1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationFoodstuffs Detection of food allergens Template for supplying information about immunological methods and molecular biological methodsPD CEN/TR 16338:2012National forewordThis Pu
2、blished Document is the UK implementation of CEN/TR 16338:2012. The UK participation in its preparation was entrusted to Technical CommitteeAW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.This publication do
3、es not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012Published by BSI Standards Limited 2012 ISBN 978 0 580 76541 4 ICS 67.050Compliance with a British Standard cannot confer immunity from legal
4、 obligations.This Published Document was published under the authority of the Standards Policy and Strategy Committee on 30 June 2012.Amendments issued since publicationAmd. No. Date Text affectedPUBLISHED DOCUMENTPD CEN/TR 16338:2012TECHNICAL REPORT RAPPORT TECHNIQUE TECHNISCHER BERICHT CEN/TR 1633
5、8 May 2012 ICS 67.050 English Version Foodstuffs - Detection of food allergens - Template for supplying information about immunological methods and molecular biological methods Produits alimentaires - Dtection des allergnes alimentaires - Modle pour la mise disposition dinformations sur les mthodes
6、danalyse immunologique et les mthodes danalyse de biologie molculaire Lebensmittel - Nachweis von Lebensmittelallergenen - Vorlage zur Bereitstellung von Informationen ber immunologische und molekularbiologische Verfahren This Technical Report was approved by CEN on 4 March 2012. It has been drawn u
7、p by the Technical Committee CEN/TC 275. 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, Pola
8、nd, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form
9、and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TR 16338:2012: EPD CEN/TR 16338:2012CEN/TR 16338:2012 (E) 2 Contents Page Foreword 31Scope 32Normative references 33Terms, definitions and abbreviations 34Necessary elements for method applications 35Estimation of measurement
10、 uncertainty .36Physical/environmental conditions .47Instrument calibration .48Safety precautions/safety measures .49Pollution prevention/waste disposal .410Appendices .4Annex A (informative) General elements for immunological method proposals .5A.1Title 5A.2Purpose, relevance and scientific basis .
11、5A.3Scope 5A.4Test principle/summary and analysis steps for immunological methods .5A.5Validation status and performance criteria/method performance for immunological methods 7Annex B (informative) General elements for molecular biological method proposals . 10B.1Title . 10B.2Purpose, relevance and
12、scientific basis 10B.3Scope . 10B.4Test principle/summary and analysis steps for molecular biological methods 10B.5Validation status and performance criteria for molecular biological methods 12Bibliography . 15PD CEN/TR 16338:2012CEN/TR 16338:2012 (E) 3 Foreword This document (CEN/TR 16338:2012) has
13、 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 i
14、dentifying any or all such patent rights. 1 Scope 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“. 2 Normative references The following documents, in whole o
15、r 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 15633-1:2009, Foodstuffs Detection of f
16、ood allergens by immunological methods Part 1: General considerations EN 15634-1:2009, Foodstuffs Detection of food allergens by molecular biological methods Part 1: General considerations. EN 15842:2010, Foodstuffs Detection of food allergens General considerations and validation of methods ISO 572
17、5-1:1994, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions 3 Terms, definitions and abbreviations For the purpose of this document, the terms and definitions given in EN 15633-1:2009, EN 15634-1:2009 and EN 15842:2010 apply. 4 Necessary
18、elements for method applications Each method should include: a) general information (e. g. title, purpose, relevance and scientific basis, scope and test principle); b) a detailed description of the method (e. g. reagents, equipment, procedure, calculations, reporting); c) validation and performance
19、 criteria. All necessary elements for immunological and molecular biological methods are given in Annex A and Annex B respectively. 5 Estimation of measurement uncertainty Uncertainty arises from many sources, including the size of the laboratory sample, sampling of the test sample from the laborato
20、ry sample, measurement of the allergen concentration in the extracts, etc. An estimate of the PD CEN/TR 16338:2012CEN/TR 16338:2012 (E) 4 measurement uncertainty for each matrix in the area of application will be estimated either from intra/inter-laboratory study data, or from estimates of the compo
21、nents as described by ISO/IEC Guide 98-3:2008 1 and ISO 5725-2:1994 2. 6 Physical/environmental conditions Specify conditions (if there are special environmental conditions for performing the analysis), such as normal laboratory conditions, or use of a cold room, performance of certain steps at spec
22、ific temperatures etc. 7 Instrument calibration Instruments should be calibrated as specified in EN ISO/IEC 17025:2005 3. 8 Safety precautions/safety measures Describe any particular safety measures (not including country/region specific issues) that shall be brought to the attention of the analyst.
23、 9 Pollution prevention/waste disposal The best, most appropriate local practices shall be adhered to. 10 Appendices The method shall include such diagrams and tables as are necessary for use by the analyst as informative data from the internal validation study and informative data from the collabor
24、ative trial. PD CEN/TR 16338:2012CEN/TR 16338:2012 (E) 5 Annex A (informative) General elements for immunological method proposals A.1 Title The title of the method should include an indication of the species and event or sequence to be detected. It is suggested that the name and address of the pers
25、on or institution submitting the method also be included in the following manner: author submitting company and independent laboratories (if participants). This information however shall not appear in the published document. A.2 Purpose, relevance and scientific basis A description on the purpose of
26、 the assay and the scientific background shall be written. A.3 Scope A description of the parameters shall be stated in the methods prepared to be a CEN Technical Specification for the immunological measurements of allergens in food. The allergen and matrix on which the method has been validated sha
27、ll be described. A.4 Test principle/summary and analysis steps for immunological methods A.4.1 General A.4.1.1 Terms and definitions For the purposes of this document, the terms and definitions given in EN 15842:2010 and EN 15633-1:2009 apply. A.4.1.2 Description of method The area of application sh
28、all be defined. A short description of the method including the method principle shall be given. EXAMPLE State which antibodies are used to determine the analyte. Include the specificity of the antibodies (polyclonal, monoclonal, recombinant, etc.). Describe against which source the antibodies are r
29、aised (defined allergen, protein, marker to be specified. Finally, state the immunoassay format used. It is mandatory to describe the design of the assay as well as a detailed description of the used antibodies (including information regarding their purification, characterised affinity and against w
30、hich substances they are raised). References to relevant scientific publications are also desirable inclusions. A.4.1.3 Sample type and matrices The material used for test calibration shall be given (i.e. whether it is whole extract, non-purified, purified, fractionated, etc.). Give also a short des
31、cription of the type of samples and matrices upon which the method has been validated and can be applied. PD CEN/TR 16338:2012CEN/TR 16338:2012 (E) 6 A.4.1.4 Interferences List conditions and materials, which are known to interfere in both a positive and a negative fashion with the method, such as s
32、amples derived from matrices that interfere with the method. A.4.2 Apparatus and equipment for immunological methods All the specific equipment used, including for example spectrophotometers, blenders or other grinding apparatus, sieves, apparatus or equipment for measuring concentrations (but exclu
33、ding general laboratory disposables and equipment), shall be stated. Examples for sources of equipments and reagents including the relevant disclaimer/non-endorsement should be stated as footnotes. A.4.3 Reagents (supplies) A.4.3.1 General List all the reagents needed. Include at least all those des
34、cribed in EN 15633-1:2009 and all additionally specified reagents. This listing shall also include all used buffers for assay completion, used reagents and solutions used for sample treatment. The quality of the reagents and the specific reagents used shall be indicated. All ingredients and componen
35、ts (for example buffers, including the chemical composition of buffer), shall be listed. A.4.3.2 Reagents preparation If some reagents need to be handled before use (e. g. diluted), the general preparation guidelines shall be given. A.4.4 Analysis steps A.4.4.1 General In this Clause, give a detaile
36、d description of each step of the analysis, from sample preparation to the immunoassay procedure, so that a trained analyst can perform the method, including all steps involved. A.4.4.2 General instructions/recommendations If there are general recommendations to assure a good test performance, this
37、shall be stated (e. g. at which temperatures the reagents should be used). A.4.4.3 Preparation of sample for immunological methods A.4.4.3.1 Sample type and amounts, including sample identification Give details of the type of laboratory and test samples required (analyte and matrix), with particular
38、 reference to issues of sampling. All samples shall be identified unambiguously. A.4.4.3.2 Sample collection, transport, preservation and storage Describe any provisions regarding sample collection and sampling, as well as storage conditions. A.4.4.3.3 Test sample preparation Outline the steps of th
39、e test sample preparation. Include such details as the grinding and sieving steps, sample amount to be weighed in, PD CEN/TR 16338:2012CEN/TR 16338:2012 (E) 7 additional reagents, amount, kind and composition of buffer, extraction time and temperature. Describe the method used to extract allergens f
40、rom the matrix; if applicable use the method from the kit insert. A.4.4.4 Immunoassay procedure/operational scheme Describe the procedure in sufficient detail that a trained analyst can perform the method, including any special steps involved. A.4.5 Reading/interpretation and test result report (cal
41、culations, reporting) for immunological methods A.4.5.1 Identification If the result of the analysis can be verified, indicate how this can be achieved. A.4.5.2 Calculations Describe any calculations or mathematical models used to derive the analytical result. Recommendations for the best curve-fitt
42、ing model shall be described (e. g. 4-parameter, cubic spline or linear regression model) for the standard curve (method dependent). Describe any calculations or models used to derive the analytical result, including conversion factors from, for example, specific of total protein amount to the amoun
43、t of allergenic component. A.4.5.3 Acceptance/rejection criteria A validated method includes criteria from which an observed measurement result can be accepted as valid. Describe the acceptance/rejection criteria for the analysis. A.4.5.4 Reporting Record keeping should be in conformity with EN ISO/
44、IEC 17025:2005 3. Describe how data should be reported. Normally the mathematical models can be calculated from the usual software delivered with each ELISA-reader. If no software is available for calculations, alternatives shall be given (e. g. semi-logarithmic mm-paper, x-axis conc. Standard mg/kg
45、, y-axis ABS standard). A.5 Validation status and performance criteria/method performance for immunological methods A.5.1 General A summary of the validated performance claims as well as the following the data collected from the internal validation (including precision, sensitivity, accuracy, specif
46、icity and ruggedness), shall be given. If a collaborative trial was also undertaken, information about such a trial (how many laboratories participated, outlier elimination, mean values, repeatability r, Sr, RSDr, reproducibility R, SR, RSDR, etc.), shall be given. A.5.2 Internal validation (manufac
47、turers in house study) Give information obtained from the intra-laboratory trial, including the RSDr. PD CEN/TR 16338:2012CEN/TR 16338:2012 (E) 8 A.5.2.1 Precision (intra- and inter-assay variation) A.5.2.1.1 Intra-assay variation Standards shall be measured from a sufficient number of repetitions.
48、Standard deviations and intra-assay CVs (coefficient of variation) should be given, including the mean CV. Spiked samples can also be measured. A.5.2.1.2 Inter-assay variation For the inter-assay variation, standards shall be measured in duplicates in a minimum of six independent runs. The mean CVs
49、should be calculated. Spiked samples can also be measured. A.5.2.2 Sensitivity A.5.2.2.1 Limit of detection (LOD) (range of application) The detection limit and range of application of the method should be given in terms of concentrations. The amount of substance in the analysed sample, in mg/kg, should be given, together with the methods used to determine these values, the matrices used. Explain also how the calculation was done (
