Foodstuffs - Detection of food allergens by immunological methods - Part 1: General considerations

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.

Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren - Teil 1: Allgemeine Betrachtungen

Dieses Dokument stellt einen Gesamtrahmen für die qualitativen und quantitativen Verfahren zur Bestimmung von Lebensmittelallergenen und allergenen Inhaltsstoffen unter Anwendung von auf Antikörpern basierenden Verfahren in Lebensmitteln zur Verfügung. Dieses Dokument legt die allgemeinen Leitlinien und Leistungskriterien für auf Antikörpern basierende Verfahren zum Nachweis und zur quantitativen Bestimmung von Proteinen fest, die als Marker für das Vorliegen von allergieauslösenden Lebensmitteln oder Lebensmittelzutaten dienen. Auch mit anderen als den beschriebenen Verfahren können die Proteine nachgewiesen und identifiziert werden. Die in diesem Dokument angeführten Leitlinien, Mindestanforderungen und Leistungskriterien sind dafür vorgesehen, sicherzustellen, dass von verschiedenen Analytikern in privaten und/oder offiziellen Kontrolllaboratorien oder bei der Durchführung von Vor-Ort-Lebensmittelanalysen reproduzierbare Ergebnisse erhalten werden.
Dieses Dokument ist für die Verwendung zusätzlich zu EN 15842 vorgesehen.
ANMERKUNG Dieses Dokument könnte auch auf andere Probentypen anwendbar sein, bei denen dieselben Grundsätze für die Verfahrensvalidierung und -überprüfung gelten würden.

Produits alimentaires - Détection des allergènes alimentaires par des méthodes d’analyse immunologiques - Partie 1 : Considérations générales

Le présent document fournit un cadre général relatif aux méthodes qualitatives et quantitatives utilisées pour déterminer les allergènes et ingrédients allergéniques alimentaires, en appliquant des méthodes basées sur des anticorps. Le présent document énonce des lignes directrices et des critères de performances généraux relatifs à des méthodes basées sur des anticorps, en vue de la détection et de la quantification de protéines qui servent de marqueurs indiquant la présence d’aliments ou d’ingrédients alimentaires à l’origine d’allergies. Il existe d’autres méthodes que celles décrites, permettant de détecter et d’identifier les protéines. Les lignes directrices, les exigences minimales et les critères de performances établis dans le présent document sont destinés à s’assurer que les analyses réalisées par différentes personnes dans des laboratoires de contrôle privés et/ou publics ou sur le terrain produiront des résultats reproductibles.
Le présent document est destiné à être utilisé conjointement avec l’EN 15842.
NOTE   Le présent document pourrait également s’appliquer à d’autres types d’échantillons dès lors que les mêmes principes régissent la validation et la vérification des méthodes.

Živila - Odkrivanje prisotnosti alergenov v živilih z imunološkimi metodami - 1. del: Splošne ugotovitve

Ta dokument podaja splošen okvir kvalitativnih in kvantitativnih metod za določanje alergenov in alergenih sestavin z uporabo metod na osnovi protiteles v živilih. Ta evropski standard določa splošne smernice in merila učinkovitosti metod na osnovi protiteles za odkrivanje in količinsko določanje beljakovin, ki služijo kot markerji za prisotnost živil ali sestavin hrane, ki izzovejo alergijo. Razen z opisanimi metodami je mogoče beljakovine zaznati in prepoznati tudi z drugimi metodami. Smernice, minimalne zahteve in merila učinkovitosti iz tega evropskega standarda so namenjena zagotavljanju, da različni analitiki v prostorih s hrano in laboratorijih pridobijo primerljive ter ponovljive rezultate.

General Information

Status
Published
Publication Date
01-Oct-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
02-Oct-2019
Completion Date
02-Oct-2019

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SLOVENSKI STANDARD
SIST EN 15633-1:2019
01-december-2019
Nadomešča:
SIST EN 15633-1:2009

Živila - Odkrivanje prisotnosti alergenov v živilih z imunološkimi metodami - 1. del:

Splošne ugotovitve

Foodstuffs - Detection of food allergens by immunological methods - Part 1: General

considerations

Lebensmittel - Nachweis von Lebensmittelallergenen mit immunologischen Verfahren -

Teil 1: Allgemeine Betrachtungen
Produits alimentaires - Détection des allergènes alimentaires par des méthodes
d’analyse immunologiques - Partie 1: Considérations générales
Ta slovenski standard je istoveten z: EN 15633-1:2019
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN 15633-1:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 15633-1:2019
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SIST EN 15633-1:2019
EN 15633-1
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 67.050 Supersedes EN 15633-1:2009
English Version
Foodstuffs - Detection of food allergens by immunological
methods - Part 1: General considerations

Produits alimentaires - Détection des allergènes Lebensmittel - Nachweis von Lebensmittelallergenen

alimentaires par des méthodes d'analyse mit immunologischen Verfahren - Teil 1: Allgemeine

immunologiques - Partie 1 : Considérations générales Betrachtungen
This European Standard was approved by CEN on 12 August 2019.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15633-1:2019 E

worldwide for CEN national Members.
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SIST EN 15633-1:2019
EN 15633-1:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 General requirements for laboratories .................................................................................................. 8

5 Procedure........................................................................................................................................................... 8

6 Interpretation and expression of results ............................................................................................ 10

7 Specific parameters which can influence results ............................................................................. 12

8 Test report ...................................................................................................................................................... 14

Bibliography ................................................................................................................................................................. 15

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SIST EN 15633-1:2019
EN 15633-1:2019 (E)
European foreword

This document (EN 15633-1:2019) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - horizontal methods”, the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by April 2020, and conflicting national standards shall be

withdrawn at the latest by April 2020.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document supersedes EN 15633-1:2009.

Significant technical changes between this standard and EN 15633-1:2009 are as follows:

a) updated terms and definitions (clause 3);
b) updated pre-requisite requirements for analysis (clause 5);
c) updated method validation parameters (clause 6);
d) updated specific influences on results (clause 7).

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
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SIST EN 15633-1:2019
EN 15633-1:2019 (E)
Introduction

A specific protein or group of proteins or peptides deriving from these proteins can serve as a marker

for the presence of food or food ingredients provoking allergic reactions. This document describes the

procedure to qualitatively detect and/or quantitate protein-derived analytes or proteins/peptides or

proteinaceous entities as a marker for potentially allergenic ingredients or constituents by analysing

the protein extracted from the sample under test. Appropriate procedures for extraction of the protein

are included in each method. The focus of this document is on antibody-based methods where a protein

or group of proteins or peptides (deriving from these proteins) representative for the allergen source is

qualitatively or quantitatively determined.
For the use of this document the term:
— ‘shall’ indicates a requirement;
— ‘should’ indicates a recommendation;
— ‘may’ indicates a permission; and
— ‘can’ indicates a possibility and/or a capability.
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SIST EN 15633-1:2019
EN 15633-1:2019 (E)
1 Scope

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.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN 15842, Foodstuffs — Detection of food allergens — General considerations and validation of methods

3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 15842 and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.1 General terms
3.1.1
denaturation of proteins

treatment (whether thermal, chemical, enzymatic or other) that affects the conformation of proteins

(i.e. the secondary, tertiary and quaternary structure) to such an extent that only the primary structure

or parts thereof remain (either intact, fragmented or hydrolysed)

Note 1 to entry: The denaturation can modify functional, enzymatic or antigenic properties of the protein.

3.1.2
cross-linkage of proteins
chemical reaction and/or physical interaction between proteins

Note 1 to entry: Cross-linkage can modify extractability of a protein within a food matrix.

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SIST EN 15633-1:2019
EN 15633-1:2019 (E)
3.2 Antibody-related terms
3.2.1
antibody

protein (immunoglobulin) produced and secreted by B lymphocytes in vertebrates in response to a

molecule recognised as foreign (antigen)

Note 1 to entry: The antibody is capable of binding to that specific antigen or related peptide structures e.g.

epitopes.
Note 2 to entry: The antigen induces an immune response.

[SOURCE: EN ISO 21572:2013, 3.2.1, modified — informative part of definition is described in Note 1 to

entry and Note 2 to entry was added]
3.2.2
antigen

substance recognised by an antibody (namely the substance that is recognised as ‘foreign’ by the

immune system and elicits an immune response)

Note 1 to entry: The antigen reacts in vivo and in vitro specifically with the generated antibodies.

Note 2 to entry: The antigen induces an immune response.
3.2.3
allergen

antigen that induces an immunoglobulin-E mediated allergic reaction (except gluten)

Note 1 to entry: An allergen within the scope of this document is of proteinaceous nature.

3.2.4
clone
population of identical cells derived from a single cell
[SOURCE: EN ISO 21572:2013, 3.2.3]
3.2.5
monoclonal antibody

antibody produced from a single hybridoma clone and directed to a single epitope on the antigen

Note 1 to entry: Antibodies produced from a B-cell clone with identical physical, biochemical and

immunological properties
3.2.6
polyclonal antibodies

antibodies produced by several B cells that recognise different epitopes of the same antigen

3.2.7
specificity of an antibody

ability of an antibody to specifically bind to a particular antigen epitope and not to other similar

structures on the same or other antigens

Note 1 to entry: The ability of antibodies to recognize and distinguish between related structures.

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SIST EN 15633-1:2019
EN 15633-1:2019 (E)
3.2.8
cross-reactivity
binding of an antibody to substances other than the antigen of primary interest

Note 1 to entry: The ability of antibodies to bind to similar epitopes present on different antigens.

Note 2 to entry: The reaction of an antibody with another antigen than those used for immunization.

[SOURCE: EN ISO 21572:2013, 3.2.4, modified — notes 1 and 2 were added]
3.2.9
epitope

region of an antigen (e.g. linear sequence or conformational) specifically recognised by an antibody or

by receptors on cells
3.2.10
conjugate
material produced by attaching two or more substances together

Note 1 to entry: Conjugates of antibodies/proteins with fluorochromes, coloured particles, gold nanoparticles,

radio-labelled substances, or enzymes are often used in immunoassays.

Note 2 to entry: A conjugate is an antibody or substance (e.g. Avidin) that is linked to a detector moiety such as

an enzyme, fluorochrome, radioactive or solid particle, allowing the production of a detectable signal. Enzyme-

linked conjugates require the addition of specific substrates to form measurable (e.g. coloured) reaction products.

3.3 Method-related terms
3.3.1
immunoblotting

transfer of proteins (i.e. the protein of interest), following electrophoretic separation, to a binding

surface (membrane) and visualisation of the antigen(s) with specific antibody-reporter conjugates

Note 1 to entry: Transfer of electrophoretically-separated proteins to a polymer sheet or nitrocellulose

membrane.
Note 2 to entry: This term is not further discussed in this document.
3.3.2
enzyme-linked immunosorbent assay
ELISA

in vitro assay for the detection of antigens that combines enzyme-linked antibodies (or antigen) and a

specific substrate to form a coloured or fluorescent reaction product

Note 1 to entry: Depending on the application, this assay can be used for qualitative or quantitative purposes.

Note 2 to entry: The ELISA assay is usually performed in the microwell plate format.

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EN 15633-1:2019 (E)
4 General requirements for laboratories
4.1 Principle

The target analyte/antigen (proteinaceous in nature) is extracted from a sample according to a matrix-

specific procedure or a general method, and a specific monoclonal or polyclonal antibody is used to

qualitatively detect or quantitatively measure the concentration of the target analyte in the sample.

4.2 Apparatus and equipment

The analyst should use maintained equipment suitable for the method employed, (standard laboratory

equipment and other items as described in the specific methods) and calibration systems for this

equipment should be available and routinely performed.
4.3 Material and reagents

During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only

de-ionized or distilled water or water of known purity. All components of a test kit are method-specific

and should only be used as a complete set and not interchanged with components of another kit. For the

correct handling of reagents such as standards, reference material, antibodies coated to a solid surface

or free in solution, controls, and samples; refer to the described method or kit instructions. Storage

conditions and shelf-life of reagents should be clearly specified in the method protocol.

5 Procedure
5.1 General

For the use of this document, general requirements of quality assurance for laboratories shall be

observed (e.g. concerning calibration of apparatus, extraction of samples and measured replicates,

blanks, use of reference materials, preparation of calibration curves, etc.). Appropriate principles can be

found in EN ISO/IEC 17025. Carefully clean all equipment coming into direct contact with the sample to

prevent cross-contamination. The scope of the method, including applicability to matrices, needs to be

clearly defined.

Before conducting food allergen analysis special considerations should be made regarding:

a) the laboratory lay-out (e.g. ideally extraction should be spatially separated from detection);

b) the current workflow (e.g. other activities in the laboratories that can increase the potential for

cross-contamination);

c) sample types handled (e.g. cross-contamination issues can undermine the capability to perform

analysis reliably);

d) equipment in the laboratories (e.g. the risk of cross contamination should be considered if

equipment is shared, use dedicated equipment where appropriate);

e) containers (e.g. disposable consumables are preferable, those that exhibit low protein binding - not

polystyrene);

f) general house-keeping tasks (e.g. effective cleaning of items and surfaces is important and should

be considered).
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SIST EN 15633-1:2019
EN 15633-1:2019 (E)
5.2 Sample preparation

To ensure reliable results sample preparation is a critical step in food allergen analysis, the objective

being to provide a homogenous and representative sample of the original lot. Equipment should be

available in the laboratory for sample homogenization - including apparatus for milling, chopping,

grinding and blending - the exact items depending upon the sample types to be routinely analysed. All

sample preparation procedures should be validated to ensure their effectiveness.

Method performance controls for the determination of recoveries, matrix effects, etc. often include the

‘spiking' of samples with known amounts of the measured allergen. Incurred control samples are

preferred and contain material produced by spiking with the relevant allergen before products are

manufactured/processed in a manner that is comparable to the test samples to be analysed. If samples

are spiked these controls can be used to monitor the repeatability and reproducibility of the method.

The spike level chosen should have relevance to the expected concentration levels. Spiking levels in the

middle of the method’s calibration curve working range usually achieve the best precision data.

A larger test portion will usually improve the potential for detection, as will reducing the particle size to

improve sample homogeneity.
5.3 Extraction

The protein is extracted according to the procedure described in the specific method protocol.

Considerations should be given to:

a) the possibility of increasing the amounts, whilst maintaining the ratio between t

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