SIST EN ISO 21572:2013
(Main)Foodstuffs - Molecular biomarker analysis - Protein-based methods (ISO 21572:2013)
Foodstuffs - Molecular biomarker analysis - Protein-based methods (ISO 21572:2013)
This 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 based 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.
Lebensmittel - Untersuchung von molekularen Biomarkern - Proteinverfahren (ISO 21572:2013)
Diese Internationale Norm legt allgemeine Leitlinien und Leistungskriterien für Verfahren zum Nachweis
und/oder zur quantitativen Bestimmung von spezifischem, aus gentechnisch verändertem (GV) pflanzlichen
Material stammenden Proteinen in einer spezifischen Matrix fest.
Diese allgemeinen Leitlinien gelten für vorhandene auf Antikörpern basierenden Verfahren. Auch andere Verfahren
als die im Anhang A oder Anhang B aufgeführten können Proteine nachweisen. Die in dieser Norm
umrissenen Kriterien werden allgemein angewendet.
Produits alimentaires - Analyse des biomarqueurs moléculaires - Méthodes basées sur les protéines (ISO 21572:2013)
L'ISO 21572:2013 énonce des lignes directrices et des critères de performances généraux pour les méthodes de détection et/ou de quantification de protéines spécifiques ou de protéine(s) d'intérêt [POI] dans une matrice donnée.
Ces lignes directrices générales concernent les méthodes basées sur des anticorps existants. Il existe d'autres méthodes que celles décrites en Annexe A ou B susceptibles de détecter des POI. Les mêmes critères que ceux répertoriés dans l'ISO 21572:2013 sont applicables de façon générale.
Živila - Analiza molekulskih biomarkerjev - Metode na osnovi proteinov (ISO 21572:2013)
Ta mednarodni standard zagotavlja splošne smernice in merila učinkovitosti za metode za odkrivanje in/ali kvantifikacijo posameznih zadevnih proteinov ali proteina(-ov) (POI) v določeni matrici. Te splošne smernice obravnavajo obstoječe metode na osnovi protiteles. Za odkrivanje zadevnih proteinov se lahko uporabljajo tudi metode, ki niso opisane v dodatku A ali B. Na splošno se uporabljajo merila iz tega mednarodnega standarda.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 21572:2013
01-junij-2013
1DGRPHãþD
SIST EN ISO 21572:2004
SIST EN ISO 21572:2004/AC:2005
Živila - Analiza molekulskih biomarkerjev - Metode na osnovi proteinov (ISO
21572:2013)
Foodstuffs - Molecular biomarker analysis - Protein-based methods (ISO 21572:2013)
Lebensmittel - Untersuchung von molekularen Biomarkern - Proteinverfahren (ISO
21572:2013)
Produits alimentaires - Analyse des biomarqueurs moléculaires - Méthodes basées sur
les protéines (ISO 21572:2013)
Ta slovenski standard je istoveten z: EN ISO 21572:2013
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN ISO 21572:2013 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 21572:2013
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SIST EN ISO 21572:2013
EUROPEAN STANDARD
EN ISO 21572
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2013
ICS 67.050 Supersedes EN ISO 21572:2004
English Version
Foodstuffs - Molecular biomarker analysis - Protein-based
methods (ISO 21572:2013)
Produits alimentaires - Analyse des biomarqueurs Lebensmittel - Untersuchung von molekularen Biomarkern -
moléculaires - Méthodes basées sur les protéines (ISO Proteinverfahren (ISO 21572:2013)
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 and 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 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 Yugoslav 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
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21572:2013: E
worldwide for CEN national Members.
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SIST EN ISO 21572:2013
EN ISO 21572:2013 (E)
Contents Page
Foreword .3
2
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SIST EN ISO 21572:2013
EN ISO 21572:2013 (E)
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 European 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 elements 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 following
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, Norway, 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.
3
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SIST EN ISO 21572:2013
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SIST EN ISO 21572:2013
INTERNATIONAL ISO
STANDARD 21572
Second edition
2013-02-01
Foodstuffs — Molecular biomarker
analysis — Protein-based methods
Produits alimentaires — Analyse des biomarqueurs moléculaires —
Méthodes basées sur les protéines
Reference number
ISO 21572:2013(E)
©
ISO 2013
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SIST EN ISO 21572:2013
ISO 21572:2013(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2013
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2013 – All rights reserved
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SIST EN ISO 21572:2013
ISO 21572:2013(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 General . 1
3.2 Terms relating to antibodies . 2
3.3 Terms relating to techniques . 2
3.4 Terms relating to control . 3
3.5 Terms relating to validation . 3
4 Principle . 6
5 Reagents . 6
6 Laboratory equipment . 6
7 Sampling . 6
8 Procedure. 7
8.1 General . 7
8.2 Preparation of sample solution . 7
8.3 Extraction . 7
8.4 Preparation of calibration curves, positive controls and reference materials . 7
8.5 Assay procedure . 7
9 Interpretation and expression of results . 8
9.1 General . 8
9.2 Quantitative and semiquantitative analysis . 8
9.3 Qualitative analysis . 8
10 Specific parameters which may influence results . 8
10.1 General . 8
10.2 Special considerations . 9
10.3 Assay applicability . 9
11 Confirmation method .10
12 Test report .10
Annex A (informative) Detection of a protein by ELISA .12
Annex B (informative) Detection of protein(s) by lateral flow devices .22
Bibliography .29
© ISO 2013 – All rights reserved iii
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SIST EN ISO 21572:2013
ISO 21572:2013(E)
Foreword
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 carried 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.
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 2.
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 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 document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 21572 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis.
This second edition cancels and replaces the first edition (ISO 21572:2004), which has been technically
revised. It also incorporates the Technical Corrigendum ISO 21572:2004/Cor. 1:2005.
iv © ISO 2013 – All rights reserved
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SIST EN ISO 21572:2013
INTERNATIONAL STANDARD ISO 21572:2013(E)
Foodstuffs — Molecular biomarker analysis — Protein-
based methods
WARNING — Follow all instructions provided by the kit/reagent manufacturers and other standard
laboratory safety procedures. Read and implement the material safety data sheets (MSDS).
1 Scope
This 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 based 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 references
The following documents, in whole or 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.
ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — General requirements and definitions
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 24276 and the following apply.
3.1 General
3.1.1
sample
subset of a population made up of one or more sampling units
[SOURCE: ISO 3534-2:2006, 1.2.17]
3.1.2
laboratory sample
sample (3.1.1) as prepared for sending to the laboratory and intended for inspection or testing
[SOURCE: ISO 78-2:1999, 3.1]
3.1.3
test sample
sample (3.1.1) as prepared for testing or analysis, the whole quantity or part of it being used for testing
or analysis at one time
[SOURCE: ISO 3534-2:2006, 5.3.11]
3.1.4
test portion
part of a test sample (3.1.3) which is used for testing or analysis at one time
[SOURCE: ISO 3534-2:2006, 5.3.12]
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SIST EN ISO 21572:2013
ISO 21572:2013(E)
3.1.5
matrix
products submitted for analysis, which might have differences in chemical composition and physical state
[SOURCE: ISO 22174:2005, 3.1.4]
3.1.6
denaturation of proteins
physical and/or (bio)chemcial treatment which destroys or modifies the structural, functional,
enzymatic, or antigenic properties of the POI or the analyte
3.2 Terms relating to antibodies
3.2.1
antibody
protein (immunoglobulin) produced and secreted by B lymphocytes in response to a molecule recognized
as foreign (antigen) and capable of binding to that specific antigen (3.2.2)
3.2.2
antigen
substance that stimulates the production of antibodies (3.2.1) and reacts with them
3.2.3
clone
population of identical cells derived from a single cell
3.2.4
cross-reactivity
binding of the antibody (3.2.1) to substances other than the analyte of primary interest
3.2.5
monoclonal antibody
antibody (3.2.1) produced from a single hybridoma clone (3.2.3) and directed to a single antigen
(3.2.2) determinant
3.2.6
polyclonal antibody
mixture of immunoglobulin molecules, secreted against a specific immunogenic substance, each
recognizing a different epitope
[SOURCE: ISO 19001:2013, 3.11]
3.2.7
selectivity of an antibody
ability of an antibody (3.2.1) to specifically bind to an antigen (3.2.2) determinant and not to other
similar structures or other antigens
3.3 Terms relating to techniques
3.3.1
conjugate
material produced by attaching two or more substances together by covalent bond via chemical groups
EXAMPLE Conjugates of antibodies with fluorochromes (e.g. chemical entity, such as a molecule or group,
which emits light that is in response to being stimulated by absorption of incident light), radiolabelled substances,
gold or enzymes are often used in immunoassays.
2 © ISO 2013 – All rights reserved
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SIST EN ISO 21572:2013
ISO 21572:2013(E)
3.3.2
western blotting protocol
protein immunoblot
transfer of a protein to a binding surface following separation by electrophoresis that may be visualised
using a variety of methods
EXAMPLE 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.
3.3.3
enzyme linked immunosorbent assay
ELISA
in vitro assay used for qualitative, semi-quantitative, or quantitative purposes that combines enzyme-
linked antibodies and a substrate to form a coloured reaction product
3.3.4
test kit
set of chemicals, materials and instructions for use, packaged together and intended for use as specified
by the manufacturer of the kit
3.3.5
lateral flow immunochromatographic assay
lateral flow device/strip
qualitative 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 test liquid to provide a measure of the POI in the liquid
Note 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 zones 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 control
3.4.1
reference material
material, sufficiently 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 properties
[SOURCE: ISO/IEC Guide 99]
3.4.2
measurement standard
measured material, measuring instrument, reference 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 analysis
3.5 Terms relating to validation
3.5.1
accuracy
closeness of agreement between a test result or measurement result and a reference value
Note 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 accuracy refers to a combination of trueness and
precision (3.5.2).
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SIST EN ISO 21572:2013
ISO 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.]
3.5.2
precision
closeness of agreement between independent test/measurement results obtained under stipulated
conditions
Note 1 to entry: Precision is normally expressed in terms of standard deviation.
[SOURCE: ISO 3534-2:2006, 3.3.4]
3.5.3
bias
difference between the expectation of a test result or measurement result and a true value
Note 1 to entry: Bias is the total 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, 3.3.2]
3.5.4
sensitivity
quotient of the change in the indication of a measuring system and the corresponding change in the
value of the quantity being measured
Note 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 distinguished from background.
[SOURCE: ISO/IEC Guide 99]
3.5.5
selectivity
extent to which a method can determine particular analyte(s) in a mixture(s) or matrice(s) without
interferences from other components of similar behaviour
Note 1 to entry: Selectivity is the recommended term in analytical chemistry to express 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.]
3.5.6
detection limit
limit of detection
LOD
lowest concentration or content of the POI per defined amount of matrix that can be consistently detected
under the experimental conditions specified in the method
[SOURCE: ISO 22174:2005, 3.1.8, modified — “LOD” has been added and “of the target organism” became
“of the POI”.]
3.5.7
determination limit
limit of quantification
LOQ
lowest concentration or content of the POI per defined amount of matrix that can be measured with
reasonable statistical certainty consistently under the experimental conditions specified in the method
4 © ISO 2013 – All rights reserved
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SIST EN ISO 21572:2013
ISO 21572:2013(E)
3.5.8
applicability range
range of quantification
range of linearity
dynamic range
upper and lower limits of quantification as expressed by a set of reference materials (or dilutions) with
a suitable level of precision and accuracy (3.5.1)
3.5.9
repeatability conditions
observation conditions where independent test/measurement results are obtained with the same
method on identical test/measurement items in the same test or measuring facility by the same operator
using the same equipment within short intervals of time
Note 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.]
3.5.10
repeatability
precision under repeatability conditions (3.5.9)
[SOURCE: ISO 3534-2:2006, 3.3.5]
3.5.11
repeatability limit
r
value less than or equal to which the absolute difference between two test results obtained under
repeatability conditions (3.5.9) may be expected to be with a probability of 95 %
[SOURCE: ISO 5725-1:1994, 3.1.6]
3.5.12
repeatability standard deviation
standard deviation of test results or measurement results obtained under repeatability conditions (3.5.9)
Note 1 to entry: It is a measure of the dispersion of the distribution of test or measurement results under
repeatability conditions.
Note 2 to entry: Similarly, “repeatability variance” and “repeatability coefficient of variation” can be defined and
used as measures of the dispersion of test or measurement results under repeatability conditions.
[SOURCE: ISO 3534-2:2006, 3.3.7]
3.5.13
reproducibility conditions
observation conditions where independent test/measurement results are obtained with the same
method on identical test/measurement items in different test or measurement facilities with different
operators using different equipment
[SOURCE: ISO 3534-2:2006, 3.3.11]
3.5.14
reproducibility
precision under reproducibility conditions (3.5.13)
[SOURCE: ISO 3534-2:2006, 3.3.10; ISO 78-2:1999, 3.13]
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SIST EN ISO 21572:2013
ISO 21572:2013(E)
3.5.15
reproducibility limit
R
value less than or equal to which the absolute difference between two test results obtained under
reproducibility conditions (3.5.13) may be expected to be with a probability of 95 %
[SOURCE: ISO 5725-1:1994, 3.20]
3.5.16
reproducibility standard deviation
standard deviation of test results or measurement results obtained under reproducibility conditions (3.5.13)
Note 1 to entry: It is a measure of the dispersion of the distribution of test or measurement results under
reproducibility conditions.
Note 2 to entry: Similarly, “reproducibility variance” and “reproducibility coefficient of variation” can be defined
and used as measures of the dispersion of test or measurement results under reproducibility conditions.
[SOURCE: ISO 3534-2:2006, 3.3.12]
4 Principle
The POI is extracted according to the procedure described for that specific matrix, and a specific
antibody is used to detect or measure the concentration of the POI in the sample. For the detection of
specific proteins in ingredients, the basic principle of a protein-based method is to:
— take a representative sample of the matrix;
— extract the proteins;
— detect and/or quantify the POI derived from the matrix under study.
5 Reagents
During the analysis, use only reagents of recognized analytical grade and only deionized or distilled
water or water that has been purified, or equivalent, unless indicated otherwise by the manufacturer of
the reagents or the kit.
Other reagents, such as antibodies, conjugates, substrates, stop solutions and buffer components are
method specific. Please refer to the method for specifics regarding reagents, such as protein standards
or reference materials, antibodies or pre-coated solid surfaces, controls and samples.
Reagents are specified in A.4.2, A.4.3, B.4.2 and B.4.3.
6 Laboratory equipment
Laboratory equipment is specified in A.5 and B.5.
7 Sampling
Sampling is not part of the method specified in this International Standard, although Annex A and
Annex B do provide sampling instructions according to the relevant methods. It is recommended that
the parties concerned come to an agreement on this subject.
6 © ISO 2013 – All rights reserved
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SIST EN ISO 21572:2013
ISO 21572:2013(E)
8 Procedure
8.1 General
Storage conditions and shelf-life of lateral flow strips, antibodies, conjugates, substrates, etc. shall be
clearly specified by the provider.
Use appropriate laboratory equipment with low protein binding capacity (e.g. polypropylene tubes) to
prevent protein adsorption during the whole procedure.
For the use of this International Standard, general requirements of quality assurance for laboratories
shall be observed (e.g. concerning calibration of apparatus, double determination, blanks, use of
reference materials, preparation of calibration curves). Carefully clean all equipment coming into direct
contact with the sample to prevent contamination. See ISO/IEC 17025 for more information.
8.2 Preparation of sample solution
Once a representative sample is obtained, specific sample preparation procedures may be found in the
annexes.
Grind samples as specified in the method before test portions are taken, if necessary. Powders/flour
might have swelling properties and may require more extraction solution if a manufacturer’s method
does not specify this information. If the sample is not immediately used, follow your laboratory’s
procedure for storage (e.g. –20 °C or below).
Laboratory samples containing high amounts of fat may be nonhomogeneous and a larger test sample
should be extracted. If applicable, instructions may be found in the annexes.
Weigh an appropriate amount (as specified in the relevant annex) of a representative test sample for
analysis to create a test portion for extraction. Add extraction solution and homogenize or mix.
8.3 Extraction
Use an extraction procedure suitable for the matrix. Details of appropriate conditions for the
extraction/dilution of the test portions, controls and reference materials are provided in Annex A for ELISA
and Annex B for lateral flow strips. Care should be taken to use extraction procedures validated for the
matrix. Extracted samples should be immediately used or treated as specified in the procedure for storage.
8.4 Preparati
...
SLOVENSKI STANDARD
oSIST prEN ISO 21572:2011
01-marec-2011
Živila - Analiza molekulskih biomarkerjev - Metode na osnovi proteinov (ISO/DIS
21572:2010)
Foodstuffs - Molecular biomarker analysis - Protein-based methods (ISO/DIS
21572:2010)
Lebensmittel - Untersuchung von molekularen Biomarkern - Proteinverfahren (ISO/DIS
21572:2010)
Produits alimentaires - Analyse des biomarqueurs moléculaires - Méthodes basées sur
les protéines (ISO/DIS 21572:2010)
Ta slovenski standard je istoveten z: prEN ISO 21572
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST prEN ISO 21572:2011 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 21572:2011
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oSIST prEN ISO 21572:2011
EUROPEAN STANDARD
DRAFT
prEN ISO 21572
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2010
ICS 67.050 Will supersede EN ISO 21572:2004
English Version
Foodstuffs - Molecular biomarker analysis - Protein-based
methods (ISO/DIS 21572:2010)
Produits alimentaires - Analyse des biomarqueurs Lebensmittel - Untersuchung von molekularen Biomarkern -
moléculaires - Méthodes basées sur les protéines (ISO/DIS Proteinverfahren (ISO/DIS 21572:2010)
21572:2010)
This draft European Standard is submitted to CEN members for parallel enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other language
made by translation under the 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN ISO 21572:2010: E
worldwide for CEN national Members.
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oSIST prEN ISO 21572:2011
prEN ISO 21572:2010 (E)
Contents Page
Foreword .3
2
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oSIST prEN ISO 21572:2011
prEN ISO 21572:2010 (E)
Foreword
This document (prEN ISO 21572:2010) 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 document is currently submitted to the parallel Enquiry.
This document will supersede EN ISO 21572:2004.
Endorsement notice
The text of ISO/DIS 21572:2010 has been approved by CEN as a prEN ISO 21572:2010 without any
modification.
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oSIST prEN ISO 21572:2011
DRAFT INTERNATIONAL STANDARD ISO/DIS 21572
ISO/TC 34SC 16 Secretariat: ANSI
Voting begins on Voting terminates on
2010-12-16 2011-05-16
INTERNATIONAL ORGANIZATION FOR STANDARDIZATION • МЕЖДУНАРОДНАЯ ОРГАНИЗАЦИЯ ПО СТАНДАРТИЗАЦИИ • ORGANISATION INTERNATIONALE DE NORMALISATION
Foodstuffs — Molecular biomarker analysis — Protein-based
methods
Produits alimentaires — Analyse des biomarqueurs moléculaires — Méthodes basées sur les protéines
(Revision of first edition of ISO 21572:2004 and of ISO 21572:2004/Cor.1:2005)
ICS 67.050
ISO/CEN PARALLEL PROCESSING
This draft has been developed within the International Organization for Standardization (ISO), and
processed under the ISO-lead mode of collaboration as defined in the Vienna Agreement.
This draft is hereby submitted to the ISO member bodies and to the CEN member bodies for a parallel
five-month enquiry.
Should this draft be accepted, a final draft, established on the basis of comments received, will be
submitted to a parallel two-month approval vote in ISO and formal vote in CEN.
To expedite distribution, this document is circulated as received from the committee
secretariat. ISO Central Secretariat work of editing and text composition will be undertaken at
publication stage.
Pour accélérer la distribution, le présent document est distribué tel qu'il est parvenu du
secrétariat du comité. Le travail de rédaction et de composition de texte sera effectué au
Secrétariat central de l'ISO au stade de publication.
THIS DOCUMENT IS A DRAFT CIRCULATED FOR COMMENT AND APPROVAL. IT IS THEREFORE SUBJECT TO CHANGE AND MAY NOT BE
REFERRED TO AS AN INTERNATIONAL STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS BEING ACCEPTABLE FOR INDUSTRIAL, TECHNOLOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON OCCASION HAVE TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL TO BECOME
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RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT, WITH THEIR COMMENTS, NOTIFICATION OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPORTING DOCUMENTATION.
© International Organization for Standardization, 2010
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oSIST prEN ISO 21572:2011
ISO/DIS 21572
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Contents Page
Foreword .vi
Introduction.vii
1 Scope.1
2 Normative references.1
3 Terms and definitions .1
3.1 General terms .1
3.2 Terms relative to antibodies.2
3.3 Terms relative to techniques.2
3.4 Terms relative to control.3
3.5 Terms relative to validation.3
4 Principle.6
5 Reagents .6
6 Apparatus and equipment .6
7 Sampling.7
8 Procedure.7
8.1 General .7
8.2 Preparation of sample solution.7
8.3 Extraction .7
8.4 Preparation of calibration curves .7
8.5 Assay procedure .7
9 Interpretation and expression of results.8
9.1 General .8
9.2 Quantitative and semi-quantitative analysis .8
9.3 Qualitative analysis.8
10 Specific parameters which may influence results .9
10.1 General.9
10.2 Special considerations.9
10.2.1 Selectivity.9
10.2.2 Extraction efficiency .9
10.2.3 Matrix effects .9
10.2.4 Parallelism/Linearity.10
10.2.5 Limits of detection.10
10.2.6 Limits of quantitation.10
11 Confirming method .10
12 Test report.10
®
Annex A (informative) Detection of CP4 EPSPS – expressing soybeans (e.g. Roundup Ready
tolerant) by ELISA .12
A.1 Introduction.12
A.2 Scope.12
A.3 Principle.12
A.4 Reagents.13
A.4.1 General.13
A.4.2 Reagents usually provided with the test kit .14
A.4.3 Chemicals not supplied with the test kit.14
A.5 Apparatus and equipment .14
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A.5.1 General. 14
A.5.2 Refrigerator, working at approximately 4ºC. 15
A.5.3 Polypropylene conical centrifuge tubes, sealable, e.g. 15 ml. 15
A.5.4 Plastic wrap or aluminium foil. 15
A.5.5 Plastic tape to prevent strip movement during plate washing . 15
A.5.6 Wash bottle, e.g. of 500 ml. 15
A.5.7 Precision micropipettes capable of delivering e.g. 20 μl to 500 μl. 15
®
A.5.8 Mixer, e.g. Vortex . 15
A.5.9 Balance capable of weighing to the nearest 0,01 g.15
A.5.10 Centrifuge capable for producing a centrifugal acceleration of at least 5 000 x g at the
outer end of the centrifuge tubes. 15
A.5.11 Microtiter plate reader capable of reading absorbance at 450 nm. 15
A.5.12 Incubator oven or water bath capable of maintaining 37 ºC . 15
A.5.13 Sieve of aperture size of 450 μm, or equivalent . 15
A.5.14 Sieve of aperture size of 150 μm (100mesh), or equivalent . 15
A.5.15 Multi-channel pipette, e.g. of 50 μl to 300 μl (optional). 15
A.5.16 Reagent reservoirs for multi-channel dispensing (optional) . 15
A.5.17 Automated plate washer (optional). 15
A.5.18 Test tube rack for 15 ml centrifuge tubes (optional). 15
A.5.19 Ultrasonic bath (optional) . 15
A.6 Procedure. 15
A.6.1 Warning and precautions. 15
A.6.2 Limitation of the procedure . 16
A.6.3 Sampling. 16
A.6.4 Sample preparation . 16
A.6.5 Measures to avoid contamination during sample preparation. 16
A.6.6 Preparation of antibody conjugate . 17
A.6.7 Preparation of wash buffer . 17
A.6.8 Assay Procedure. 17
A.6.9 Test performance. 18
A.7 Flowcharts. 20
A.8 Evaluation. 21
A.9 Accept/reject criteria . 21
A.10 Status. 22
Annex B (informative) Detection of proteins by lateral flow devices. 24
B.1 Introduction. 24
B.2 Scope. 24
B.3 Principle. 25
B.4 Test Kit Contents . 25
B.4.1 General. 25
B.4.2 Reagents usually provided with a test kit . 26
B.4.3 Reagents not supplied with a test kit . 26
B.5 Apparatus and equipment . 26
B.5.1 General. 26
B.5.2 Refrigerator, that can maintain approximately 4 ºC (not always necessary if the kits are
stored at room temperature, which is usually defined as not exceeding 25 ºC). 27
B.5.3 Laboratory balance. 27
®
B.5.4 Mixer, e.g. Vortex (optional) . 27
B.5.5 Pipettes. 27
B.5.6 LFD strip reader (optional). 27
B.5.7 Test tube rack for sample tubes. 27
B.6 Procedure. 27
B.6.1 Warning or precautions . 27
B.6.2 Limitation of the procedure . 27
B.6.3 Sampling. 28
B.6.4 Sampling shall be carried out according this Standard, section 7. 28
B.6.5 Sample preparation . 28
B.6.6 Measures to avoid contamination during sample preparation. 28
B.6.7 Assay procedure. 29
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B.6.8 Test performance .29
B.7 Statistical applications for threshold analyses.30
B.8 Status.30
Bibliography.33
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Foreword
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 carried 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. 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 2.
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 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 document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 21572 was prepared by Technical Committee ISO/TC 34, Food Products, Subcommittee SC 16,
Molecular Biomarker Analysis.
This second/third/. edition cancels and replaces the first/second/. edition (), [clause(s) / subclause(s) /
table(s) / figure(s) / annex(es)] of which [has / have] been technically revised.
Other standards dealing with methods of analysis for the detection of genetically modified traits and derived
products in foodstuffs are the following:
EN ISO 21571 Foodstuffs – Methods of analysis for the detection of genetically modified organisms and
derived products – Nucleic acid extraction
EN ISO 21569 Foodstuffs – Methods of analysis for the detection of genetically modified organisms and
derived products – Qualitative nucleic acid based methods
EN ISO 21570 Foodstuffs – Methods of analysis for the detection of genetically modified organisms and
derived products – Quantitative nucleic aced based methods
Further information about definitions and general items involving the steps cited above are collected in:
EN ISO 24276 Foodstuffs – Nucleic acid based methods of analysis for the detection of genetically modified
organisms and derived products – General requirements and definitions
Annex A is informative.
Annex B is informative.
This document may touch copyrights and patents: for further information, contact your National
Standardisation Institute.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this International Standard: Austria, Belgium, Czech Republic, Denmark,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway,
Portugal, Slovakia, Spain, Sweden, Switzerland, and the United Kingdom.
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Introduction
Analysis to detect genetically modified traits (GMs) and derived products can either be performed to screen,
identify or quantify GMs and their derived products in a given matrix.
For the detection of the transgenic origin of ingredients, the basic principle of a protein-based method is to:
Take a representative sample of the matrix;
Extract the proteins;
Detect and/or quantify the specific protein derived from the GM(s) under study.
As new methods become validated and accepted, they will be annexed to this standard.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 21572
Foodstuffs — Molecular biomarker analysis — Protein-based
methods
1 Scope
This International Standard provides general guidelines and performance criteria for methods for the detection
and/or quantification of specific proteins derived from genetically modified (GM) plant material in a specified
matrix.
These general guidelines address existing antibody based methods. Methods other than those described in
Annex A or Annex B may also detect the protein. The same criteria as outlined in this standard generally apply.
2 Normative references
This International Standard incorporates by dated or undated reference, provisions from other publications.
These normative references are cited at the appropriate places in the text and the publications are listed
hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to
this International Standard only when incorporated in it by amendment or revision. For undated references the
latest edition of the publication referred to applies (including amendments).
3 Terms and
...
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