CEN/TC 275/WG 12 - Food allergens

This document specifies minimum performance requirements for methods that quantify the food allergens milk, egg, peanut, hazelnut, almond, brazil nut, macadamia nut, cashew, pistachio nut, walnut, pecan nut, lupine, sesame, mustard, soy, celery, fish, molluscs, crustaceans, and wheat in raw and processed foodstuffs. Within the scope of this document, minimum requirements for an LOQ (Limit of Quantification) will be derived from threshold data of allergic consumers. For quantitative antibody-based methods, a normative annex will describe what specific information the method developer needs to deliver and how performance characteristics shall be validated. Regarding PCR and LC-MS/MS, information on performance characteristics are in parts covered by EN 15634-1 and EN 17644. This document does not apply to fragmented or hydrolysed food allergens, such as casein hydrolysates or soy sauce. It also does not apply to methods that deliver qualitative results only. Methods that cover gluten-containing cereals (wheat, rye, and barley) with regard to coeliac disease are covered by EN 17254.

This document specifies a method for the detection of hazelnut (Corylus avellana) in chocolate.
Real-time PCR (Polymerase chain reaction) detection of hazelnut is based on an152 bp (base pair) sequence from the corA 1 gene of hazelnut.

This document specifies a procedure for the qualitative detection of species specific DNA from white mustard (Sinapis alba) and soya (Glycine max) in cooked sausages using singleplex real-time PCR based on the genes MADS-D (mustard) and lectin (soya).

This document specifies a method for the detection of peanut (Arachis hypogaea) in chocolate.
Real-time PCR (Polymerase Chain Reaction) detection of peanut is based on an 86 bp (base pair) sequence from the Ara h 2 gene of peanut.

This document establishes an overall framework covering qualitative and quantitative methods for the determination of food allergens and allergenic ingredients using mass spectrometry-based methods for the determination of specific peptides/proteins. This document provides general guidelines and performance criteria applicable to this methodology. Guidelines, minimum requirements and performance criteria laid down in this document are intended to ensure that comparable and reproducible results are obtained by different analysts, instrumentation and laboratories.

This document specifies minimum method performance requirements for enzyme-linked immunosorbent assays that quantify non-fragmented or fragmented gluten from wheat (e.g. Triticum aestivum), rye, and barley in raw and processed foodstuffs.
This document is intended to be used in addition to EN 15842.

This document specifies a method for the detection of celery (Apium graveolens) in emulsion-type sausages (e.g. Frankfurter, Wiener).
Real-time PCR (polymerase chain reaction) detection of celery is based on an 101 bp (base pair) sequence from the gene of the mannitol dehydrogenase (GenBank Acc. No. AF067082 ) of celery (Apium graveolens).
The method has been validated on emulsion-type sausages (Bavarian “Leberkäse”) spiked with celery. For this purpose meat batter containing mass fractions of 50 % pork meat, 25 % pork fat, 23 % crushed ice and 1,8 % of a mixture of sodium chloride, nitrite, nitrate, phosphates and ascorbates was prepared according to a standard procedure for emulsion-type sausage. The meat batter was spiked with either ground celery seeds or celery root powder to 1000 mg/kg. Lower spiking levels were obtained by diluting with celery-free meat batter. The batter was stuffed into casings and heated at 65 °C for 60 min [1].
This document is intended to be used in addition to EN 15842 and FprEN 15634 1.

This document provides an overall framework covering qualitative and quantitative methods for the determination of food allergens and allergenic ingredients using antibody-based methods in foods. This document specifies general guidelines and performance criteria for antibody-based methods for the detection and quantification of proteins that serve as markers for the presence of allergy provoking foods or food ingredients. Other methods than those described can also detect and identify the proteins. Guidelines, minimum requirements and performance criteria laid down in this document are intended to ensure that reproducible results are obtained by different analysts in private and/or official control laboratories or when conducting onsite food testing.
This document is intended to be used in addition to EN 15842.
NOTE   This document could also be applicable to other sample types where the same principles for method validation and verification would apply.

This document provides the overall framework for detection of sequences corresponding to species containing allergens using the polymerase chain reaction (PCR). It relates to the requirements for the specific amplification of target nucleic acid sequences (DNA) and for the confirmation of the identity of the amplified nucleic acid sequence.
Guidelines, minimum requirements and performance criteria laid down in European Standards are intended to ensure that comparable and reproducible results are obtained in different laboratories. This document has been established for food matrices.
This document is intended to be used in addition to EN 15842.

This document specifies how to use the standards for immunoassays, nucleic based and chromatographic methods and their relationship in the analysis of food allergens; and contains general definitions, requirements and guidelines for laboratory set-up, method validation requirements, description of methods, and test reports.
This document also specifies general guidelines for the requirements and use of reference materials for the determination of allergenic commodities in food products. The term "reference materials" in this document includes certified reference materials as well as quality control materials. Currently only a limited number of reference materials for food allergen determination are available. As new materials become accepted and validated, they can be appended as an annex to this document.
This document does not deal with sampling issues. It simply details processes involved from receipt of the laboratory sample to the end result.

This Technical Specification specifies an enzyme linked immunsorbent assay (ELISA) method for the determination of hazelnut from food samples. In the ELISA the antibodies bind to hazelnut proteins from the food sample. The result of the ELISA is given in mg hazelnut/kg (ppm) because the calibrators consist of an extract of whole hazelnut.
Matrices like cereals, ice cream, cookies, chocolate, sausage, cottage cheese, yogurt and salad dressing were validated by spiking experiments with a carboxymethylcellulose-suspension containing hazelnut paste [2].
The monoclonal antibodies, raised against the whole aqueous extract of hazelnut, detect proteins with approximate molecular weights of 14 kDa, 18 kDa, and 42 kDa. The antibodies detect the major thermostable allergen Cor a9 (11S storage protein). Both antibodies were evaluated by western blots with partially purified hazelnut extracts and purified allergenic proteins.
The ELISA test method is commercially available ). The performance has been validated by an in house validation performed by the manufacturer. All parameters of interest are indicated.
In addition, the ELISA was successfully validated by a collaborative study in order to determine the interlaboratory reproducibility. This ring trial was organised by the working group established by the Federal Office of Consumer Protection and Food Safety (BVL) for the execution of § 64 of the German Food and Feed Code (LFGB) for the determination of hazelnut content in dark chocolate. Fourteen German laboratories participated in this collaborative study.

This Technical Specification specifies an ELISA-method for the determination of hazelnut concentration in food samples.
Spiking experiments using diluted ground hazelnut have been used to validate the method’s use on food matrices such as mixed grain cereals, dark chocolate (45 % cocoa) and ice cream. The range of the method is 0,5 mg to 5,0 mg hazelnut protein per kg of food sample. As hazelnut kernels typically contain between 12 % to 15 % protein [2], [3], this equates to approximately 3,7 mg to 37 mg hazelnut kernel per kg of food sample. The upper limit of the range of quantitation can be extended, if required, by further dilution of sample extracts.
The method is commercially available ) and has been validated in-house by the manufacturer. The data is included in A.2.
The method has been successfully validated by a collaborative study. The study was organized by the Working Group established by the Federal Office of Consumer Protection and Food Safety (BVL) for the execution of § 64 of the German Food and Feed Code (LFGB) for the determination of hazelnut content in dark chocolate. Thirteen German laboratories participated. The data are included in A.3.

This Technical Report describes necessary information for method providers which needs to be provided with proposals for new work items for consideration in CEN/TC 275/WG 12 "Food allergens".

This Technical Specification specifies a procedure for the qualitative detection of species specific DNA from white mustard (Sinapis alba) and soya (Glycine max) in cooked sausages using singleplex real-time PCR based on the genes MADS-D (mustard) and lectin (soya) [1]. A mustard content of 10 mg/kg or greater and a soya content of 10 mg/kg or greater can be detected with a probability of > 95 %.

This Technical Specification describes a procedure for the qualitative detection of peanut (Arachis hypogaea) in chocolate using real-time PCR based on the gene for the peanut allergen Ara h 2 [1], [2].

This Technical Specification describes a procedure for the qualitative detection of hazelnut (Corylus avellana) in chocolate. DNA is extracted from the chocolate and a specific DNA sequence for hazelnut detected from the gene for corA 1 [4], [5].

This Technical Specification specifies a method for the qualitative detection of celery (Apium graveolens) in emulsion-type sausages (e.g. frankfurter, wiener).
Real-time PCR detection of celery is based on a 101 bp (base pair) sequence from the gene of the mannitol dehydrogenase (GenBank acc. no. AF067082) of celery (Apium graveolens).
The method has been validated on emulsion-type sausages (Bavarian "Leberkäse") spiked with celery. For this purpose meat batter containing mass fractions of 50 % pork meat, 25 % pork fat, 23 % crushed ice and 1,8 % of a mixture of sodium chloride, nitrite, nitrate, phosphates and ascorbates was prepared according to a standard procedure for emulsion-type sausage. The meat batter was spiked with ground celery seeds or alternatively celery root powder, both at a 1000 mg/kg level. Lower spiking levels were obtained by diluting with celery-free meat batter. The batter was stuffed into casings and heated at 65 °C for 60 min [2].

This European Standard specifies how to use the standards for immunoassays, nucleic based and chromatographic methods and their relationship in the analysis of food allergens; and contains general definitions, requirements and guidelines for laboratory set-up, method validation requirements, description of methods, and test reports.
This document also specifies general guidelines for the requirements and use of reference materials for the determination of allergenic commodities in food products. The term "reference materials" in this document includes certified reference materials as well as quality control materials. Currently only a limited number of reference materials for food allergen determination are available. As new materials become accepted and validated, they may be appended as an annex to this document.
This document does not deal with sampling issues. It simply details processes involved from receipt of the laboratory sample to the end result.

This European Standard provides the overall framework for detection of sequences corresponding to species containing allergens using the polymerase chain reaction (PCR). It relates to the requirements for the specific amplification of target nucleic acid sequences (DNA) and for the confirmation of the identity of the amplified nucleic acid sequence.
Guidelines, minimum requirements and performance criteria laid down in the European Standard are intended to ensure that comparable and reproducible results are obtained in different laboratories. This European Standard has been established for food matrices.

This European Standard provides the overall framework of qualitative and quantitative methods for the determination of allergens and allergenic ingredients in foodstuffs using antibody-based methods. This European Standard specifies general guidelines and performance criteria for antibody-based methods for the detection and quantification of proteins that serve as a marker for the presence of allergy provoking foods or food ingredients. Other methods than those described may also detect and identify the proteins. Guidelines, minimum requirements and performance criteria laid down in the European Standard are intended to ensure that comparable and reproducible results are obtained in different laboratories. This European Standard has been established for food matrices.