1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationFoodstuffs Molecular biomarker analysis Protein-based methodsBS EN ISO 21572:2013National forewordThis British Standard is the UK implementation of EN ISO 21572:2013. Itsupersede
2、s BS EN ISO 21572:2004 which is withdrawn.The UK participation in its preparation was entrusted to Technical CommitteeAW/275, Food analysis - Horizontal methods.A list of organizations represented on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 onrequest to its secretary.This publication does not purport to inclu
3、de all the necessary provisions of acontract. Users are responsible for its correct application. The British Standards Institution 2013Published by BSI Standards Limited 2013ISBN 978 0 580 62705 7ICS 67.050Compliance with a British Standard cannot confer immunity fromlegal obligations.This British S
4、tandard was published under the authority of the StandardsPolicy and Strategy Committee on 28 February 2013.Amendments issued since publicationAmd. No. Date Text affectedBRITISH STANDARDBS EN ISO 21572:2013EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 21572 February 2013 ICS 67.050 Supers
5、edes EN ISO 21572:2004English Version Foodstuffs - Molecular biomarker analysis - Protein-based methods (ISO 21572:2013) Produits alimentaires - Analyse des biomarqueurs molculaires - Mthodes bases sur les protines (ISO 21572:2013) Lebensmittel - Untersuchung von molekularen Biomarkern -Proteinverfa
6、hren (ISO 21572:2013) This European Standard was approved by CEN on 12 January 2013. 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 a
7、nd 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
8、 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, Former Yugosl
9、av 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 COMIT EUROPEN D
10、E NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 21572:2013: EBS EN ISO 21572:2013EN ISO 21572:2013 (E) 2 Contents Page For
11、eword 3 BS EN ISO 21572:2013EN ISO 21572:2013 (E) 3 Foreword This document (EN ISO 21572:2013) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held by DIN. This Eu
12、ropean Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by August 2013, and conflicting national standards shall be withdrawn at the latest by August 2013. Attention is drawn to the possibility that some of the ele
13、ments 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 document supersedes EN ISO 21572:2004. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the follo
14、wing countries are bound to implement this European Standard: 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, N
15、orway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 21572:2013 has been approved by CEN as EN ISO 21572:2013 without any modification. BS EN ISO 21572:2013ISO 21572:2013(E) ISO 2013 All rights reserved ii
16、iContents Page1 Scope . 12 Normative references 13 Terms and definitions . 13.1 General . 13.2 Terms relating to antibodies . 23.3 Terms relating to techniques 23.4 Terms relating to control 33.5 Terms relating to validation 34 Principle 65 Reagents 66 Laboratory equipment . 67 Sampling 68 Procedure
17、. 78.1 General . 78.2 Preparation of sample solution 78.3 Extraction 78.4 Preparation of calibration curves, positive controls and reference materials . 78.5 Assay procedure 79 Interpretation and expression of results 89.1 General . 89.2 Quantitative and semiquantitative analysis . 89.3 Qualitative
18、analysis . 810 Specific parameters which may influence results . 810.1 General . 810.2 Special considerations 910.3 Assay applicability . 911 Confirmation method .1012 Test report 10Annex A (informative) Detection of a protein by ELISA .12Annex B (informative) Detection of protein(s) by lateral flow
19、 devices .22Bibliography .29BS EN ISO 21572:2013INTERNATIONAL STANDARD ISO 21572:2013(E)Foodstuffs Molecular biomarker analysis Protein-based methodsWARNING Follow all instructions provided by the kit/reagent manufacturers and other standard laboratory safety procedures. Read and implement the mater
20、ial safety data sheets (MSDS).1 ScopeThis International Standard provides general guidelines and performance criteria for methods for the detection and/or quantification of specific proteins or protein(s) of interest POI(s) in a specified matrix.These general guidelines address existing antibody bas
21、ed methods. Methods other than those described in Annex A or Annex B can also detect the POI. The same criteria as outlined in this International Standard apply generally.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispens
22、able 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.ISO 24276, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products
23、General requirements and definitions3 Terms and definitionsFor the purposes of this document, the terms and definitions given in ISO 24276 and the following apply.3.1 General3.1.1samplesubset of a population made up of one or more sampling unitsSOURCE: ISO 3534-2:2006, 1.2.173.1.2laboratory samplesa
24、mple (3.1.1) as prepared for sending to the laboratory and intended for inspection or testingSOURCE: ISO 78-2:1999, 3.13.1.3test samplesample (3.1.1) as prepared for testing or analysis, the whole quantity or part of it being used for testing or analysis at one timeSOURCE: ISO 3534-2:2006, 5.3.113.1
25、.4test portionpart of a test sample (3.1.3) which is used for testing or analysis at one timeSOURCE: ISO 3534-2:2006, 5.3.12 ISO 2013 All rights reserved 1BS EN ISO 21572:2013ISO 21572:2013(E)3.1.5matrixproducts submitted for analysis, which might have differences in chemical composition and physica
26、l stateSOURCE: ISO 22174:2005, 3.1.43.1.6denaturation of proteinsphysical and/or (bio)chemcial treatment which destroys or modifies the structural, functional, enzymatic, or antigenic properties of the POI or the analyte3.2 Terms relating to antibodies3.2.1antibodyprotein (immunoglobulin) produced a
27、nd secreted by B lymphocytes in response to a molecule recognized as foreign (antigen) and capable of binding to that specific antigen (3.2.2)3.2.2antigensubstance that stimulates the production of antibodies (3.2.1) and reacts with them3.2.3clonepopulation of identical cells derived from a single c
28、ell3.2.4cross-reactivitybinding of the antibody (3.2.1) to substances other than the analyte of primary interest3.2.5monoclonal antibodyantibody (3.2.1) produced from a single hybridoma clone (3.2.3) and directed to a single antigen (3.2.2) determinant3.2.6polyclonal antibodymixture of immunoglobuli
29、n molecules, secreted against a specific immunogenic substance, each recognizing a different epitopeSOURCE: ISO 19001:2013, 3.113.2.7selectivity of an antibodyability of an antibody (3.2.1) to specifically bind to an antigen (3.2.2) determinant and not to other similar structures or other antigens3.
30、3 Terms relating to techniques3.3.1conjugatematerial produced by attaching two or more substances together by covalent bond via chemical groupsEXAMPLE Conjugates of antibodies with fluorochromes (e.g. chemical entity, such as a molecule or group, which emits light that is in response to being stimul
31、ated by absorption of incident light), radiolabelled substances, gold or enzymes are often used in immunoassays.2 ISO 2013 All rights reservedBS EN ISO 21572:2013ISO 21572:2013(E)3.3.2western blotting protocolprotein immunoblottransfer of a protein to a binding surface following separation by electr
32、ophoresis that may be visualised using a variety of methodsEXAMPLE One example of such a method is with a specific radiolabelled or enzyme-conjugated antibody followed by the addition of an enzyme-specific substrate to form a coloured reaction product.3.3.3enzyme linked immunosorbent assayELISAin vi
33、tro assay used for qualitative, semi-quantitative, or quantitative purposes that combines enzyme-linked antibodies and a substrate to form a coloured reaction product3.3.4test kitset of chemicals, materials and instructions for use, packaged together and intended for use as specified by the manufact
34、urer of the kit3.3.5lateral flow immunochromatographic assaylateral flow device/stripqualitative or semiquantitative, simple rapid assay formats intended to detect the presence (or absence) of a POI in a sample where an antibody (3.2.1) or an analyte is coated to a solid surface and dipped into a te
35、st liquid to provide a measure of the POI in the liquidNote 1 to entry: The test sample flows along a solid substrate via capillary action. After the liquid sample enters the test strip, it encounters a coloured reagent which mixes with the sample and transits the substrate encountering lines or zon
36、es which have been pretreated with an antibody or antigen. Depending on the analytes present in the sample the coloured reagent can become bound at the test line or zone. These assays can operate as either competitive or sandwich assays.3.4 Terms relating to control3.4.1reference materialmaterial, s
37、ufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process or in examination of nominal propertiesSOURCE: ISO/IEC Guide 993.4.2measurement standardmeasured material, measuring instrument, ref
38、erence material (3.4.1) or measuring system intended to define, realize, conserve or reproduce one or more values to serve as a reference or preparation of known characteristics used to standardize the analysis3.5 Terms relating to validation3.5.1accuracycloseness of agreement between a test result
39、or measurement result and a reference valueNote 1 to entry: The term “accuracy”, when applied to a set of test results or measurement results, involves a combination of random components and a common systematic error or bias (3.5.3) component.Note 2 to entry: When applied to a test method, the term
40、accuracy refers to a combination of trueness and precision (3.5.2). ISO 2013 All rights reserved 3BS EN ISO 21572:2013ISO 21572:2013(E)SOURCE: ISO 3534-2:2006, 3.3.1, modified Notes 1 and 2 differ from the original and there is no Note 3.3.5.2precisioncloseness of agreement between independent test/
41、measurement results obtained under stipulated conditionsNote 1 to entry: Precision is normally expressed in terms of standard deviation.SOURCE: ISO 3534-2:2006, 3.3.43.5.3biasdifference between the expectation of a test result or measurement result and a true valueNote 1 to entry: Bias is the total
42、systematic error as contrasted to random error. There may be one or more systematic error components contributing to the bias. A larger systematic difference from the true value is reflected by a larger bias value.SOURCE: ISO 3534-2:2006, 3.3.23.5.4sensitivityquotient of the change in the indication
43、 of a measuring system and the corresponding change in the value of the quantity being measuredNote 1 to entry: The sensitivity can depend on the value of the quantity being measured. Sensitivity usually is meant as the smallest quantity or concentration of the analyte that can be reliably distingui
44、shed from background.SOURCE: ISO/IEC Guide 993.5.5selectivityextent to which a method can determine particular analyte(s) in a mixture(s) or matrice(s) without interferences from other components of similar behaviourNote 1 to entry: Selectivity is the recommended term in analytical chemistry to expr
45、ess the extent to which a particular method can determine analyte(s) in the presence of other components. Selectivity can be graded.SOURCE: Pure Appl. Chem.3.5.6detection limitlimit of detectionLODlowest concentration or content of the POI per defined amount of matrix that can be consistently detect
46、ed under the experimental conditions specified in the methodSOURCE: ISO 22174:2005, 3.1.8, modified “LOD” has been added and “of the target organism” became “of the POI”.3.5.7determination limitlimit of quantificationLOQlowest concentration or content of the POI per defined amount of matrix that can
47、 be measured with reasonable statistical certainty consistently under the experimental conditions specified in the method4 ISO 2013 All rights reservedBS EN ISO 21572:2013ISO 21572:2013(E)3.5.8applicability rangerange of quantificationrange of linearitydynamic rangeupper and lower limits of quantifi
48、cation as expressed by a set of reference materials (or dilutions) with a suitable level of precision and accuracy (3.5.1)3.5.9repeatability conditionsobservation conditions where independent test/measurement results are obtained with the same method on identical test/measurement items in the same t
49、est or measuring facility by the same operator using the same equipment within short intervals of timeNote 1 to entry: Repeatability conditions include: the same measurement procedure or test procedure; the same operator; the same measuring or test equipment used under the same conditions; the same location and repetition over a short period of time.SOURCE: ISO 3534-2:2006, 3.3.6, modified the Note has been deleted.3.5.10repeatabilityprecision under repeatability conditions (3.5.9)SOURCE: ISO
