1、April 2009DEUTSCHE NORM English price group 9No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.050!$Vo;“1517624www.di
2、n.deDDIN EN 15633-1Foodstuffs Detection of food allergens by immunological methods Part 1: General considerationsEnglish version of DIN EN 15633-1:2009-04Lebensmittel Nachweis von Lebensmittelallergenen mit immunologischen Verfahren Teil 1: Allgemeine BetrachtungenEnglische Fassung DIN EN 15633-1:20
3、09-04www.beuth.deDocument comprises 14 pagesDIN EN 15633-1:2009-04 2 National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Leb
4、ensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-05 AA Lebensmittelallergene. Further parts of this standards series will be developed in the future. EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15633-1January 2009ICS
5、 67.050English VersionFoodstuffs - Detection of food allergens by immunologicalmethods - Part 1: General considerationsProduits alimentaires - Dtection des allergnesalimentaires par des mthodes danalyse immunologiques -Partie 1: Considrations gnralesLebensmittel - Nachweis von Lebensmittelallergenen
6、 mitimmunologischen Verfahren - Teil 1: AllgemeineBetrachtungenThis European Standard was approved by CEN on 1 December 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard wit
7、hout any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language
8、made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,Franc
9、e, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORM
10、UNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15633-1:2009: EEN 15633-1:2009 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Normative references 53 Terms and d
11、efinitions .54 General laboratory requirements .85 Procedure .86 Interpretation and expression of results .97 Specific parameters which may influence results 108 Test report . 11Bibliography . 12DIN EN 15633-1:2009-04 EN 15633-1:2009 (E) 3 Foreword This document (EN 15633-1:2009) has been prepared b
12、y 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 July 2009, and conflicting national
13、standards shall be withdrawn at the latest by July 2009. 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. According to the CEN/CENELEC
14、Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg
15、, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15633-1:2009-04 EN 15633-1:2009 (E) 4 Introduction A specific protein or group of proteins or peptides deriving from these proteins can serve as a marker for the pre
16、sence of food or food ingredients provoking allergic reactions. This European Standard describes the procedure to qualitatively detect and/or quantitate a protein as a marker for potentially allergenic ingredients or constituents by analysing the protein extracted from the sample under study. Approp
17、riate procedures for extraction of the protein are included in each method. The main focus of this European Standard will be on antibody based methods. DIN EN 15633-1:2009-04 EN 15633-1:2009 (E) 5 1 Scope This European Standard provides the overall framework of qualitative and quantitative methods f
18、or 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
19、 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 dif
20、ferent laboratories. This European Standard has been established for food matrices. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of th
21、e referenced document (including any amendments) applies. prEN 15842:2008, 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 prEN 15842:2008 and the following apply. 3.
22、1 General terms 3.1.1 denaturation of proteins treatment (thermal or chemical), which destroys or modifies the secondary and/or tertiary structure of a protein NOTE The denaturation may modify functional, enzymatic or antigenic properties of the protein. 3.2 Terms relative to antibodies 3.2.1 antibo
23、dy protein (immunoglobulin) produced and secreted by B lymphocytes in response to a molecule recognised as foreign (antigen) adapted from EN ISO 21572:2004 1 NOTE 1 The antibody is capable of binding to that specific antigen. NOTE 2 Antibodies recognise specific areas in the antigen called epitopes.
24、 3.2.2 antigen substance that is recognised as foreign by the immune system and elicits an immune response DIN EN 15633-1:2009-04 EN 15633-1:2009 (E) 6 EN ISO 21572:2004 1 NOTE 1 The antigen reacts in vivo and in vitro specifically with the generated antibodies. NOTE 2 The antigen elicits an immune
25、response to that antigen. 3.2.3 allergen antigen with special properties to induce an allergic reaction 3.2.4 clone population of identical cells derived from a single cell line EN ISO 21572:2004 1 3.2.5 cross-reactivity binding of the antibody to substances other than the analyte of primary interes
26、t EN ISO 21572:2004 1 NOTE 1 The ability of antibodies to bind to similar epitopes present on different antigens. NOTE 2 The reaction of an antibody with another antigen than those used for immunisation. 3.2.6 monoclonal antibody antibody produced from a single hybridoma clone and directed to a sing
27、le antigen determinant EN ISO 21572:2004 1 NOTE Antibodies produced from a B-cell clone with identical physical, biochemical and immunological properties. 3.2.7 polyclonal antibodies antibodies produced by several clones of lymphocytes EN ISO 21572:2004 1 NOTE Antibodies produced by several B-cells
28、that recognise different epitopes in the same antigen. 3.2.8 specificity of an antibody ability of an antibody to specifically bind to an antigen determinant and not to other similar structures on that or other antigens EN ISO 21572:2004 1 NOTE The ability of antibodies to recognise and distinguish
29、between related structures. 3.2.9 epitope molecular structure of an antigen specifically recognised by antibodies or receptors on cells DIN EN 15633-1:2009-04 EN 15633-1:2009 (E) 7 3.3 Terms relative to methods 3.3.1 conjugate material produced by attaching two or more substances together EN ISO 215
30、72:2004 1 NOTE 1 Conjugates of antibodies with fluorochromes (e.g. coloured particles), radio-labelled substances, or enzymes are often used in immunosassays. NOTE 2 A conjugate (including conjugated antibody) is an antibody or substance (e.g. Avidin), that is linked to an enzyme, fluorochrome, radi
31、oactive or solid particle. 3.3.2 western blotting transfer of an antigen (i.e. the protein of interest), following electrophoretic separation, to a binding surface and visualisation of the antigen with a specific radio-labelled or enzyme-conjugated antibody EN ISO 21572:2004 1 NOTE Transfer of elect
32、rophoretically separated proteins to a polymer sheet. 3.3.3 ELISA enzyme linked immunosorbent assay in vitro assay that combines enzyme-linked antibodies and substrate to form a coloured reaction product, whereas depending on the application, this assay can be used for qualitative or quantitative pu
33、rposes adapted from EN ISO 21572:2004 1 NOTE 1 In vitro assay by use of enzyme-linked antibodies or antigens and a substrate that forms a coloured (or fluorescent) reaction product. NOTE 2 The ELISA assay is usually performed in the microwell plate format. 3.3.4 immunochromatographic methods rapid i
34、mmunoassay formats including lateral flow devices (LFDs), strips and cards, where an antibody or an analyte is coated to a solid surface 3.3.5 RIE rocket immunoelectrophoresis in vitro assay where the antigen moves electrophoretically in an antibody containing gel NOTE Precipitation zones are obtain
35、ed in the shape of rockets at equivalence between the antigen and the corresponding antibody. 3.3.6 dip stick format qualitative and rapid assay format, where an antibody or an analyte is coated to a solid surface adapted from EN ISO 21572:2004 1 3.3.7 biosensor equipment designed to study the affin
36、ity or avidity of interacting molecules such as antigens, allergens and antibodies DIN EN 15633-1:2009-04 EN 15633-1:2009 (E) 8 3.3.8 protein microchip antibody (micro- or nano-)chip miniaturised and automated device based on ELISA principles NOTE The chip may consist of multiple spots of immobilise
37、d antigens, allergens or antibodies, coating the inner part of the reaction chamber and allowing the simultaneous recognition and quantitation of antigens, allergens or antibodies. 4 General laboratory requirements 4.1 Principle The target analyte is extracted according to the procedure described fo
38、r that specific matrix or in the specific method, and a specific antibody is used to detect or measure the concentration of the target in the sample. 4.2 Apparatus and equipment The laboratory should use properly maintained equipment suitable for the method employed, e.g. according to the requiremen
39、ts outlined by EN ISO/IEC 17025 2. In addition to standard laboratory equipment, additional apparatuses are described in the specific methods. Apparatus and equipment shall be maintained according to manufacturers instructions. Calibration systems shall be available and calibration routinely perform
40、ed for equipment. 4.3 Material and reagents During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and only de-ionised or distilled water or water that has been purified, or equivalent. Other reagents, such as antibodies, conjugated antibodies, substrates, sto
41、p solutions and buffer components are method specific. Please refer to the method for specifics regarding reagents such as standards or reference material, antibodies coated to a solid surface or free, controls and samples. Storage conditions and shelf-life of reagents should be clearly specified. 5
42、 Procedure 5.1 General For the use of this European Standard, general requirements of quality assurance for laboratories shall be observed (e.g. concerning calibration of apparatus, concerning extraction of samples and measured replicates, blanks, use of reference materials, preparation of calibrati
43、on curves, etc.). 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. 5.2 Preparation of sample Further information can be found in prEN 15842:2008 and in t
44、he respective used assay protocol. 5.3 Extraction The protein is extracted according to the procedure described in the specific assay method. The protein is then allowed to react with the antibody. The detection of the antigen-antibody complex can be performed by addition of a conjugate or other mea
45、ns. The addition of a conjugate leads to a detectable signal. See details in the instructions for use of the specific assay system. DIN EN 15633-1:2009-04 EN 15633-1:2009 (E) 9 5.4 Calibration curves Further information can be found in prEN 15842:2008 and in the Instructions for use of the respectiv
46、e used assay system. 5.5 Assay procedure The assay procedures as given in the respective used assay protocol shall be followed. 6 Interpretation and expression of results 6.1 General remarks Further information can be found in prEN 15842:2008 and follow the protocol of the respective used assay proc
47、edure. Antibodies in different kits are generated against different targets and therefore have different specificities and sensitivities. The antibody specificity (antigenic target protein) shall be defined for the method. Analytical sensitivity (Limit of Detection) should be calculated according to
48、 established analytical principles and quoted for the method. In addition, the target for the antibody (and any conversion factor) needs to be clearly defined: e.g. if Ara h1 or total peanut protein is measured, the final calculated figure needs to state the amount of allergenic component per kg sam
49、ple. Results of the method should be expressed in terms of either the total amount (mg/kg) of the allergenic foodstuff or in terms of protein with a suitable conversion factor(s) to convert to total weight of allergenic food. The parameters to interpret vary depending on whether the assay is qualitative or quantitative. 6.2 Quantitative results The following parameters are evaluated: raw data (continuous numerical values) of sample test solution; of blank (zero standard);
