EN ISO 14675-2003 en Milk and milk products - Guidelines for a standardized description of competitive enzyme immunoassays - Determination of aflatoxin M1 content《牛奶和牛奶制品 竞争酵素免疫分析的.pdf

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1、BRITISH STANDARD BS EN ISO 14675:2003 Milk and milk products Guidelines for a standardized description of competitive enzyme immunoassays Determination of aflatoxin M 1content The European Standard EN ISO 14675:2003 has the status of a British Standard ICS 67.100.10 BS EN ISO 14675:2003 This British

2、 Standard was published under the authority of the Standards Policy and Strategy Committee on 29 April 2003 BSI 29 April 2003 ISBN 0 580 41637 2 National foreword This British Standard is the official English language version of EN ISO 14675:2003. It is identical with ISO 14675:2003. The UK particip

3、ation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or Euro

4、pean publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include a

5、ll the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries

6、on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN ISO title page, the EN ISO foreword page, t

7、he ISO title page, pages ii to v, a blank page, pages 1 to 5 and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM ENISO14675 January200

8、3 ICS67.100.10 Englishversion MilkandmilkproductsGuidelinesforastandardized descriptionofcompetitiveenzymeimmunoassays DeterminationofaflatoxinM1content(ISO14675:2003) LaitetproduitslaitiersLignesdirectricespourune descriptionnormalisedestestsimmunoenzymatiques DterminationdelateneurenaflatoxineM1(I

9、SO 14675:2003) MilchundMilchprodukteLeitfadenfreinevereinheitlichte BeschreibungkompetitiverEnzymImmunoassays BestimmungdesGehaltsanAflatoxinM1(ISO 14675:2003) ThisEuropeanStandardwasapprovedbyCENon6December2002. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditi

10、onsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementCentreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Avers

11、ioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Hungary,Iceland,Ireland,I

12、taly,Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,SwitzerlandandUn ited Kingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2003CEN Allrightsofexploitationinanyformandbyanymeansreser

13、ved worldwideforCENnationalMembers. Ref.No.ENISO14675:2003EINESO41:5762003(E) 2 Foreword Thisdocument(ENISO14675:2003)hasbeenpreparedbyTechnicalCommitteeISO/TC34 “Agriculturalfoodproducts“incollaborationwithTechnicalCommitteeCEN/TC302“Milkand milkproductsMethodsofsamplingandanalysis“,thesecretariato

14、fwhichisheldbyNEN. ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationof anidenticaltextorbyendorsement,atthelatestbyJuly2003,andconflictingnationalstandards shallbewithdrawnatthelatestbyJuly2003. AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizat

15、ionsof thefollowingcountriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,Czech Republic,Denmark,Finland,France,Germany,Greece,Hungary,Iceland,Ireland,Italy, Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,Switzerlandand theUnitedKingdom. NOTEFROMCMC Theforewordissuscept

16、ibletobeamendedonreceptionoftheGerman languageversion.Theconfirmedoramendedforeword,andwhenappropriate,thenormative annexZAforthereferencestointernationalpublicationswiththeirrelevantEuropean publicationswillbecirculatedwiththeGermanversion. Endorsementnotice ThetextofISO14675:2003hasbeenapprovedbyC

17、ENasENISO14675:2003withoutany modifications. ENISO14675:2003 Reference numbers ISO 14675:2003(E) IDF 186:2003(E)INTERNATIONAL STANDARD ISO 14675 IDF 186 First edition 2003-01-15 Milk and milk products Guidelines for a standardized description of competitive enzyme immunoassays Determination of aflat

18、oxin M 1content Lait et produits laitiers Lignes directrices pour une description normalise des tests immuno-enzymatiques Dtermination de la teneur en aflatoxine M 1ENISO14675:2003ENISO14675:2003iiIS:57641 O3002(E) ID:681 F3002(E) I SO dna ID 3002 F All irhgts seredevr iiiForeword ISO (the Internati

19、onal Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been

20、 established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standa

21、rdization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for

22、voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of patent rights. ISO shall not be held responsible for identifyi

23、ng any or all such patent rights. ISO 14675 IDF 186 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separa

24、tely by AOAC International. ENISO14675:2003iiiIS:57641 O3002(E) ID:681 F3002(E) vi I SO dna ID 3002 F All irhgts seredevrForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the

25、 right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing

26、 Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of National Committees casting a vote. International Standard ISO 14675 IDF 186 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC

27、5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team, Organic contaminants, of the Standing

28、Committee on Analytical methods for additives and contaminants, under the aegis of its project leader, Dr E. Mrtlbauer (DE). ENISO14675:2003ivIS:57641 O3002(E) ID:681 F3002(E) I SO dna ID 3002 F All irhgts seredevr vIntroduction Proprietary methods such as ELISA methods cannot be described in separa

29、te International Standards. Therefore, this International Standard is intended to provide guidelines on basic parameters required for evaluation/validation of competitive enzyme immunoassays for the quantitative determination of aflatoxin M 1in milk and milk products. Currently several quantitative

30、immunochemical test formats are commercially available, which all share the basic principles of the competitive enzyme immunoassay. However, since the test format of the 96-well microtitre plate assay is most commonly used for quantitative measurement purposes, the parameters given in this Internati

31、onal Standard are specifically adopted to this test format, and may not necessarily apply in full to a different test format. ENISO14675:2003vNITERNATNOIAL STANDARD IS:57641 O3002(E) ID:681 F3002(E)I SO dna ID 3002 F All irhgts seredevr 1Milk and milk products Guidelines for a standardized descripti

32、on of competitive enzyme immunoassays Determination of aflatoxin M 1content 1 Scope This International Standard give guidelines on the use of screening methods used for the determination of aflatoxin M 1content in milk and milk products, based upon competitive enzyme immunoassays. For legal purposes

33、, positive enzyme immunoassay results require confirmation by an accepted reference method. However, depending on whether the test complies with the specifications given hereafter, enzyme immunoassays can be used for routine quality control, especially when the absence of aflatoxin M 1above the regu

34、latory limit needs to be documented. 2 Principle Immunochemical methods are based on the ability of antibodies to bind to specific substances. The reversible association between antibodies and their corresponding antigens is called the immunological reaction. The binding forces involved are weak mol

35、ecular interactions, such as Coulomb and van der Waals forces, as well as hydrogen bonds and hydrophobic binding. The antigen-antibody reaction is based on the law of mass action and the amount of antigen or antibody present in the reaction mixture can be inferred from the extent of the reaction. Th

36、e “quality” of any immunoassay is a function of the immunochemical principle of the method, the properties of the reagents, the assay design and the experimental errors. These basic principles determine the sensitivity, specificity, precision and accuracy of the assay. Concerning the principle of th

37、e method, a distinction exists between competitive methods and non-competitive methods. For practical reasons, these methods need either labelled antigen or labelled antibody to permit observation of the antigen-antibody reaction. Competitive methods are based on the competition of free (A g ) and l

38、abelled (A g *) antigen for a limited number of antibody-combining sites (AB). Schematically, this immunochemical principle may be presented according to the following formula: gg g g gg * A A AB A AB A AB A A +=+ + In most cases, the assay response represents the bound-labelled antigen, but any mea

39、sure of the distribution of the labelled antigen is, in principle, possible. For the detection of low molecular weight compounds like mycotoxins, which possess only one antibody binding site (epitope), the competitive assay format is mandatory. To provide a distinction between unreacted and complexe

40、d reactants, most assays use either antibody (direct competitive assay) or antigen (indirect competitive assay) bound to a solid-phase as immunosorbent. So all the reagents that are not bound by the antibody can be easily removed by “washing” the solid phase. ENISO14675:20031IS:57641 O3002(E) ID:681

41、 F3002(E) 2 I SO dna ID 3002 F All irhgts seredevr3 Aflatoxin M 1enzyme immunoassay Based on the information given in the general principles of immunoassays (see clause 2), it is recommended that an ELISA-method for aflatoxin M 1should comply with the specifications given in Table 1. Table 1 Specifi

42、cation of assay parameters Parameter Specification Antibody Source Polyclonal or monoclonal Labelled antigen Marker enzyme Horseradish peroxidase Design Aflatoxin B 1or M 1 -oxime-horseradish peroxidase Assay format Immunochemical principle Competitive enzyme immunoassay Design 96-well microtitre pl

43、ate assay Time 3 h to 4 h Assay sensitivity AFM 1concentration giving relative absorbanceaof: 80 % 80 % for the range from 10 ng/kg to 50 ng/kg (milk)caAbsorbance standard or sample/absorbance zero standard 100. bSee statistical parameters in 6.3. cCorrection for recovery usually not necessary.ENISO

44、14675:20032IS:57641 O3002(E) ID:681 F3002(E) I SO dna ID 3002 F All irhgts seredevr 34 Sensitivity The potential sensitivity of any immunoassay is directly related to the affinity of the antibody and can be calculated, if the equilibrium constant is known (see reference 2). Since the antigen-antibod

45、y reaction may be described using reaction kinetics as well as thermodynamic equations, time and temperature also have an influence on the assay sensitivity. 5 Specificity Next to sensitivity, the specificity of an immunochemical method is important for the performance of the assay. In principle, a

46、specific reaction in immunology may be defined as follows: In the presence of different molecules, the specific antibodies must complex only one kind of molecule. The probability of forming a “wrong” complex determines the specificity of the reaction. In other words, specificity is determined by the

47、 sterix (three-dimensional) match of an antigen and antibody as well as by the number of molecular interactions taking place between both molecules. Discussion of specificity requires that both the structure of the antigen and the homogeneity or heterogeneity of the antibodies should be considered.

48、An antibody preparation is homogenic if all the antibodies bind only the one and same epitope, although with different affinity. This condition is fulfilled by monoclonal antibodies, but also by antisera against compounds of low molecular weight (haptenes). On the other hand, an antibody preparation is heterogenic if it co