EN ISO 21572:2013

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
Foreword .3
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.

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
ISO 21572:2013(E)
© 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
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Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
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Published in Switzerland
ii © ISO 2013 – All rights reserved

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 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

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]
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

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).
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

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]
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

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 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 routinely required for qualitative application such as lateral
flow strips, 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, measurement standards, and add each of them at minimum in duplicate,
properly diluted to the assay.
For a qualitative test or semiquantitative test, select the required number of test (e.g. lateral flow strips
or ELISA) needed for the test portions to be analysed. The stability of the final signal can 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 relevant
ISO 21572:2013(E)
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 CV of calculated concentrations
resulting from replicate measurements of a sample test solution, in general, should not exceed 20 %.
If the CV limit is exceeded, the analyses should be repeated on freshly prepared sample test solution.
To establish a CV, in this case, at least three determinations shall be carried out (e.g. values from three
microtitre 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 semiquantitative analysis
The following parameters shall be evaluated: raw data of sample test solution, blanks, reference materials
or measurement standards, and negative controls; percentage CV between replicates, percentage CV of
standard and percentage CV of control samples.
According to ISO/IEC 17025:2005, 5.10.3.1 c), 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
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 which 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. CV 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
8 © ISO 2013 – All rights reserved

ISO 21572:2013(E)
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 has to 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) shall account for incomplete extraction.
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, 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.
10.3 Assay applicability
10.3.1 General
Food processing generally leads 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.
10.3.2 Hook effect
In an antibody-based lateral flow device and plate format assay, a hook (saturation) effect can 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.
ISO 21572:2013(E)
10.3.3 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 shall 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 described as a quadratic
or four-parameter function.
The number of calibration points supplied should be at least four and reflect the usable portion of the curve.
10.3.4 Limits of detection
Results should not be interpreted below the limit of detection. In this case, reporting of results shall be
stated according to applicable method as described in 9.1 to 9.3.
10.3.5 Limits of quantification
The limits of quantification for each set of calibrants (or dilution) shall be stated explicitly.
The estimated concentration of unknown sample test solutions shall be interpolated and not extrapolated.
Results shall not be extrapolated below the limit of quantification or above the highest or below the
lowest calibration points.
11 Confirmation method
To establish the credibility of assays, another method, such as western blot, HPLC or functional assay
can be used to measure split analytical samples of known concentration. The results of both methods
are then qualitatively/quantitatively compared. This is especially important for immunoassays, since
antibodies can cross react with other analytes present in a matrix.
12 Test report
The test report shall contain at least the following information:
a) all the information needed to identify the laboratory;
b) all the information needed to identify the laboratory sample;
c) reference to this International Standard, i.e. ISO 21572, and to the method used, and an indication
of whether it was a qualitative, quantitative or semiquantitative method;
d) limit of detection;
e) lower and/or upper limits of quantification;
f) date and type of sampling procedure used (if known);
g) date of sample receipt;
h) analysis start date or other appropriate documentation;
i) amount of the test portion;
j) amount of the test sample;
k) results and the units used to report them;
l) any special points observed during testing;
10 © ISO 2013 – All rights reserved

ISO 21572:2013(E)
m) if known, limitations, such as possible cross-reactants or selectivity of each method;
n) any operation not specified in this method or considered to be optional, but that can have an effect
on the results.
ISO 21572:2013(E)
Annex A
(informative)
Detection of a protein by ELISA
A.1 ELISA methods
ELISAs, have been successfully and routinely used for qualitative or quantitative detection of antigens,
including novel proteins expressed in crops derived from modern biotechnology. The expressed proteins
generally confer agronomic or quality characteristics, such as tolerance to specified herbicide (e.g.
glyphosate, glufosinate) or resistance to insects (e.g. Cry-1Ab, Cry3Bb1, Cry1F). The proteins produced
by the biotechnology derived plants may be detected using ELISA-based methods.
Most of the commercial ELISA methods for modern biotechnology are applicable to samples where little
or no treatment and processing have been performed, and, thus, the POIs have not been denatured.
Heating, steaming, drying, are just a few examples to which food ingredients are subjected during
processing which will denature proteins, and thus impact or greatly diminish the ability of the
immunological reagents used in the ELISAs to bind to the specific proteins in its non-native states.
Selectivity of the method should be reported, in certain cases ELISAs will also react with similar proteins
that are produced in various varieties of the biotechnology derived crops, such as Cry1Ab in Bt11 corn.
The ELISA methods are routinely utilized in a qualitative manner for presence or absence testing as
well as in a quantitative manner. The limit of detection (LOD) for ELISA kit is usually established by
the kit manufacturer to be consistent with the expression in the lowest expressing variety. Cross-
reactivity with other proteins specific for biotechnology derived crops is examined. The ELISA kits are
commercially available worldwide. Applications for other (i.e. non-grain) protein-containing matrices,
such as leaf, are clearly identified by the kit manufacturer in the product inserts that are part of the kit
and should be supported by manufacturer’s method validation data.
A.2 Procedure
This annex outlines a generic example of a procedure for using ELISA to determine if the POI is present,
and to quantitate the amount of the POI present in the sample. The method is applicable to samples
where little or no treatment or processing has been carried out, and, thus, the POI is not denatured. For
example, high temperatures at which food ingredients are processed may impact the ability to detect
the POI. Each manufacturer shall supply the ELISA procedure with the kit and specify which matrix
can be tested using the ELISA along with acceptance and rejection criteria that have been established
through development and validation of the method.
A.3 Principle
A direct sandwich enzyme linked immunosorbent assay (ELISA) is used for detection of a POI as shown
in Figures A.1 to A.4.
12 © ISO 2013 – All rights reserved

ISO 21572:2013(E)
The surface of a microtitre plate is coated with a specific monoclonal capture antibody.
Figure A.1 — Step 1
When the sample of interest is added, the capture antibody binds the antigen. Unbound components of
the sample are removed by washing.
Figure A.2 — Step 2
After washing, a polyclonal antibody, covalently linked (for example) to horseradish peroxidase (HRP)
is added, which is specific for a second antigenic site on the bound POI.
Figure A.3 — Step 3
ISO 21572:2013(E)
After washing, a tetramethylbenzidine (TMB) chromogenic substrate for horseradish peroxidase is
added. The horseradish peroxidase generates a colour signal which is proportional to the concentra-
tion of the antigen in a linear range. To stop the colour development, a stop solution is added. The
degree of colour produced is measured at a wavelength of 450 nm.
Figure A.4 — Step 4
A.4 Reagents
A.4.1 General
During the
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