Professor Clare Mills

Precautionary Allergen (“may contain”) Labelling (PAL) is used by industry to communicate potential risk to food-allergic individuals posed by unintended allergen presence (UAP). In 2014, the World Allergy Organization (WAO) highlighted that PAL use was increasing, but often applied inconsistently and without regulation — which reduces its usefulness to consumers with food allergy and those purchasing food for them. WAO proposed the need for a regulated, international framework to underpin application of PAL. In 2019, the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) of the United Nations convened an expert consultation to address the issue of PAL, the outputs of which are now being considered by the Codex Committee on Food Labelling (CCFL). To summarise the latest data to inform the application of PAL in a more systematic way, for implementation into global food standards. A non-systematic review of issues surrounding precautionary labelling and food allergens in pre-packaged products. Approximately, 100 countries around the world have legislation on the declaration of allergenic ingredients. Just a few have legislation on UAP. Given the risks that UAP entails, non-regulated PAL creates inconvenience in real life due to its unequal, difficult interpretation by patients. The attempts made so far to rationalize PAL present lights and shadows. At a time when CCFL is considering the results of the FAO/WHO Expert Consultation 2020–2023, we summarise the prospects to develop an effective and homogeneous legislation at a global level, and the areas of uncertainty that might hinder international agreement on a regulated framework for PAL of food allergens.

•First industrially, clinically relevant multi-allergen matrix RM traceable to SI.•Egg white, skimmed milk, almond, hazelnut, walnut incurred at 10 mg kg−1 protein.•Matrix blank and allergen raw materials are also available as RMs.•ISO 17034 accredited and allows comparability and harmonisation of measurements.•Homogeneity and stability assured, RMs now available to the analytical community. Measurement of food allergen protein concentrations against thresholds can improve allergen risk management and precautionary allergen labelling. Such measurement suffers well known problems which could be ameliorated by well characterised reference materials (RMs) providing meaningful information for risk assessors. We investigated the preparation and characterisation of the first consensus informed industrially and clinically relevant multi-allergen matrix RM kit for five priority allergens. It is a medium analytical difficulty processed food chocolate paste matrix (a) devoid of allergens, and (b) incurred with five allergens at the clinically relevant concentration of 10 mg kg−1 expressed as protein. The allergen raw materials: hens’ egg white powder, skimmed cows’ milk powder, almond powder (full fat), hazelnut powder (partially defatted), and walnut powder (partially defatted), are also available as RMs. The preparation, gravimetric traceability to the SI, homogeneity, and stability were found to be fit-for-purpose and the RMs are now available to the analytical community.

[Display omitted] Celery root is known to cause severe allergic reactions in patients sensitized to mugwort pollen. We studied clinically well-characterized patients with celery allergy by IgE testing with a comprehensive panel of celery allergens to disentangle the molecular basis of what is known as the celery–mugwort syndrome. Patients with suspected food allergy to celery underwent a standardized interview. Main inclusion criteria were a positive food challenge with celery or an unambiguous case history of severe anaphylaxis. IgE to celery allergens (rApi g 1.01, rApi g 1.02, rApi g 2, rApi g 4, nApi g 5, rApi g 6, rApi g 7) and to mugwort allergens (rArt v 1, rArt v 3, rArt v 4) were determined. IgE levels ≥0.35 kUA/L were regarded positive. Seventy-nine patients with allergy to celery were included. Thirty patients had mild oral or rhinoconjunctival symptoms, and 49 had systemic reactions. Sixty-eight percent had IgE to celery extract, 80% to birch pollen, and 77% to mugwort pollen. A combination of Api g 1.01, 1.02, 4, 5, and 7 increased the diagnostic sensitivity for celery allergy to 92%. The lipid transfer proteins Api g 2 and Api g 6 were not relevant in our celery-allergic population. IgE to Api g 7, detected in 52% of patients, correlated closely (r = 0.86) to Art v 1 from mugwort pollen. Eleven of 12 patients with monosensitization to Api g 7 were IgE negative to celery extract. The odds ratio for developing a severe anaphylactic reaction rather than only mild oral symptoms was about 6 times greater (odds ratio, 5.87; 95% confidence interval, 1.08-32.0; P = .0410) for Api g 7–sensitized versus –nonsensitized subjects. There is an urgent need for routine diagnostic tests to assess sensitization to Api g 7, not only to increase test sensitivity but also to identify patients at risk of a severe allergic reaction to celery.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µg /g ), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µg /g , for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µg /g , respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.

With a society increasingly demanding alternative protein food sources, new strategies for evaluating protein safety issues, such as allergenic potential, are needed. Large-scale and systemic studies on allergenic proteins are hindered by the limited and non-harmonized clinical information available for these substances in dedicated databases. A missing key information is that representing the symptomatology of the allergens, especially given in terms of standard vocabularies, that would allow connecting with other biomedical resources to carry out different studies related to human health. In this work, we have generated the first resource with a comprehensive annotation of allergens’ symptomatology, using a text-mining approach that extracts significant co- mentions between these entities from the scientific literature (PubMed, ~36 million abstracts). The method identifies statistically significant co-mentions between the textual descriptions of the two types of entities in the literature as indication of relationship. 1,180 clinical signs extracted from the Human Phenotype Ontology, the Medical Subject Heading terms of PubMed together with other allergen-specific symptoms, were linked to 1,036 unique allergens annotated in two main allergen-related public databases via 14,009 relationships. This novel resource, publicly available through an interactive web interface, could serve as a starting point for future manually curated compilation of allergen symptomatology.   

Sensitisation to the lipid transfer protein Pru p 3 is associated with severe allergic reactions to peach, the proteins stability being thought to play a role in its allergenicity. Lipid binding increases susceptibility of Pru p 3 to digestion and so the impact of bile salts on the in vitro gastrointestinal digestibility of Pru p 3 was investigated and digestion products mapped by SDS-PAGE and mass spectrometry. Bile salts enhanced the digestibility of Pru p 3 resulting in an ensemble of around 100 peptides spanning the protein’s sequence which were linked by disulphide bonds into structures of ~ 5–6 kDa. IgE binding studies with a serum panel from peach allergic subjects showed digestion reduced, but did not abolish, the IgE reactivity of Pru p 3. These data show the importance of including bile salts in vitro digestion systems and emphasise the need to profile of digestion in a manner that allows identification of immunologically relevant disulphide-linked peptide aggregates.

Rapid and comprehensive genetic sequencing has shed light on the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and allowed timely implementation of PCR tests to determine the presence of viral RNA. PCR tests for SARS-CoV-2 are some way from being reliably qualitative and will never indicate how the disease might progress in an individual. As COVID-19 becomes endemic, there is a concomitant need for accurate serological assays to detect antibodies to SARS-CoV-2 antigens and ultimately tests for prognostic markers to target treatment options.1,2 With this considerable genetic insight, and the emerging structural information, comes associated questions regarding the molecular descriptors that contribute to disease progression, especially when we consider spread across different populations. The power of mass spectrometry to generate rapid, precise, and reproducible diagnostic information that complements genomic information and accelerates our understanding of the disease, is now becoming a reality.