SIST EN ISO 21572:2020
(Main)Foodstuffs - Molecular biomarker analysis - Immunochemical methods for the detection and quantification of proteins (ISO 21572:2019)
Foodstuffs - Molecular biomarker analysis - Immunochemical methods for the detection and quantification of proteins (ISO 21572:2019)
This Standard specifies performance criteria for immunochemical methods for the detection and/or quantification of a specific protein or protein(s) of interest [POI(s)] in a specified matrix. The methods discussed are applicable to the analysis of proteins from a variety of sample types. Some uses for these methods include, but are not limited to, analysing proteins involved in crop and food production, food processing, food marketing, food safety, biotechnology or disease indexing.
Lebensmittel - Untersuchung auf molekulare Biomarker - Proteinverfahren (ISO 21572:2019)
Dieses Dokument legt allgemeine Leistungskriterien für immunochemische Verfahren zum Nachweis und/oder zur quantitativen Bestimmung eines spezifischen Proteins oder von mehreren Proteinen von Interesse (en: protein(s) of interest, POI) in einer spezifischen Matrix fest.
Die hier besprochenen Verfahren sind für die Untersuchung von Proteinen in einer Vielzahl von Probentypen anwendbar. Einige dieser Verfahren dienen zur Untersuchung von Proteinen, die beim Anbau von Feldfrüchten, bei der Herstellung, Verarbeitung, Vermarktung und Sicherheit von Lebensmitteln, in der Biotechnik oder zur Krankheitsbestimmung relevant sind, sind aber nicht darauf beschränkt.
Produits alimentaires - Analyse des biomarqueurs moléculaires - Méthodes immunochimiques pour la détection et la quantification des protéines (ISO 21572:2019)
Le présent document énonce des critères de performances pour les méthodes immunochimiques de détection et/ou de quantification d'une protéine spécifique ou de protéine(s) d'intérêt (POI) dans une matrice donnée.
Les méthodes décrites sont applicables à l'analyse de protéines provenant de divers types d'échantillons. Certaines utilisations de ces méthodes comprennent, entre autres, l'analyse des protéines impliquées dans la production végétale et agroalimentaire, la transformation des aliments, la commercialisation des produits alimentaires, la sécurité des aliments, les biotechnologies ou l'indexation des maladies.
Živila - Analiza molekulskih biomarkerjev - Imunokemijske metode za odkrivanje prisotnosti in kvantifikacijo beljakovin (ISO 21572:2019)
Ta standard določa merila uspešnosti imunokemijskih metod za odkrivanje in/ali kvantifikacijo specifičnih zadevnih beljakovin ali (POI) v določeni matrici. Obravnavane metode so uporabne za analizo beljakovin iz različnih vrst vzorcev. Nekatere uporabe teh metod med drugim vključujejo analizo beljakovin, vključenih v pridelavo pridelkov in hrane, predelavo hrane, trženje hrane, varnost hrane, biotehnologijo ali indeksiranje bolezni.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 21572:2020
01-januar-2020
Nadomešča:
SIST EN ISO 21572:2013
Živila - Analiza molekulskih biomarkerjev - Imunokemijske metode za odkrivanje
prisotnosti in kvantifikacijo beljakovin (ISO 21572:2019)
Foodstuffs - Molecular biomarker analysis - Immunochemical methods for the detection
and quantification of proteins (ISO 21572:2019)
Lebensmittel - Untersuchung auf molekulare Biomarker - Proteinverfahren (ISO
21572:2019)
Produits alimentaires - Analyse des biomarqueurs moléculaires - Méthodes
immunochimiques pour la détection et la quantification des protéines (ISO 21572:2019)
Ta slovenski standard je istoveten z: EN ISO 21572:2019
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN ISO 21572:2020 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 21572:2020
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SIST EN ISO 21572:2020
EN ISO 21572
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 67.050 Supersedes EN ISO 21572:2013
English Version
Foodstuffs - Molecular biomarker analysis -
Immunochemical methods for the detection and
quantification of proteins (ISO 21572:2019)
Produits alimentaires - Analyse des biomarqueurs Lebensmittel - Untersuchung auf molekulare
moléculaires - Méthodes immunochimiques pour la Biomarker - Proteinverfahren (ISO 21572:2019)
détection et la quantification des protéines (ISO
21572:2019)
This European Standard was approved by CEN on 30 September 2019.
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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21572:2019 E
worldwide for CEN national Members.
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SIST EN ISO 21572:2020
EN ISO 21572:2019 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 21572:2020
EN ISO 21572:2019 (E)
European foreword
This document (EN ISO 21572:2019) 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 April 2020, and conflicting national standards shall be
withdrawn at the latest by April 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 21572:2013.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 21572:2019 has been approved by CEN as EN ISO 21572:2019 without any modification.
3
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SIST EN ISO 21572:2020
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SIST EN ISO 21572:2020
INTERNATIONAL ISO
STANDARD 21572
Third edition
2019-10
Foodstuffs — Molecular biomarker
analysis — Immunochemical methods
for the detection and quantification of
proteins
Produits alimentaire — Analyse des biomarqueurs moléculaires —
Méthodes immunochimiques pour la détection et la quantification des
protéines
Reference number
ISO 21572:2019(E)
©
ISO 2019
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SIST EN ISO 21572:2020
ISO 21572:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 21572:2020
ISO 21572:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Laboratory equipment . 2
7 Sampling . 2
8 Procedure. 2
8.1 General . 2
8.2 Preparation of sample solution . 2
8.3 Extraction . 3
8.4 Preparation of calibration curves, positive controls, and reference materials . 3
8.5 Assay procedure . 3
9 Interpretation and expression of results . 3
9.1 General . 3
9.2 Quantitative and semi-quantitative analysis . 4
9.3 Qualitative analysis . 4
10 Specific parameters that can influence results . 4
10.1 General . 4
10.2 Special considerations . 5
10.2.1 Selectivity . 5
10.2.2 Extraction efficiency . 5
10.2.3 Matrix effects. 5
10.2.4 Assay applicability . 5
10.2.5 Hook effect . 5
10.2.6 Parallelism/linearity . 5
10.2.7 Limits of detection . 6
10.2.8 Limits of quantification . 6
11 Confirming method . 6
12 Test report . 6
Annex A (informative) Detection of a protein by ELISA . 8
Annex B (informative) Detection of a protein or proteins by lateral flow devices .19
Bibliography .26
© ISO 2019 – All rights reserved iii
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SIST EN ISO 21572:2020
ISO 21572:2019(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso
.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis.
This third edition cancels and replaces the second edition (ISO 21572:2013), which has been technically
revised. The main changes compared with the previous edition are as follows:
— the title has been changed to specify that the document is focused on immunochemical protein
detection methods;
— an introduction has been added;
— terms, definitions and references have been updated;
— the text has been modified to improve the document’s applicability to general protein analysis
applications.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
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SIST EN ISO 21572:2020
ISO 21572:2019(E)
Introduction
Analytical techniques based on highly specific immunochemical-binding interactions have become
key tools for analysing many different chemical and macromolecular analytes, including proteins.
Methods utilizing these techniques are widely accepted in the scientific and regulatory communities.
Immunochemical assay methods are most commonly used to detect (presence or absence) and/
or quantify specific protein analytes such as allergenic proteins, disease marker proteins or newly
expressed proteins in biotech crops.
Prior to analysis, samples generally need to be ground or processed in a manner that facilitates
extraction of the analyte from the sample matrix. An important step in analytical method development
is therefore the selection of a suitable extraction buffer that does not interfere with the analytical
method performance and that ensures an appropriate level of analyte stability during the analytical
process.
The immunochemical assay process generally incorporates at least two steps:
— binding or capturing the analyte of interest present in samples with an antibody targeted specifically
to the analyte;
— detection of the antibody-analyte complex using a technique that signals the specific interaction.
Once an analytical method has been developed and optimized, it should be validated to demonstrate
that its performance is reliable and suitable for the intended use and to characterize the method
limitations. This involves performing several experiments with real samples to evaluate parameters
such as accuracy, precision, sensitivity, selectivity and the detection or quantification limits. Validation
also allows for the establishment of method performance criteria, against which routine analytical
performance can be compared to ensure that acceptable analytical results are consistently reported.
This document provides a set of general procedures and analytical considerations for using
immunochemical techniques to analyse target proteins. It discusses aspects of sample processing,
extraction, assay set-up, interpretation and reporting of results, and relevant assay performance
parameters. Two annexes are included containing example procedures that can be followed when
analysing a protein of interest (POI) in a variety of background matrices using methods based on
enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices (LFDs).
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SIST EN ISO 21572:2020
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SIST EN ISO 21572:2020
INTERNATIONAL STANDARD ISO 21572:2019(E)
Foodstuffs — Molecular biomarker analysis —
Immunochemical methods for the detection and
quantification of proteins
1 Scope
This document specifies performance criteria for immunochemical methods for the detection and/or
quantification of a specific protein or protein(s) of interest [POI(s)] in a specified matrix.
The methods discussed are applicable to the analysis of proteins from a variety of sample types. Some
uses for these methods include, but are not limited to, analysing proteins involved in crop and food
production, food processing, food marketing, food safety, biotechnology or disease indexing.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 16577, Molecular biomarker analysis — Terms and definitions
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
conjugate
material produced by attaching two or more substances together by covalent bond via chemical groups
Note 1 to entry: Conjugates of antibodies with fluorochromes (e.g. chemical entity, such as a molecule or group,
that emits light in response to excitation by absorbed incident light), radiolabelled substances, gold or enzymes
are often used in immunoassays.
4 Principle
The target protein 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 specific protein derived from the matrix under study.
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SIST EN ISO 21572:2020
ISO 21572:2019(E)
5 Reagents
During the analysis, use only reagents of recognized analytical grade and only de-ionized 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. 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 document, though Annex A and Annex B do provide
sampling instructions as per the relevant methods. It is recommended that the parties concerned come
to an agreement on this subject.
8 Procedure
8.1 General
Storage conditions and the shelf-life of LFDs, antibodies, conjugate, substrate, 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 document, 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, prepare the sample solution. Sample preparation procedures
are described in Annexes A and B.
Grind samples as specified in the method before test portions are taken, if necessary. Powders/flour can
have swelling properties and could require more extraction solution if a manufacturer’s method does
not specify this information. If the sample is not immediately used, follow the laboratory’s procedure
for storage (e.g. −20 °C or below).
Weigh an appropriate amount of a representative test sample (as specified in A.6.6.1 and B.6.2.1) for
analysis to create a test portion for extraction. Add extraction solution and homogenize or mix.
Laboratory samples containing high amounts of fat can be non-homogeneous and a larger test portion
should be extracted to ensure that it is representative. If applicable, instructions can be found in the
sample preparation sections of Annexes A and B.
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SIST EN ISO 21572:2020
ISO 21572:2019(E)
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 LFDs. 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 Preparation of calibration curves, positive controls, and reference materials
For the preparation of calibration curves, positive controls and reference materials for Annex A, it
is recommended to use matrix-matched reference materials or reference materials that have been
validated for the matrix. Calibration curves are not required for qualitative application such as LFDs.
However, positive and negative controls can be prepared at the discretion of the analyst.
8.5 Assay procedure
For a quantitative test, select the required number of wells, (e.g. in ELISA) for the test portion(s) to be
analysed, including blanks, positive controls and negative controls, and add each of them, at minimum
in duplicate and properly diluted so as to be within the range of the assay.
For a qualitative test or semi-quantitative test, select the required number of tests (e.g. ELISA or LFDs)
needed for the test portions to be analysed, including blanks, positive controls and negative controls. The
stability of the final signal can vary. Read the results in a timely manner as specified in Annexes A and B.
According to the method chosen, follow the instructions of each method for sample analyses, including
blanks, reference materials and/or measurement standards (if necessary). Allow the reaction to
occur at a specified temperature range and time. If necessary, terminate the reaction according to the
method described in A.6.6.2.7 and B.6.4.2. For example, if the ELISA method requires acquiring data
on a spectrophotometer, perform this step. In the case of qualitative tests, follow the kit instructions.
Generally, these are interpreted visually.
9 Interpretation and expression of results
9.1 General
The parameters to interpret vary depending on whether the assay is qualitative, semi-quantitative or
quantitative.
For quantitative methods, the coefficient of variation (C ) of optical density values resulting from
V
replicate measurements of a sample test solution, in general, should not exceed 15 %. The coefficient of
variation of calculated concentrations resulting from replicate measurements of a sample test solution,
in general, should not exceed 20 %.
If the coefficient of variation limit is exceeded, the analyses should be repeated on freshly prepared
sample test solution. To establish a coefficient of variation, in this case, at least three determinations
shall be carried out (e.g. values from three micro-titre wells).
Negative results shall be reported as “negative at the limit of detection” and the limit of detection (LOD)
shall be reported.
Positive results below the limit of quantification shall be reported as “positive above the limit of
detection, but below the limit of quantification”. The limits of quantification and detection shall be
reported.
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SIST EN ISO 21572:2020
ISO 21572:2019(E)
9.2 Quantitative and semi-quantitative analysis
For quantitative and semi-quantitative analysis, the following parameters shall be evaluated:
— raw data of the sample test solution;
— blanks;
— reference materials or measurement standards;
— negative controls;
— % C between replicates;
V
— % C of standards;
V
— % C of control samples.
V
In accordance with ISO/IEC 17025, measurement uncertainty should be reported, where applicable.
Quantitative results shall not be reported by extrapolating above the highest or below the lowest
calibration point.
9.3 Qualitative analysis
For qualitative tests, including all applications thereof, the corresponding parameters are described in
Annexes A and B. The LOD shall always be reported. Negative results shall be reported as “negative at
the limit of detection”.
Positive results shall also report the LOD.
10 Specific parameters that can influence results
10.1 General
The performance criteria listed in the method of Annex A are a set of performance specifications
established for each method during the development, validation and routine use of the method. These
parameters shall be estimated and evaluated for each method to ensure they are reliable and of
consistently high quality. Each time a method is implemented, the data generated shall be evaluated
and compared with the established method performance criteria.
When a value (e.g. coefficient of variation of replicate determinations) does not agree with the assay
specifications, it signals that the result is atypical and warrants closer evaluation of the data. The list
of specifications shall be taken as whole. In certain instances, individual parameters may not meet the
specifications but the data are still perfectly acceptable. If any of the criteria are not met, this should,
however, be acknowledged in writing and the data evaluated to determine if the analysis of results
should be adjusted, or if a particular sample or a set of samples should be repeated. These decisions
should be based on the judgement of the technical expert interpreting the entire set of criteria.
In contrast to the method described in Annex A, the performance criteria of LFD assays as described
in Annex B are evaluated during the development of the method by the manufacturer of the kit. The
method should be evaluated for repeatability in the laboratory prior to use on test samples. Non-
performing kits shall not be used.
4 © ISO 2019 – All rights reserved
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SIST EN ISO 21572:2020
...
SLOVENSKI STANDARD
oSIST prEN ISO 21572:2019
01-marec-2019
Živila - Analiza molekulskih biomarkerjev - Metode na osnovi proteinov (ISO/DIS
21572:2019)
Foodstuffs - Molecular biomarker analysis - Protein-based methods (ISO/DIS
21572:2019)
Lebensmittel - Untersuchung auf molekulare Biomarker - Proteinverfahren (ISO/DIS
21572:2019)
Produits alimentaires - Analyse des biomarqueurs moléculaires - Méthodes basées sur
les protéines (ISO/DIS 21572:2019)
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 21572:2019 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 21572:2019
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oSIST prEN ISO 21572:2019
DRAFT INTERNATIONAL STANDARD
ISO/DIS 21572
ISO/TC 34/SC 16 Secretariat: ANSI
Voting begins on: Voting terminates on:
2019-01-24 2019-04-18
Foodstuffs — Molecular biomarker analysis — Protein-
based methods
Produits alimentaires — Analyse des biomarqueurs moléculaires — Méthodes basées sur les protéines
ICS: 67.050
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
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
ISO/CEN PARALLEL PROCESSING
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 STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 21572:2019(E)
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. ISO 2019
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oSIST prEN ISO 21572:2019
ISO/DIS 21572:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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oSIST prEN ISO 21572:2019
ISO/DIS 21572:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Laboratory equipment . 2
7 Sampling . 2
8 Procedure. 2
8.1 General . 2
8.2 Preparation of sample solution . 2
8.3 Extraction . 2
8.4 Preparation of calibration curves, positive controls, and reference materials . 3
8.5 Assay procedure . 3
9 Interpretation and expression of results . 3
9.1 General . 3
9.2 Quantitative and semi-quantitative analysis . 3
9.3 Qualitative analysis . 4
10 Specific parameters that may influence results . 4
10.1 General . 4
10.2 Special considerations . 4
10.2.1 Selectivity . 4
10.2.2 Extraction efficiency . 4
10.2.3 Matrix effects. 5
10.2.4 Assay applicability . 5
10.2.5 Hook effect . 5
10.2.6 Parallelism/linearity . 5
10.2.7 Limits of detection . 5
10.2.8 Limits of quantification . 5
11 Confirming method . 5
12 Test report . 6
Annex A (informative) Detection of a protein by ELISA . 7
Annex B (informative) Detection of a protein(s) by lateral flow devices .17
Bibliography .24
© ISO 2019 – All rights reserved iii
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oSIST prEN ISO 21572:2019
ISO/DIS 21572:2019(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso
.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 034, Food Products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis.
This third edition cancels and replaces the second edition (ISO 21572:2013), which has undergone a
minor revision.
The main changes compared to the previous edition are as follows:
— The title was changed to specify that this document is focused on immunochemical protein detection
methods. An introduction was added. The text was modified to improve applicability to general
protein analysis applications. Terms, definitions and references were updated. The document was
reformatted according to the current ISO template. Additional minor revisions were incorporated
for clarity and grammar.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
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Introduction
Analytical techniques based on highly specific immunochemical-binding interactions have become
key tools for analysing many different chemical and macromolecular analytes, including proteins, and
methods utilising these techniques are widely accepted in the scientific and regulatory communities.
Immunochemical assay methods are most commonly used to detect (presence or absence) and/
or quantify specific protein analytes such as allergenic proteins, disease marker proteins or newly
expressed proteins in biotech crops.
Prior to analysis, samples generally need to be ground or processed in a manner that facilitates
extraction of the analyte from the sample matrix. An important step in analytical method development
is therefore the selection of a suitable extraction buffer that does not interfere with the analytical
method performance and that ensures an appropriate level of analyte stability during the analytical
process.
The immunochemical assay process generally incorporates at least two steps; binding or capturing
the analyte of interest present in samples with an antibody targeted specifically to the analyte and
detection of the antibody-analyte complex using a technique that signals the specific interaction.
Once an analytical method has been developed and optimized it should be validated to demonstrate
that its performance is reliable and suitable for the intended use and to characterize the method
limitations. This involves performing several experiments to evaluate parameters such as accuracy,
precision, sensitivity, selectivity and the detection or quantitative limits. Validation also allows for the
establishment of method performance criteria, against which routine analytical performance can be
compared to ensure that acceptable analytical results are consistently reported.
This International Standard provides a set of general procedures and analytical considerations for
using immunochemical techniques to analyze target proteins. It discusses aspects of sample processing,
extraction, assay set-up, interpretation and reporting of results, and relevant assay performance
parameters. Two informative appendices are included that contain example procedures to follow when
analysing a protein of interest (POI) in a variety of background matrices using methods based on
enzyme-linked immunosorbent assays (ELISA) and lateral flow devices (LFD).
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DRAFT INTERNATIONAL STANDARD ISO/DIS 21572:2019(E)
Foodstuffs — Molecular biomarker analysis — Protein-
based methods
1 Scope
This International Standard provides general guidelines and performance criteria for immunochemical
methods for the detection and/or quantification of a specific protein or protein(s) of interest [POI(s)]
in a specified matrix. The methods discussed are applicable to analysis of a variety of different types
of proteins. Some uses for these methods include, but are not limited to, analysing proteins involved in
biotechnology or disease indexing.
2 Normative references
The following documents are referred to in the text in such a way that some or all their content
constitutes requirements of this document. 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
[1]
For the purposes of this document, the terms and definitions given in ISO 16577 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
conjugate
material produced by attaching two or more substances together by covalent bond via chemical groups
Note 1 to entry: Conjugates of antibodies with fluorochromes (e.g. chemical entity, such as a molecule or group,
that emits light that is in response to being stimulated by absorption of incident light), radiolabelled substances,
gold or enzymes are often used in immunoassays.
4 Principle
The target protein 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 specific protein derived from the matrix under study.
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5 Reagents
During the analysis, use only reagents of recognized analytical grade and only de-ionized 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, though Annex A and
Annex B do provide sampling instructions as per the relevant methods. It is recommended that the
parties concerned come to an agreement on this subject.
8 Procedure
8.1 General
Storage conditions and shelf-life of lateral flow devices, antibodies, conjugate, substrate, 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 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, etc.) Carefully clean all equipment coming into direct
[2]
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 non-homogeneous 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 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
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Annex B for lateral flow devices. 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 Preparation of calibration curves, positive controls, and reference materials
For preparation of calibration curves, positive controls, and reference materials for Annex A, it is
recommended to use matrix matched reference materials or reference materials that have been
validated for the matrix. Calibration curves are not routinely required for qualitative application such
as lateral flow devices; however, positive and negative controls can be prepared at the discretion of the
analyst.
8.5 Assay procedure
For a quantitative test select the required number of wells, (e.g., in ELISA) for the test portion(s) to
be analysed, including blanks, positive and negative standards, and add each of them at minimum in
duplicate, properly diluted so as to be within the range of the assay.
For a qualitative test or semi-quantitative test select the required number of tests (e.g., lateral flow
devices or ELISA) needed for the test portions to be analysed. The stability of the final signal may vary.
Read the results in a timely manner as specified in the Annexes.
According to the method chosen, follow the instructions of each method for sample analyses,
including blanks and measurement standards (if necessary). Allow the reaction to occur at a specified
temperature range and time. If necessary, terminate the reaction according to the method described in
the Annex. For example, if ELISA method requires acquiring data on a spectrophotometer, perform this
step. In the case of qualitative tests, generally these are interpreted visually, follow the kit instructions.
9 Interpretation and expression of results
9.1 General
The parameters to interpret vary depending on whether the assay is qualitative, semiquantitative or
quantitative.
For quantitative methods, the coefficient of variation (CV) of optical density values resulting from
replicate measurements of a sample test solution, in general, should not exceed 15 %. The coefficient of
variation of calculated concentrations resulting from replicate measurements of a sample test solution,
in general, should not exceed 20 %.
If the coefficient of variation limit is exceeded, the analyses should be repeated on freshly prepared
sample test solution. To establish a coefficient of variation, in this case, at least three determinations
shall be carried out (e.g. values from three micro-titer wells).
Negative results shall be reported as “negative at the limit of detection” and the limit of detection shall
be reported.
Positive results below the limit of quantification shall be reported as “positive above the limit of
detection, but below the limit of quantification”. The limits of quantification and detection shall be
reported.
9.2 Quantitative and semi-quantitative analysis
The following parameters shall be evaluated: raw data of sample test solution, blanks, reference
materials or measurement standards, and negative controls; % CV between replicates, % CV of
standards and % CV of control samples.
[2]
In accordance with ISO/IEC 17025 , measurement uncertainty should be reported where applicable.
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Quantitative results shall not be reported by extrapolating above the highest or below the lowest
calibration point.
9.3 Qualitative analysis
For qualitative tests, including all applications thereof, the corresponding parameters are described in
the annexes. The limit of detection shall always be reported, and negative results shall be reported as
"negative at the limit of detection".
Positive results shall also report the limit of detection.
10 Specific parameters that may influence results
10.1 General
The performance criteria listed in the method of Annex A are a set of performance specifications
established for each method during the development, validation and routine use of the method. These
parameters shall be estimated and evaluated for each method and are reliable and of consistently high
quality. Each time a method is implemented the data generated shall be evaluated and compared with
the established method performance criteria.
When a value (e.g. coefficient of variation of replicate determinations) does not agree with the assay
specifications, it signals that the result is atypical and warrants closer evaluation of the data. The list
of specifications shall be taken as whole, individual parameters may in certain instances not meet the
specifications, but the data may still be perfectly acceptable. If any of the criteria are not met, it should,
however be acknowledged in writing and the data evaluated to determine if the analysis of results
should be adjusted, or if a particular sample or a set of samples should be repeated. These decisions
should be based on the judgement of the technical expert interpreting the entire set of criteria.
10.2 Special considerations
10.2.1 Selectivity
Adequate selectivity of the assay for a particular analyte shall be demonstrated for each POI or analyte
(protein) to be measured in each matrix to be tested. Where appropriate, cross-reactivity should be
evaluated for analogues (proteins with a similar sequence or structure). To test for the absence of
the POI in non-POI sample, assay the non-POI containing sample and POI-containing sample at the
appropriate dilutions and compare.
This is generally done during the development and validation of the method and is not necessary during
routine analysis of samples for which the method has previously been validated. Selectivity of the test
kits, either ELISA or lateral flow device-based methods, should be addressed by the manufacturer of
the kit (e.g. listed in the manufacturer’s product inserts).
10.2.2 Extraction efficiency
Special care shall be taken to assess the influence of process parameters applied for the production of a
given laboratory sample.
In order to provide for the greatest sensitivity of the immunoassay, extraction efficiency should be
as high as possible, especially for quantitative methods. The assay performance is matrix dependent.
Extraction efficiency should be determined and documented for each matrix.
The extraction procedure shall be demonstrated to be reproducible and the method of calibration (if
applicable) should account for incomplete extraction.
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10.2.3 Matrix effects
The scope of application clearly and exactly defines the matrices for which the given immunoassay
is applicable. The use of matrix matched reference materials allows for direct comparison between
reference materials and samples. However, if samples are to be analysed against reference materials
which are not the same matrix, then the matrix effects will have to be evaluated.
For example, prepare a negative extract for each sample (matrix) to be analysed by the method and
an extract of a positive control of known concentration. Prepare a series of dilutions of the positive
control in the negative extract and compare the resulting dose response curve with the calibration
curve from the method. If the two curves are different, then there is a matrix effect. Use a matrix that
most closely represents the true samples that will be tested. A dilution curve with a positive control of
known concentration should also be included as a reference. The shape of the calibration curve should
not change due to a matrix effect.
10.2.4 Assay applicability
Food processing will generally lead to degradation or denaturation of the POI, which may result in a
substantial change in immunoreactivity. Immunoassays should be evaluated for applicability to the POI
in processed products.
10.2.5 Hook effect
In an antibody-based lateral flow device and plate format assay, a hook (saturation) effect could lead to
a false negative result. A thorough demonstration that the working concentration range comfortably
covers the practical need of POI test samples is necessary.
10.2.6 Parallelism/linearity
For quantitative analyses, the expected dynamic range of the immunoassay should be explicitly
stated in the scope of applications for all matrices covered by it. The relationship of the instrument
response to known POI concentration may not be linear and must be established for each quantitative
immunoassay method by the manufacturer. It can be linear in a very narrow range of the POI, but in
most cases, immunoassays show a more complex relationship which has been previously describes as a
quadratic or 4-parameter function.
The number of calibration poi
...
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