to guide the sampling process. In fact, you are now seeking to maximise the chance of a detected allergen result if it poses a risk to your consumer. Achieving this goal will need more detailed planning and information.
Specifically, we need an allergen risk assessment. This would include a review of all production processes and documents, such as raw materials, inventories, site layouts, recipes, process flows, production plans, etc. Although the scope of this article does not cover the specifics of an allergen’s risk assessment, it should generate the following:
- Product table – listing of all allergens intentionally on site (including form, eg, wheat flour – powder, pasteurised egg – liquid and expected amounts)
- List of potential adulteration/contamination risks for allergens to be monitored in the supply chain (eg, milk – liquid, packaged in the egg supplier facility)
- Potential transmission/contamination routes (eg, flour settling from air, potential carry over from previous product, or PPE)
- Cleaning validation sampling plan (specifying the allergen, sample points and frequency)
- Cleaning verification sampling plan (specifying the allergen, sample points and frequency).
When developing the sampling plan, it is important to include even small details like the impact of submitting multiple items as a composite sample. This is because submitting a beginning, middle and end-of-run product as a single item to be combined could mask a positive result if only one item had low levels of the allergen. Combining the three may dilute the result to below the reporting limit of the allergen test. If the purpose was to detect any carryover allergen from the previous run, it may be more appropriate to submit the beginning-of-run sample alone as it would have the highest risk of being contaminated.
When conducting sampling it is also important not to introduce new allergen hazards through sampling (eg, PPE unchanged by the quality control (QC) technician between areas). Routine sample types may include raw materials, intermediate products, final products, surface swabs and rinse waters. Where cleaning products may remain on surfaces, confirmation exercises could be undertaken via a swab type sample to ensure the cleaning agent is not interfering with the test (eg, submitting positive control swabs spiked with cleaning agents) and masking positive results.
Communication is a two-way street
Allergen testing is a collaborative venture between the food manufacturer and laboratory. Once the risk assessment is complete, communication with the testing laboratory can assist in the selection of appropriate testing, documentation and packaging of samples in the correct format for analysis. This will reduce the risk of delays in transport, registration and testing of your samples.
Sample information provided to the laboratory should be as detailed as possible, specifying the ingredients and any processing. The ingredient list will allow the laboratory to identify components that may cross-react with or even inhibit the test, causing inaccurate results. Processing may mean the sample requires an alternate test method for you to receive the most accurate result.
There is also information that you will want from your laboratory service. Typically, this would include test reporting limits, any legislative or regulatory recommended method information, accreditation details (eg, UKAS), confirmation of participation in external proficiency test schemes with satisfactory performance, details of the specific reporting form of the allergen (eg, ‘egg white protein mg/kg’ and not ‘egg’), uncertainty measurement for the method, and the amount of sample required for testing.
It is important to document any information received that relates to decisions on testing. Justifications for specific testing may seem obvious immediately after conducting the risk assessments but may become less clear 12 months later, especially when required to justify the sampling/testing plan during an audit.
Analysis
The most common detection techniques for food allergens are ELISA, lateral flow and PCR. Each method has strengths and limitations. Liquid chromatography with tandem mass‑spectroscopy (LC-MS/MS) continues to be an emerging technology in the allergen testing arena but is not commonly used for routine food compliance analysis due to the high cost.
ELISA
The most widely used method for detection of allergens is enzyme-linked immunosorbent assay (ELISA). ELISA techniques use antibodies to selectively target allergens or specific marker proteins within a food product for detection. The technique is applicable to a wide range of sample matrices. The strengths and limitations of this technique are outlined below.
Strengths:
- Highly specific for the analyte
- More environmentally friendly as it does not produce large volumes of waste solvents
- Provides rapid detection of allergens.
Limitations:
- Sample processing (eg, heat, drying, fermentation) may affect test sensitivity
- Inconsistency in results between labs due to different test kit manufacturers
- Requires specialist equipment.
Lateral flow assay (dip-stick test)
Lateral flow is a widely used method for on‑site verification of cleaning or food materials (when validated) for allergens. The lateral flow assay technique also uses antibodies to selectively target allergens or specific marker proteins within a food product. However, the lateral flow format requires no specialist equipment and can be performed with minimal training.
Strengths:
- Easy to use and can be performed at the sampling location
- Inexpensive when compared with other test options
- Suitable to verify cleaning before use of equipment.
Limitations:
- Qualitative result (detected/not detected – line appears on the strip)
- Not suitable for all sample types (eg, may give a false negative result for highly positive results).
PCR
PCR is a DNA-based detection method – it amplifies and detects specific DNA sequences unique to the target allergen. This technique requires analytical equipment to perform the analysis and quantify/read results.
Strengths:
- Improved specificity over ELISA – detects unique DNA sequences for the allergen
- Simultaneous multiple allergen detection
- DNA may be more heat tolerant in some processing circumstances
- Suitable for low protein allergens (eg, celery).
Limitations:
- Qualitative/semi-quantitative; values extrapolated from DNA content and not allergen protein
- Not tissue specific (eg, egg and chicken have the same DNA)
- DNA may be degraded by some processing environments.
Results interpretation
Understanding results is also a collaborative effort. Ultimately, the food manufacturer is the ‘expert’ when it comes to the products or samples submitted. The laboratory can investigate the validity of any results provided regarding the testing performed. When results do not appear as expected, all avenues should be investigated both at the sample production site and testing laboratory. A mistake can occur at any point within the testing process.
We highly recommend that our clients follow a preprepared procedure when an unexpected positive result is retuned. Timelines for product recalls or product hold action can be very narrow. Organisations such as the Allergens Bureau3 have set out warning and action levels for allergens based on the lowest amount of allergen which may cause harm. The information was collated from the latest allergen information and enables companies to link their risk assessments with real-world data concerning risk to individuals.
In summary, a carefully planned and well‑integrated allergen testing programme can be a powerful tool in assuring the quality of products. In addition, communication with your testing laboratory is key to ensuring the correct selection of testing which, in turn, will help to generate relevant and meaningful results.
Luke Manning
Luke is a Laboratory Manager and a part of the Food Consultancy team at ALS Food and Pharmaceutical UK. He gained an interest in food allergens while studying biomedical sciences at the University of Chester and has worked in the food testing industry for over 12 years.
References
- ‘Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011’, https://webarchive.nationalarchives.gov.uk/eu-exit/https://eur-lex.europa.eu/legal-
content/EN/TXT/?uri=CELEX:02011R1169-20180101. https://www.legislation.gov.uk/eur/2011/1169/contents
- Wearne S. ‘FOOD ALLERGY AND INTOLERANCE PROGRAMME – Report’. Food Standards Agency, Mar. 15, 2017. [Online]. Available: chrome-extension://oemmndcbldboiebfnladdacbdfmadadm/
https://www.food.gov.uk/sites/default/files/media/document/fsa170306.pdf
- ‘VITAL® Voluntary Incidental Trace Allergen Labelling’, VITAL. https://vital.allergenbureau.net/
