1、BS EN15633-1:2009ICS 67.050NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Detectionof food allergensby immunologicalmethodsPart 1: General considerationsThis British Standardwas published under theauthority of the StandardsPolicy and StrategyCommitte
2、e on 31 January2009 BSI 2009ISBN 978 0 580 58240 0Amendments/corrigenda issued since publicationDate CommentsBS EN 15633-1:2009National forewordThis British Standard is the UK implementation of EN 15633-1:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/-/3, Food a
3、nalysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard c
4、annot confer immunityfrom legal obligations.BS EN 15633-1:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15633-1January 2009ICS 67.050English VersionFoodstuffs - Detection of food allergens by immunologicalmethods - Part 1: General considerationsProduits alimentaires - Dtection des allergnesa
5、limentaires par des mthodes danalyse immunologiques -Partie 1: Considrations gnralesLebensmittel - Nachweis von Lebensmittelallergenen mitimmunologischen Verfahren - Teil 1: AllgemeineBetrachtungenThis European Standard was approved by CEN on 1 December 2008.CEN members are bound to comply with the
6、CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or
7、to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language 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 version
8、s.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain,
9、Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement 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.
10、Ref. No. EN 15633-1:2009: EBS EN 15633-1:2009EN 15633-1:2009 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Normative references 53 Terms and definitions .54 General laboratory requirements .85 Procedure .86 Interpretation and expression of results .97 Specific parameters which may influenc
11、e results 108 Test report . 11Bibliography . 12BS EN 15633-1:2009EN 15633-1:2009 (E) 3 Foreword This document (EN 15633-1:2009) 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
12、 status of a national standard, either by publication of an identical text or by endorsement, at the latest by July 2009, and conflicting national 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 subj
13、ect 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 Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bu
14、lgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15633-1:2009EN 1563
15、3-1:2009 (E) 4 Introduction A specific protein or group of proteins or peptides deriving from these proteins can serve as a marker for the presence of food or food ingredients provoking allergic reactions. This European Standard describes the procedure to qualitatively detect and/or quantitate a pro
16、tein as a marker for potentially allergenic ingredients or constituents by analysing the protein extracted from the sample under study. Appropriate procedures for extraction of the protein are included in each method. The main focus of this European Standard will be on antibody based methods. BS EN
17、15633-1:2009EN 15633-1:2009 (E) 5 1 Scope 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 pe
18、rformance 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 pe
19、rformance 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. 2 Normative references The following referenced documents are indispensable for
20、 the application 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. prEN 15842:2008, Foodstuffs Detection of food allergens General considerations and validation of methods
21、3 Terms and definitions For the purposes of this document, the terms and definitions given in prEN 15842:2008 and the following apply. 3.1 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 NOT
22、E The denaturation may modify functional, enzymatic or antigenic properties of the protein. 3.2 Terms relative to antibodies 3.2.1 antibody 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
23、1 The antibody is capable of binding to that specific antigen. NOTE 2 Antibodies recognise specific areas in the antigen called epitopes. 3.2.2 antigen substance that is recognised as foreign by the immune system and elicits an immune response BS EN 15633-1:2009EN 15633-1:2009 (E) 6 EN ISO 21572:200
24、4 1 NOTE 1 The antigen reacts in vivo and in vitro specifically with the generated antibodies. NOTE 2 The antigen elicits an immune 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
25、cell line EN ISO 21572:2004 1 3.2.5 cross-reactivity binding of the antibody to substances other than the analyte of primary interest 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 antige
26、n than those used for immunisation. 3.2.6 monoclonal antibody antibody produced from a single hybridoma clone and directed to a single 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 polyclo
27、nal antibodies antibodies produced by several clones of lymphocytes EN ISO 21572:2004 1 NOTE Antibodies produced by several B-cells 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
28、 other similar structures on that or other antigens EN ISO 21572:2004 1 NOTE The ability of antibodies to recognise and distinguish between related structures. 3.2.9 epitope molecular structure of an antigen specifically recognised by antibodies or receptors on cells BS EN 15633-1:2009EN 15633-1:200
29、9 (E) 7 3.3 Terms relative to methods 3.3.1 conjugate material produced by attaching two or more substances together EN ISO 21572:2004 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 conju
30、gate (including conjugated antibody) is an antibody or substance (e.g. Avidin), that is linked to an enzyme, fluorochrome, radioactive 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 visua
31、lisation of the antigen with a specific radio-labelled or enzyme-conjugated antibody EN ISO 21572:2004 1 NOTE Transfer of electrophoretically 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 fo
32、rm a coloured reaction product, whereas depending on the application, this assay can be used for qualitative or quantitative purposes 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
33、product. NOTE 2 The ELISA assay is usually performed in the microwell plate format. 3.3.4 immunochromatographic methods rapid immunoassay 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 immunoelectrophores
34、is in vitro assay where the antigen moves electrophoretically in an antibody containing gel NOTE Precipitation zones are obtained 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
35、 an analyte is coated to a solid surface adapted from EN ISO 21572:2004 1 3.3.7 biosensor equipment designed to study the affinity or avidity of interacting molecules such as antigens, allergens and antibodies BS EN 15633-1:2009EN 15633-1:2009 (E) 8 3.3.8 protein microchip antibody (micro- or nano-)
36、chip miniaturised and automated device based on ELISA principles NOTE The chip may consist of multiple spots of immobilised antigens, allergens or antibodies, coating the inner part of the reaction chamber and allowing the simultaneous recognition and quantitation of antigens, allergens or antibodie
37、s. 4 General laboratory requirements 4.1 Principle The target analyte is extracted according to the procedure described for 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
38、The laboratory should use properly maintained equipment suitable for the method employed, e.g. according to the requirements 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 ma
39、intained according to manufacturers instructions. Calibration systems shall be available and calibration routinely performed 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
40、or water that has been purified, or equivalent. Other reagents, such as antibodies, conjugated antibodies, substrates, stop 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
41、 solid surface or free, controls and samples. Storage conditions and shelf-life of reagents should be clearly specified. 5 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 apparat
42、us, concerning extraction of samples and measured replicates, blanks, use of reference materials, preparation of calibration 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 mat
43、rices, needs to be clearly defined. 5.2 Preparation of sample Further information can be found in prEN 15842:2008 and in the 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
44、 with the antibody. The detection of the antigen-antibody complex can be performed by addition of a conjugate or other means. The addition of a conjugate leads to a detectable signal. See details in the instructions for use of the specific assay system. BS EN 15633-1:2009EN 15633-1:2009 (E) 9 5.4 Ca
45、libration curves Further information can be found in prEN 15842:2008 and in the Instructions for use of the respective 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
46、remarks Further information can be found in prEN 15842:2008 and follow the protocol of the respective used assay procedure. Antibodies in different kits are generated against different targets and therefore have different specificities and sensitivities. The antibody specificity (antigenic target pr
47、otein) shall be defined for the method. Analytical sensitivity (Limit of Detection) should be calculated according to 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
48、 peanut protein is measured, the final calculated figure needs to state the amount of allergenic component per kg sample. 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 c
49、onvert 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); or of reference material or analytical standard; of analytical kit controls (negative and/or positive samples). The following values should be evaluated: RSD between replicates; RSD
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