CEN/TS 15633-3:2012

Foodstuffs - Detection of food allergens by immunological methods - Part 3: Quantitative determination of hazelnut with an enzyme immunoassay using polyclonal antibodies and Lowry protein detection

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.

Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren - Teil 3: Quantitative Bestimmung von Haselnuss mit einem Enzym-Immunoassayverfahren unter Verwendung von polyklonalen Antikörpern und Proteindetektion nach Lowry

Diese Technische Spezifikation legt ein ELISA Verfahren (enzymgebundener Immunosorbent-Test, en: Enzyme-linked immunosorbent assay) zur Bestimmung der Haselnusskonzentration in Lebensmittel¬proben fest.
Zur Validierung der Anwendung des Verfahrens auf Lebensmittelmatrices, wie z. B. Mehrkorn Zerealien, feinherbe Schokolade (45 % Kakao) und Eiscreme, wurden Aufstockversuche unter Verwendung von verdünnter gemahlener Haselnuss durchgeführt. Der Bereich des Verfahrens reicht von 0,5 mg bis 5,0 mg Hasel¬nussprotein je kg Lebensmittelprobe. Da Haselnusskerne üblicherweise zwischen 12 % bis 15 % Protein enthalten [2], [3], entspricht das etwa 3,7 mg bis 37 mg Haselnusskernen je kg Lebensmittelprobe. Die obere Grenze des Quantifizierungsbereichs kann bei Bedarf durch weitere Verdünnung von Probenextrakten erweitert werden.
Das Verfahren ist im Handel erhältlich ) und wurde vom Hersteller intern validiert. Die Daten sind in Anhang A.2 enthalten.
Das Verfahren wurde in einem Ringversuch erfolgreich validiert. Der Versuch wurde von der Arbeitsgruppe organisiert, die vom Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL) zur Durchführung von § 64 des Lebensmittel-, Bedarfsgegenstände- und Futtermittelgesetzbuches (LFGB) für die Bestimmung des Haselnussgehaltes in feinherber Schokolade eingerichtet wurde. Es nahmen 13 deutsche Laboratorien teil. Die Daten sind in Anhang A.3 enthalten.

Produits alimentaires - Détection des allergènes alimentaires par des méthodes d'analyse immunologiques - Partie 3: Détermination quantitative de la présence de noisette par un immuno-essai enzymatique à l'aide d'anticorps polyclonaux et détection des protéines par la méthode de Lowry

Cette Spécification Technique décrit un essai d'immuno-absorption enzymatique (ELISA) utilisé pour la
détermination de la concentration de noisette dans des échantillons de produits alimentaires.
Des expériences d’addition réalisées avec des noisettes moulues et diluées ont permis de valider l’utilisation
de la méthode avec des matrices alimentaires telles que des mélanges de céréales, du chocolat noir (45 % de
cacao) et de la crème glacée. L’étendue de la méthode se situe entre 0,5 mg et 5,0 mg de protéine de
noisette par kg d’échantillon de produit alimentaire. Comme les cerneaux de noisettes contiennent
habituellement entre 12 % et 15 % de protéine [2], [3], ceci équivaut à environ 3,7 mg à 37 mg de cerneau de
noisette par kg d’échantillon de produit alimentaire. Si nécessaire, la limite supérieure de la gamme de
quantification peut être repoussée, en diluant davantage les extraits d’échantillons.
Cette méthode est disponible sur le marché 1) et a été validée en interne par le fabricant. Les données
correspondantes figurent en Annexe au point A.2.
La méthode a été validée avec succès via une étude collaborative. L’étude a été organisée par le Groupe de
Travail établi par le Bureau Fédéral de la Protection des Consommateurs et de la Sécurité alimentaire (BVL)
en vue de la mise en application du § 64 du Code Allemand sur les produits alimentaires et la nourriture
(LFGB) pour la détermination de la teneur en noisette dans le chocolat noir. Treize laboratoires allemands y
ont participé. Les données correspondantes figurent en Annexe au point A.3.

Živila - Odkrivanje prisotnosti alergenov v živilih z imunološkimi metodami - 3. del: Kvantitativno določanje lešnika z encimsko-imunološko metodo z uporabo poliklonalnih protiteles in detekcijo beljakovin po Lowryju

Ta tehnična specifikacija določa metodo ELISA za določitev koncentracije lešnikov v vzorcih hrane. Preizkusi z dodatkom lešnikov z razredčenim semenom so bili uporabljeni za potrditev uporabe metode pri matricah, kot so mešane žita v zrnju, temna čokolada (45 % kakava) in sladoled. Razpon te metode je od 0,5 mg do 5,0 mg beljakovin lešnikov na kg vzorca hrane. Ker jedrca lešnikov običajno vsebujejo od 12 % do 15 % beljakovin, to ustreza približno od 3,7 mg do 37 mg lešnikovih jedrc na kg vzorca hrane. Zgornja meja količinskega razpona se po potrebi lahko zviša, in sicer z dodatnim redčenjem ekstrakta vzorca. Ta metoda je na voljo na tržišču in jo proizvajalec interno potrdi. Ti podatki so vključeni v Dodatek A.2. Ta metoda je uspešno potrjena z medlaboratorijsko študijo. Študijo je organizirala delovna skupina, ki jo je ustanovil Zvezni urad za zaščito potrošnikov in varnost živil za izvedbo ? 64 nemškega zakonika za hrano in krmo (LFGB) za določitev vsebnosti lešnikov v temni čokoladi. V medlaboratorijski študiji je sodelovalo trinajst nemških laboratorijev. Ti podatki so vključeni v Dodatek A.3.

General Information

• Status: Published
• Publication Date: 07-Aug-2012
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 12 - Food allergens
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Completion Date: 01-Mar-2023
• Ref Project: SIST-TS CEN/TS 15633-3:2012 - Foodstuffs - Detection of food allergens by immunological methods - Part 3: Quantitative determination of hazelnut with an enzyme immunoassay using polyclonal antibodies and Lowry protein detection

Standards Content (Sample)

SLOVENSKI STANDARD
01-december-2012
äLYLOD2GNULYDQMHSULVRWQRVWLDOHUJHQRYYåLYLOLK]LPXQRORãNLPLPHWRGDPLGHO
.YDQWLWDWLYQRGRORþDQMHOHãQLND]HQFLPVNRLPXQRORãNRPHWRGR]XSRUDER
SROLNORQDOQLKSURWLWHOHVLQGHWHNFLMREHOMDNRYLQSR/RZU\MX
Foodstuffs - Detection of food allergens by immunological methods - Part 3: Quantitative
determination of hazelnut with an enzyme immunoassay using polyclonal antibodies and
Lowry protein detection
Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren -
Teil 3: Quantitative Bestimmung von Haselnuss mit einem Enzym-
Immunoassayverfahren unter Verwendung von polyklonalen Antikörpern und
Proteindetektion nach Lowry
Produits alimentaires - Détection des allergènes par des méthodes immunologiques -
Partie3 : Détermination quantitative de la présence de noisette par un immuno-essai
enzymatique à l'aide d'anticorps polyclonaux et détection des protéines par la méthode
de Lowry
Ta slovenski standard je istoveten z: CEN/TS 15633-3:2012
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL SPECIFICATION
CEN/TS 15633-3
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
August 2012
ICS 67.050
English Version
Foodstuffs - Detection of food allergens by immunological
methods - Part 3: Quantitative determination of hazelnut with an
enzyme immunoassay using polyclonal antibodies and Lowry
protein detection
Produits alimentaires - Détection des allergènes par des
Lebensmittel - Nachweis von Lebensmittelallergenen mit
méthodes immunologiques - Partie3 : Détermination immunologischen Verfahren - Teil 3: Quantitative
quantitative de la présence de noisette par un immuno- Bestimmung von Haselnuss mit einem Enzym-
essai enzymatique à l'aide d'anticorps polyclonaux et Immunoassayverfahren unter Verwendung von
détection des protéines par la méthode de Lowry polyklonalen Antikörpern und Proteindetektion nach Lowry
This Technical Specification (CEN/TS) was approved by CEN on 4 March 2012 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15633-3:2012: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 Principle .5
5 Reagents .6
5.1 Reagents usually supplied with the kit, as standard .6
5.2 Preparation of Working Strength Solutions from Concentrates .7
5.3 Reagents not supplied with the test kit .7
6 Apparatus and equipment .8
7 General instructions/recommendations/preparation .8
7.1 General .8
7.2 Preparation of sample .9
7.3 Immunoassay procedure/Operational Scheme . 10
7.4 Reading/Interpretation and test result report (Calculations, Reporting) . 11
8 Estimation of measurement uncertainty . 13
Annex A (informative) Validation status and performance criteria/method performance . 14
A.1 General . 14
A.2 Internal validation (manufacturer´s in house study). . 14
A.3 Collaborative study. 31
Bibliography . 33

Foreword
This document (CEN/TS 15633-3:2012) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This Technical Specification consists of the following parts:
 CEN/TS 15633-1, Foodstuffs — Detection of food allergens by immunological methods — Part 1: General
considerations
 FprCEN/TS 15633-2, Foodstuffs — Detection of food allergens by immunological methods — Part 2:
Quantitative determination of hazelnut with an enzyme immunoassay using monoclonal antibodies and
bicinchoninic acid-protein detection
 CEN/TS 15633-3, Foodstuffs — Detection of food allergens by immunological methods — Part 3:
Quantitative determination of hazelnut with an enzyme immunoassay using polyclonal antibodies and
Lowry protein detection
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany,
Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Introduction
The hazelnut (Corylus avellana) belongs to a group of foods commonly referred to as tree nuts. Allergic
reactions to hazelnut ranging from oral allergy syndrome to severe anaphylaxis have been widely reported.
Food labelling legislation requires the presence of hazelnut in food to be declared in numerous nations of the
European Union, North America, Asia and Australasia.
IgE binding studies from the sera of sensitized patients have revealed a number of allergens, including both
pollen related and non-pollen related allergens [1]. Threshold dose studies have reported provocative doses
as low as 1 mg of hazelnut protein [1].
Hazelnut material may occur unintentionally in foods for several reasons. It may be present in contaminated
ingredients or cross contamination may occur during food manufacture. Consequently there is a need for
sensitive and reliable tests for the detection of hazelnut in food samples.
1 Scope
This Technical Specification specifies an enzyme-linked immunosorbent assay (ELISA)-method for the
determination of hazelnut concentration in food samples.
Spiking experiments with 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.
1)
The method is commercially available and has been validated in-house by the manufacturer. These data are
included in Annex 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 in the collaborative study. These data are included
in Annex A.3.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
EN 15633-1:2008, Foodstuffs – Detection of food allergens by immunological methods – Part 1: General
considerations
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 15633-1 apply.
4 Principle
A direct sandwich ELISA is used for detection of hazelnut protein.
A 13 kDa to 14 kDa protein band common to both raw and roasted hazelnuts was identified using polyacryl-
amide gel electrophoresis. This protein marker was purified by high performance liquid chromatography and
used to raise rabbit anti-hazelnut polyclonal anti-sera. The IgG fraction of this antiserum was purified by
affinity chromatography then used to develop a direct sandwich ELISA as outlined below:
1) Soluble hazelnut protein is extracted from a food sample.
2) The extract is then added to micro-titre wells coated with the anti-hazelnut capture antibody. The sample
is allowed to react before thorough washing.

1) ELISA Systems Hazelnut Residue ELISA is the trade name of a product supplied by ELISA Systems Pty Ltd, Brisbane, Australia.
This information is given for the convenience of users of this Technical Specification and does not constitute an endorsement by CEN of
the product named. Equivalent products may be used if they can be shown to lead to the same results.
3) The conjugate (horseradish peroxidase labelled anti-hazelnut antibodies) is then added and allowed to
react before thorough washing. Hazelnut protein captured in step 2 will bind the conjugate to form a sandwich.
4) Tetramethylbenzidine (TMB), the chromogenic substrate, is added and allowed to react. Hazelnut protein-
antibody sandwiches will cause development of a blue colour.
5) The reaction is stopped by the addition of 1 mol/l phosphoric acid which causes a colour change to
yellow.
A spectrophotometer (450 nm primary wavelength and 620 nm to 650 nm reference wavelength) is used to
measure the optical density (OD) of each well.
The OD produced is determined by the hazelnut protein concentration. The hazelnut concentration of a
sample can be calculated by comparison of the OD it generates with the OD generated by standards. The
standards are calibrated in hazelnut protein mg/kg. They were prepared from an aqueous extract of
commercial hazelnuts (Corylus avellana). The protein concentration was determined by the Lowry method.
5 Reagents
5.1 Reagents usually supplied with the kit, as standard
5.1.1 Extraction Solution
The routine extraction solution is phosphate buffered saline with Tween 20 (PBST).The solution is supplied as
a 20-fold concentrate and requires dilution to working strength prior to use (see 5.2.1).
Table 1 — Composition of ELISA Systems Working Strength PBST
Compound Working
Concentration
KH PO 0,2 g/l
2 4
NaH PO 1,2 g/l
2 4
Tween 20 2 ml/l
NaCl 8 g/l
KCl 0,2 g/l
Ethyl(2-mercaptobenzoato-(2-)-O,S)mercurate(1-) sodium 0,2 g/l
(Thiomersal (INN))
5.1.2 ESADDSOL (Enhanced Extraction Solution for Samples containing Polyphenols)
A specialized extraction solution should be used for foods containing polyphenols. The solution is supplied as
a concentrate and requires dilution prior to use (see 5.2.2). The working strength s
...