1、BRITISH STANDARD BS EN ISO 14675:2003 Incorporating Corrigendum No. 1 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 B
2、S EN ISO 14675:2003 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 29 April 2003 BSI 15 May 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 I
3、SO 14675:2003. The UK participation 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 i
4、mplement international or European 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
5、 does not purport to include all 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/Eur
6、opean committee any enquiries 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 pag
7、e, the EN ISO foreword page, the 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 Comments 14482 Corrigendum No. 1 15 May 2003 R
8、eplacing the EN ISO foreword pageEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM ENISO14675 January2003 ICS67.100.10 Englishversion MilkandmilkproductsGuidelinesforastandardized descriptionofcompetitiveenzymeimmunoassays DeterminationofaflatoxinM1content(ISO14675:2003) LaitetproduitslaitiersLignesdir
9、ectricespourune descriptionnormalisedestestsimmunoenzymatiques DterminationdelateneurenaflatoxineM1(ISO 14675:2003) MilchundMilchprodukteLeitfadenfreinevereinheitlichte BeschreibungkompetitiverEnzymImmunoassays BestimmungdesGehaltsanAflatoxinM1(ISO 14675:2003) ThisEuropeanStandardwasapprovedbyCENon6
10、December2002. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheManagementC
11、entreortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheManagementCentrehasthesamestatusasthe official versions. CENmembersarethenationalstandardsb
12、odiesofAustria,Belgium,CzechRepublic,Denmark,Finland,France,Germany,Greece, Hungary,Iceland,Ireland,Italy,Luxembourg,Malta,Netherlands,Norway,Portugal,Slovakia,Spain,Sweden,SwitzerlandandUn ited Kingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG Mana
13、gementCentre:ruedeStassart,36B1050Brussels 2003CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.ENISO14675:2003ECORRECTED20030430 Foreword Thisdocument(ENISO14675:2003)hasbeenpreparedbyTechnicalCommitteeISO/TC34 “Agriculturalfoodproducts“incollaboratio
14、nwithTechnicalCommitteeCEN/TC302“Milkand milkproductsMethodsofsamplingandanalysis“,thesecretariatofwhichisheldbyNEN. ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationof anidenticaltextorbyendorsement,atthelatestbyJuly2003,andconflictingnationalstandards shallbewithdraw
15、natthelatestbyJuly2003. AccordingtotheCEN/CENELECInternalRegulations,thenationalstandardsorganizationsof thefollowingcountriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,Czech Republic,Denmark,Finland,France,Germany,Greece,Hungary,Iceland,Ireland,Italy, Luxembourg,Malta,Netherlands,Norwa
16、y,Portugal,Slovakia,Spain,Sweden,Switzerlandand theUnitedKingdom. Endorsementnotice ThetextofISO14675:2003hasbeenapprovedbyCENasENISO14675:2003withoutany modifications. ENISO14675:2003 Reference numbers ISO 14675:2003(E) IDF 186:2003(E)INTERNATIONAL STANDARD ISO 14675 IDF 186 First edition 2003-01-1
17、5 Milk and milk products Guidelines for a standardized description of competitive enzyme immunoassays Determination of aflatoxin M 1content Lait et produits laitiers Lignes directrices pour une description normalise des tests immuno-enzymatiques Dtermination de la teneur en aflatoxine M 1ENISO14675:
18、2003ENISO14675:2003iiIS:57641 O3002(E) ID:681 F3002(E) I SO dna ID 3002 F All irhgts seredevr iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally car
19、ried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been 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.
20、 ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. 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 Interna
21、tional Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for 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
22、elements of this International Standard may be the subject of patent rights. ISO shall not be held responsible for identifying 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 Internation
23、al Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately 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
24、worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the 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
25、 of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing 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 v
26、ote. International Standard 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 separately b
27、y AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team, Organic contaminants, of the Standing 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)
28、I SO dna ID 3002 F All irhgts seredevr vIntroduction Proprietary methods such as ELISA methods cannot be described in separate International Standards. Therefore, this International Standard is intended to provide guidelines on basic parameters required for evaluation/validation of competitive enzym
29、e immunoassays for the quantitative determination of aflatoxin M 1in milk and milk products. Currently several quantitative 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-wel
30、l microtitre plate assay is most commonly used for quantitative measurement purposes, the parameters given in this International 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 O3
31、002(E) ID:681 F3002(E)I SO dna ID 3002 F All irhgts seredevr 1Milk and milk products Guidelines for a standardized description 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 det
32、ermination of aflatoxin M 1content in milk and milk products, based upon competitive enzyme immunoassays. For legal purposes, positive enzyme immunoassay results require confirmation by an accepted reference method. However, depending on whether the test complies with the specifications given hereaf
33、ter, enzyme immunoassays can be used for routine quality control, especially when the absence of aflatoxin M 1above the regulatory limit needs to be documented. 2 Principle Immunochemical methods are based on the ability of antibodies to bind to specific substances. The reversible association betwee
34、n antibodies and their corresponding antigens is called the immunological reaction. The binding forces involved are weak molecular 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 acti
35、on and the amount of antigen or antibody present in the reaction mixture can be inferred from the extent of the reaction. The “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
36、basic principles determine the sensitivity, specificity, precision and accuracy of the assay. Concerning the principle of the method, a distinction exists between competitive methods and non-competitive methods. For practical reasons, these methods need either labelled antigen or labelled antibody t
37、o permit observation of the antigen-antibody reaction. Competitive methods are based on the competition of free (A g ) and labelled (A g *) antigen for a limited number of antibody-combining sites (AB). Schematically, this immunochemical principle may be presented according to the following formula:
38、 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 measure 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 antibod
39、y binding site (epitope), the competitive assay format is mandatory. To provide a distinction between unreacted and complexed 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 tha
40、t are not bound by the antibody can be easily removed by “washing” the solid phase. ENISO14675:20031IS:57641 O3002(E) ID:681 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
41、is recommended that an ELISA-method for aflatoxin M 1should comply with the specifications given in Table 1. Table 1 Specification of assay parameters Parameter Specification Antibody Source Polyclonal or monoclonal Labelled antigen Marker enzyme Horseradish peroxidase Design Aflatoxin B 1or M 1 -ox
42、ime-horseradish peroxidase Assay format Immunochemical principle Competitive enzyme immunoassay Design 96-well microtitre plate 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 s
43、ample/absorbance zero standard 100. bSee statistical parameters in 6.3. cCorrection for recovery usually not necessary.ENISO14675:20032IS: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 affin
44、ity of the antibody and can be calculated, if the equilibrium constant is known (see reference 2). Since the antigen-antibody 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
45、 to sensitivity, the specificity of an immunochemical method is important for the performance of the assay. In principle, a 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 probabili
46、ty of forming a “wrong” complex determines the specificity of the reaction. In other words, specificity is determined by the 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 r
47、equires that both the structure of the antigen and the homogeneity or heterogeneity of the antibodies should be considered. 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 antib
48、odies, but also by antisera against compounds of low molecular weight (haptenes). On the other hand, an antibody preparation is heterogenic if it contains different antibody populations, specific for different epitopes. On a molecular basis “true” cross-reactivity describes the case in which at least two different antigens compete for the same antibody-binding site.
