Tropomyosin, a heat-stable protein, was previously identified as the only allergen in oyster, reacting with IgE antibody
AOAC Food Allergen Community
NEWSLETTER Volume 8 | Issue 1
2017
IN THIS ISSUE How do you know a rose?
1
Editorial Comment
News
2
How do you know a rose?
Why (validated) Reference Materials for Food Allergen Analysis are so important
2
Editorial Comment
Scientific Developments 3 Gluten Protein Types from Wheat, Rye, Barley and Oats for Use as Reference Materials 3 Genome-derived proteomic reveals the hidden allergens of the Pacific oyster (Crassostra gigas) 4
Upcoming Events 5 2nd Food Allergen Management Symposium5
How do you recognise a rose if you do not know what a rose looks like? While rose allergy was described by Demir et al. in 2002, this is beside the point. You will need to have a reference (rose) to recognise a rose. And for food allergens, the problem – and possible solution - described in this issue evolve around the question how to produce appropriate and widely available reference materials for food allergens. While it seems straightforward to obtain peanuts to use for controls in peanut assays, peanuts in USA and China can be significantly different in their composition, including the (allergenic) protein content. And an ELISA measures differently from a PCR and differently again from LC-MS/ MS. So how do you generate a reference material that reflects what is currently traded on the market and used to produce foods, still considering the possible analytical tools used to test for the allergen? Two articles in this issue of our Food Allergen Newsletter shed some light onto this: Roland Poms (MoniQA Association) describes what has been done so far to produce commutable reference materials and explains which materials can already be obtained. The second article by Katharina Scherf focuses on gluten reference materials from wheat, rye, barley and oats. The article also contains a direct download link to her recently published peer-reviewed article. And last but not least: it is oyster time! However, those allergic to seafood may seek to avoid them. The article by Roni Nugraha & Andreas Lopata from the James Cook University describes how they revealed the hidden allergens using bioinformatic tools. Enjoy the read! Bert Popping Editorial Board Member
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Editor in Chief & Graphic Design: Carmen Diaz-Amigo
Editorial Members: James Roberts , Bert Popping , Samuel Godefroy Terry Koerner, Jupiter Yeung This newsletter is a public document and can be distributed. Partial reproduction is permitted with the proper indication of the author and its source. For questions contact us at
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NEWSLETTER Volume 8 | Issue 1
2017
News
Why (validated) Reference Materials for Food Allergen Analysis are so important
Effective food allergen risk assessment and food allergen management are important to protect allergic consumers and to comply with allergen labelling regulations. Such approaches require reliable analytical tools for the detection of allergens in food. Due to the nature of the analytes and their susceptibility to various processing effects, reliability and comparability of results have posed a great challenge. Both reference methods and reference materials are urgently needed to assure the quality, reliability and comparability of analytical results obtained with different methods. Being an important component of this analytical quality assurance, reference materials contribute to reliable and accurate results. Ensuring the correctness of analytical results is crucial to laboratories, since incorrect results may trigger decisions that can cause economic damage or pose a risk to public health. The use of reference materials: Validated reference materials/ quality control materials and certified reference materials are indispensable for:
Method development Method calibration Calibration of instruments Validation of methods Method verification Proficiency testing Process control and quality assurance in laboratory routine Required use by ISO/IEC 17025
The quality of reference materials is critical for accuracy and comparability of analysis results. Reference materials must be sufficiently homogenous, stable and traceable. Usually extensive material characterisation and testing for homogeneity and stability of the material precede the availability of reference materials. Ideally a certified reference material (CRM) shall be used, which has been validated by accredited institutions and is subject to strict quality testing. Certified reference materials usually come with a certificate with information on the methods used for validation/assigning a value, the measurement uncertainty and traceability of the numerical value of the analyte’s concentration in the material or the analyte’s purity. According to ISO/IEC 17025, accredited laboratories are required to use certified reference material. At this point the currently available knowledge base and methodological abilities do not allow to certify food allergen reference materials according to international standards requirements, however, for the currently available internationally validated materials the international task force led by MoniQA Association is discussing appropriate
procedures for the certification of the offered food allergen reference materials according to ISO Standards. The first validated Reference Materials for Food Allergen Analysis are now available and can be ordered from MoniQA Association www.moniqa.org. The first set of materials includes testing materials for milk allergen analysis comprising a Positive Control (SMP-MQA 092014, characterized dried skim milk powder, validated protein content), Negative Control (BLANK-MQA 082015, based on a gluten free cookie), and 2 Incurred Materials: LOW-MQA 102016 (SMP incurred in gluten free cookies, milled, concentration approx. 3.5 ppm milk protein, validated) and HIGH-MQA 082016 (SMP incurred in gluten free cookies, milled, concentration approx. 35 ppm milk protein, validated). The materials were produced by Trilogy Laboratories USA and have been commercially available starting 01 January 2017. All materials come with a data sheet and a reference certificate to the analytical results, a measurement uncertainty and validation information. Distribution and shipment of the materials is subcontracted to Authorized Distributors among the MoniQA Member Institutions. Further reference materials are in preparation (gluten, egg, soy) and will become available shortly. The initiative – who is behind the project: The design and production of these materials was in response to the urgent need for reference materials expressed by the food industry and food analytical laboratories, as well as providers of food allergen test kits and other methodologies, and national authorities. An international initiative (since 2013) led by MoniQA Association discussed and agreed upon the requirements for food allergen reference materials. For this purpose MoniQA has liaised with the EU funded project iFAAM, the Prolamin Working Group, Health Canada, FARRP, Australia’s Allergen Bureau (Vital), and others. The initial group of 15 experts from the global analytical community grew over time to a group of some 50 institutions contributing to the design of the materials and giving scientific and technological input during the testing and production phase of the materials. Roland Ernest Poms MoniQA Association
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NEWSLETTER Volume 8 | Issue 1
2017
Scientific Developments Gluten Protein Types from Wheat, Rye, Barley and Oats for Use as Reference Materials The consumption of wheat, rye, barley and, in rare cases, oats may be harmful to genetically predisposed persons, because specific proteins may trigger hypersensitivities such as wheat allergy, celiac disease (CD) and non-celiac gluten sensitivity (NCGS). The major immunogenic agents are the storage proteins (gluten) of these grains, but other proteins such as lipid-transfer-proteins, puroindolines and amylase-trypsininhibitors (ATIs) also show immunoreactivity. Cereal grains contain hundreds of different proteins which are traditionally classified into four so-called Osborne fractions: albumins soluble in water, globulins soluble in salt solution, prolamins soluble in aqueous alcohol and insoluble glutelins, which are only alcohol-soluble in the presence of reducing agents. Albumins and globulins (ALGL) constitute about 20 - 25% of grain proteins whereas prolamins and glutelins are the two components of gluten and make up about 75 - 80% of grain proteins. The gluten proteins are called gliadins (prolamins) and glutenins (glutelins) in wheat, secalins in rye, hordeins in barley and avenins in oats. The Osborne fractions can be further subdivided into gluten protein types (GPT), i.e., ω5-gliadins, ω1,2-gliadins, α-gliadins, γ-gliadins and high-molecular-weight (HMW) and lowmolecular-weight (LMW) glutenin subunits (GS) from wheat, ω-secalins, γ-75k-secalins, γ-40k-secalins and HMW-secalins from rye, C-hordeins, γ-hordeins, B-hordeins and D-hordeins from barley and avenins from oats. These GPT contain different numbers of single proteins, e.g., HMW-GS with 3 - 5 proteins and α-gliadins and LMW-GS with more than 20 proteins. Well-defined proteins are essential for clinical studies, diagnosis of hypersensitivities and as reference materials (RM) for food analysis, such as the Prolamin Working Group (PWG)gliadin. Different RM have been used in various studies, but a
thorough characterization of the protein source, content and composition often is either missing or inaccessible, because the material is proprietary to a company. Gluten and gliadin preparations frequently used for both clinical and analytical purposes often differ in protein content and proportions of ALGL, prolamin and glutelin fractions and lack reproducibility, sometimes leading to questionable and contradictory conclusions. This study developed and applied a comprehensive strategy to isolate well-defined gluten protein fractions and GPT from wheat, rye, barley and oat flours suitable as RM for clinical assays and gluten-free compliance testing. First, the gluten protein fractions were extracted from the flours, dialyzed and lyophilized. Then, the GPT of each fraction were separated on preparative reversed-phase high-performance liquid chromatography (RP-HPLC), collected from several runs, pooled and lyophilized. The isolated GPT were extensively characterized using analytical RP-HPLC (Figure 1), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), N-terminal sequencing, electrospray-ionization quadrupole time-of-flight MS (LC-ESI-QTOF-MS) and untargeted liquid chromatography-mass spectrometry (LCMS/MS) of chymotryptic hydrolyzates of the single GPT. Taken together, the analytical methods confirmed that all GPT were reproducibly isolated in high purity from the flours and were suitable to be used as RM, e.g., for calibration of LCMS/MS methods or enzyme-linked immunosorbent assays (ELISAs). Katharina Anne Scherf German Institute for Food Chemistry Reference: Schalk K, Lexhaller B, Koehler P, Scherf KA. (2017) Isolation and characterization of gluten protein types from wheat, rye, barley and oats for use as reference materials. PLoS One, 12(2): e0172819. Download
Figure 1. RP-HPLC chromatograms of the isolated gliadin fraction and the gluten protein types ω5-gliadins, ω1,2-gliadins, α-gliadins and γ-gliadins. AU¬210, absorbance units at 210 nm
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NEWSLETTER Volume 8 | Issue 1
2017
Genome-derived proteomic reveals the hidden allergens of the Pacific oyster (Crassostra gigas) Oysters are famous for their delicacy; served raw and cooked. However, for up to 2% of the population, some oyster proteins are recognised by the immune system as harmful substances and provoke adverse immunological, known as allergy. Currently, little is known about which proteins are responsible for allergic reactions to oyster. Tropomyosin, a heat-stable protein, was previously identified as the only allergen in oyster, reacting with IgE antibody from shellfish allergic patients. This allergenic protein was however identified from cooked oyster meat. This type of sample preparation eliminates many proteins and leaves only heat-stable proteins. Since most people consume oysters raw, this cooking method reduces the chance of identifying relevant allergenic proteins. Furthermore, the traditional method for allergen identification based on IgE reactivity has two major drawbacks. Firstly, this approach may not necessarily detect allergens with low abundance, and secondly, IgE recognition patterns are highly dependent on the patient group recruited for the study and may skew the outcomes depending on the demographics of the cohort. The inclusion of bioinformatic analysis can help to overcome those limitations by taking the advantages of the growing allergen database and amino acid sequence comparison to assess protein allergenicity. The high degree of sequence conservation among allergenic proteins can give valuable information in predicting potential allergenic cross-reactivity between a protein and known allergens. Several algorithms including a full-length sequence alignment using FASTA or BLASTP program, a sliding 80mer FASTA and/or exact match of 8 amino acids have been recommended to evaluate protein allergenicity. We therefore combined the bioinformatics analysis with IgE reactivity assay to comprehensively identify potential allergens of the Pacific oyster (Figure 1). A repertoire of 25,982 genome-derived proteins from the Pacific oyster was aligned with 2117 allergen sequences obtained from two primary allergen database – IUIS/WHO Allergen Nomenclature Subcommittee and FARRP. Comparative analysis of those proteins
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Figure 1. Workflow of allergen identification from Pacific oyster (C. gigas) using a combination of bioinformatics, allergenomics and proteomics.
resulted in 831 proteins showing identity with known allergens and of those, 95 proteins were potentially allergenic, based on amino acid identity of over 50%. Several proteins and their respective isoforms have very high amino acid identity (>70%) with known allergens. Furthermore, their high identity is not only restricted to invertebrate proteins but is also extended to fish, edible plants and pollen. Immunoblotting against IgE of shellfish allergic patients confirmed 25 out of 95 potential allergens were presented in the IgE-reactive spots. The identified proteins share high amino acid identity with allergens from different organisms and with different routes of sensitisation; responsible for immunological cross reactivity of allergic patients to oyster and other organisms. The combination of genomics coupled with proteomics and patient IgE reactivity profiling is a powerful method for the identification of novel allergens from any food source. Using this combined approach, we were able to expand the knowledge on IgE reactivity to several proteins of the Pacific oyster. These newly identified allergens and the knowledge of their gene structure improves the current diagnostics and immunotherapy research on oyster allergy. Roni Nugraha & Andreas Lopata James Cook University
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NEWSLETTER Volume 8 | Issue 1
2017
Upcoming Events
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2nd Food Allergen Management Symposium
AOAC Food Allergen Community Newsletter Contribute with articles, news items or suggestions.
Global Harmonisation of Food Allergy Management Collaboration, innovation, science and communication May 21-24, 2017 Sydney, Australia
Submission deadline for the 2nd issue of 2017: July 21 Send your articles to
[email protected] Topics for publication 99 Regulatory Updates
FAMS2017 provides a unique opportunity for food industry, researchers, dieticians, clinicians and consumer groups to come together to address the needs of the food allergic consumer. In bringing together a diverse mix of key stakeholders, the FAMS2017 program will allow information sharing, new learnings and stimulate collaborative and innovative approaches to the future harmonisation of food allergen management
99 Food Industry Initiatives
FAMS2017 includes the Second Asia Pacific Food Allergen Management Workshop
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The AOAC Food Allergen Community is a forum serving the scientific community working on Food Allergens: The community aims to help AOAC INTERNATIONAL in its consensus-based scientific and advisory capacity on methods of analysis for allergens in foods and other commodities. It is also meant to serve the broader Stakeholder Community whose objectives it is to enhance the protection of food allergic consumers worldwide. Contact us at
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