Why are analysis and allergen RMs important?
To recap: allergen QRAs are based on clinically derived reference dose amounts of the allergen protein that elicit an objective reaction in a given percentage of the population with the allergy. The ED01 is the eliciting dose for one percent of the allergic population, ED05 is the eliciting dose for five percent, and so on. For example, the ED01 for cow’s milk is generally agreed to be around 0.2mg of total protein from cow’s milk.2 If the amount of food eaten unexpectedly contains around 0.2mg of milk protein, a reaction in one out of a hundred people with a milk allergy is predicted. Analysis of the food will tell us the concentration of milk protein in the food in milligrams per kilogram (mg/kg). The result will either be a positive number – eg, ‘milk protein: 25 mg/kg’, or a limit of quantification (LoQ), eg, ‘milk protein not detected, less than 0.4 mg/kg’. To convert these concentrations into amounts to compare with the reference dose, we need to know the most likely amount of food eaten. The amount eaten is not always the portion size (think of a packet of biscuits, how many biscuits would you eat?).
But can we rely on the analytical result? Allergen measurement, which has come a long way, still has some drawbacks including that quantification of allergens in foods using different test methods can give rise to conflicting results. That is where allergen RMs can help by anchoring individual laboratory findings to an external reference, thus providing meaningful information for risk assessors.
Which allergen RMs are available?
RM and certified reference material (CRM) are well defined terms within an associated international infrastructure. Food allergen RMs should comply with this infrastructure. RMs produced by national measurement institutes or related designated institutes exhibit the highest standards. RMs currently available take two different forms: raw ingredients and incurred processed food matrices. Some were not originally intended as allergen protein RMs but have been used in the absence of anything better. The European Commission’s Joint Research Centre (JRC) and the US National Institute of Standards and Technology (NIST) are major providers of RMs in general, including some that have been used for allergens. The MoniQA association made available the first validated reference material for food allergen analysis, milk powder cookies at two agreed concentrations. Proficiency Test (PT) providers often make available allergen quality control (QC) or reference materials subsequent to the PT round.
The multi-allergen RM kit
Our multi-allergen RM kit consists of a processed food chocolate paste matrix incurred with each of the following allergens at the clinically relevant concentration of 10 mg/kg expressed as protein. The allergens are:
- chicken egg white powder
- skimmed cow’s milk powder
- almond powder (full fat)
- hazelnut powder (partially defatted)
- walnut powder (partially defatted).
As well as the incurred material, a blank of the same matrix devoid of the above allergens is provided, as are the individual raw materials .
To research and produce the allergen RMs, LGC led a consortium with the University of Manchester and Romer Laboratories Ltd. The work was funded by the UK Food Standards Agency (FSA) (Project FS101206) following an open competitive tender and was supported by the UK Department for Business, Energy and Industrial Strategy (as was).
A recently published report on the FSA website describes the governance of the project,2 stakeholder engagement to optimise the format and contents of the reference material kit and its preparation and characterisation. More detailed information is available in three peer reviewed publications referenced in the report.
Why is the RM kit clinically and industrially relevant?
The incurred concentration of 10 mg/kg of each allergen expressed as protein is clinically relevant because it approximates to clinically derived eliciting doses and typical food intakes. Moreover, the matrix was prepared in line with the EuroPrevall standardised low-dose double‑blind placebo-controlled challenge vehicle. The matrix ingredients and allergen raw materials were commercial food ingredients obtained from reputable suppliers based on extensive previous experience of sourcing for clinical challenge studies (EuroPrevall and iFAAM). The matrix is a processed food matrix of medium analytical difficulty, owing in particular to the polyphenol-containing cocoa powder. Chocolate is a well-known challenging matrix for allergen analysis. The work for this allergen RM kit was based on earlier studies that produced a similar peanut QC material. A partial pre-print of this work has been published.3
How can we be sure the allergen RM kit is fit for purpose?
The preparation, gravimetric traceability to the SI, homogeneity and short-term (transportation) stability and long-term stability of the RM have been demonstrated and are fully described in a peer reviewed publication.4 Long-term stability continues to be monitored. These data together with species characterisation by Sanger sequencing and allergen profiling in the raw materials by a combination of immunoblotting and discovery mass spectrometry demonstrate the suitability of the reference materials. The reference materials have been confirmed within the scope of LGC’s ISO 17034 accreditation. Assigned values are compared with independently obtained data from two ELISA platforms and statements of measurement have been published.
How should the allergen RM kit be used?
The applications include method and ELISA or PCR kit development, validation studies of analytical methods and competency assessments of analytical service providers and staff. The allergen RM kit may be used to verify in-house QC materials as it is impossible to make RMs for the required wide variety of matrices that are analysed. In doing so, the raw material allergen RMs can be used to prepare fortified (‘spiked’) matrices to assess recovery in real life situations. The blank matrix can be used as a ‘no-template’ control to provide assurance of absence of in-lab allergen cross contamination (either environmentally, from personnel or in reagents) and to calculate method limit of detection (LoD) or limit of quantitation (LoQ).
What does all this mean for the food industry?
While analysis is not the only way to verify allergen QRAs, it is an important step. It is vital that risk assessors and risk managers can rely on analytical results. The use of properly characterised and assessed allergen RMs is essential to the validation and continued quality assurance of allergen analyses. Risk assessment can be thrown off track by unreliable results. These can be false positives or false negatives, as a result of matrix interferences or testing for the wrong allergen, eg, casein instead of β-lactoglobulin.4 They can also be over- or under‑reported allergen concentrations through different test methods, giving rise to conflicting results. The regular use of allergen RMs (or an in‑house QC material anchored to an RM) gives the laboratory and the customer added confidence in the data produced. If an unusual result is flagged by the RM, in one of the above categories, what can be done? My advice is to discuss it with the method provider (the kit manufacturer) and with the customer. This almost always resolves the issue, though further complementary analysis may be required, eg, a different kit or method. The harmonisation of precautionary allergen labelling (PAL) may be within our grasp. Allergen QRA and analysis will have a crucial role in deciding if PAL should be applied or not – but both must be done correctly.
- Food allergies – are we at the crux? [Internet]. New Food Magazine. [cited 2023 Oct 30]. Available from: https://www.newfoodmagazine.com/article/168081/food-allergies-are-we-at-the-crux/
- Blom WM, Westerhout J, Baumert JL, et al. Updated full range of Eliciting Dose values for Cow’s milk for use in food allergen risk assessment. Food and Chemical Toxicology, 168, p.113381.
- Development of Reference Materials: Executive Summary [Internet]. [cited 2023 Oct 26]. Available from: https://www.food.gov.uk/research/development-of-reference-materials-executive-summary
- Holcombe G, Walker M, Singh M, et al. , Clinically and industrially relevant incurred reference materials to improve analysis of food allergens, milk, egg, almond, hazelnut and walnut, Food Chemistry, 434, 2024, 137391, https://doi.org/10.1016/j.foodchem.2023.137391 .