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

Status
Published
Public Enquiry End Date
24-Mar-2019
Publication Date
10-Dec-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
21-Nov-2019
Due Date
26-Jan-2020
Completion Date
11-Dec-2019

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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

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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.

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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

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;
— % C of standards;
— % C of control samples.

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.
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SIST EN ISO 21572:2020
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