ISO 15089:2000

Water quality — Guidelines for selective immunoassays for the determination of plant treatment and pesticide agents

This International Standard specifies a guide for the selective quantitative analysis by immunoassays of environmental chemicals such as pesticides (including insecticides) or their metabolites in drinking, ground and surface water. The application range of the procedure for the analysis of pesticides in drinking water applies to mass concentrations W 0,05 _g/l. Therefore, the determination limit should be in this case u 0,05 _g/l.

Qualité de l'eau — Lignes directrices relatives aux dosages immunologiques sélectifs pour la détermination des agents de traitement des plantes et des pesticides

La présente Norme internationale spécifie des lignes directrices pour l'analyse quantitative sélective, par des dosages immunologiques, des produits chimiques utilisés dans l'environnement, tels que les pesticides (y compris les insecticides) ou leurs métabolites présents dans l'eau potable, l'eau souterraine et l'eau de surface. Le présent mode opératoire pour l'analyse des pesticides dans l'eau potable est applicable à des concentrations en masse W 0,05 μg/l. En conséquence, il convient que, dans ce cas, la limite de détermination soit u 0,05 μg/l.

Kakovost vode - Smernice za selektivno imunoanalizo za določevanje aktivnih snovi v sredstvih za zaščito rastlin in v pesticidih

Ta mednarodni standard določa vodilo za selektivne kvantitativne analize z imunoanalizo okoljskih kemikalij, kot so pesticidi (vključno insekticidi) ali njihovi metaboliti v pitni, podtalni ali površinski vodi. Razpon uporabe postopka za analizo pesticidov v pitni vodi velja za koncentracije mas  . Potemtakem bi morala v tem primeru biti meja določevanja u  .

General Information

• Status: Published
• Publication Date: 22-Mar-2000
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: ISO/TC 147/SC 2 - Physical, chemical and biochemical methods
• Drafting Committee: ISO/TC 147/SC 2 - Physical, chemical and biochemical methods
• Current Stage: 9093 - International Standard confirmed
• Start Date: 05-Aug-2021
• Completion Date: 14-Feb-2026
• Ref Project: SIST ISO 15089:2010 - Water quality - Guidelines for selective immunoassays for the determination of plant treatment and pesticide agents

Overview

ISO 15089:2000 - "Water quality - Guidelines for selective immunoassays for the determination of plant treatment and pesticide agents" provides guidance for using selective immunoassays (antibody-based assays such as ELISA) to quantify pesticides, insecticides and their metabolites in drinking, ground and surface water. The standard addresses procedural principles, sampling and sample preparation, reagents and apparatus, assay validity and reporting. For drinking-water analysis the document indicates an application range around 0.05 µg/l and specifies that the method’s determination limit should be at this level.

Key topics and technical requirements

• Scope and purpose: Guidance for selective quantitative immunoassays applied to environmental contaminants (pesticides/haptens) in water.
• Analytical principle: Competitive immunoassays (heterogeneous solid-phase formats) using antibodies, tracers and calibration with analyte standards (EIA/ELISA, fluorescence or luminescence variants).
• Assay components: Antibodies/antisera, coating (hapten–carrier) conjugates, enzyme or fluorescence tracers, buffered washing solutions and stopping reagents (acid/base).
• Sampling & sample prep: Conformance with ISO 5667 series for sampling design, techniques and preservation; raw water is typical, with optional concentration or cleanup for matrix interference removal.
• Interferences & matrix effects: Identification and mitigation of adsorption, humic substances, salinity, solvents and pH effects; use of spike recovery and cross-absorption where necessary.
• Apparatus & consumables: Solid phases (microplates, beads, magnetic particles), multipipettes, magnetic racks, calibrated dispensers.
• Quality criteria: Calibration, determination limits (noted at ~0.05 µg/l for drinking water), validity criteria, precision (interlaboratory data included) and reporting requirements.
• Supplementary information: Informative annexes include a flowchart/pipetting scheme and interlaboratory precision data.

Applications and who uses it

ISO 15089 is intended for:

• Environmental and public-health laboratories performing pesticide screening in water.
• Drinking-water utilities and regulators monitoring compliance and early warning.
• Manufacturers of immunoassay kits and assay developers validating antibody specificity and cross-reactivity.
• Research groups applying antibody-based methods for rapid field screening prior to confirmatory instrumental analyses (e.g., LC-MS/MS).

Practical uses include rapid, cost-effective screening of water for pesticide residues, routine monitoring programs, and pre-screening to prioritize samples for confirmatory analysis.

Related standards (referenced)

• ISO 5667-1, -2, -3 (water sampling design, techniques, preservation)
• ISO/TR 13530 (analytical quality control for water analysis)

Keywords: ISO 15089:2000, water quality, immunoassays, ELISA, pesticides, drinking water, pesticide residues, antibody assays, competitive immunoassay, detection limit 0.05 µg/l.

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2010
.DNRYRVWYRGH6PHUQLFH]DVHOHNWLYQRLPXQRDQDOL]R]DGRORþHYDQMHDNWLYQLK
VQRYLYVUHGVWYLK]D]DãþLWRUDVWOLQLQYSHVWLFLGLK
Water quality - Guidelines for selective immunoassays for the determination of plant
treatment and pesticide agents
Qualité de l'eau - Lignes directrices relatives aux dosages immunologiques sélectifs pour
la détermination des agents de traitement des plantes et des pesticides
Ta slovenski standard je istoveten z: ISO 15089:2000
ICS:
13.060.50 3UHLVNDYDYRGHQDNHPLþQH Examination of water for
VQRYL chemical substances
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

INTERNATIONAL ISO
STANDARD 15089
First edition
2000-03-15
Water quality — Guidelines for selective
immunoassays for the determination of
plant treatment and pesticide agents
Qualité de l'eau — Lignes directrices relatives aux dosages
immunologiques sélectifs pour la détermination des agents de traitement
des plantes et des pesticides
Reference number
©
ISO 2000
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not
be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this
file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this
area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters
were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event
that a problem relating to it is found, please inform the Central Secretariat at the address given below.
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body
in the country of the requester.
ISO copyright office
Case postale 56 � CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 734 10 79
E-mail [e-mail]
Web www.iso.ch
Printed in Switzerland
ii © ISO 2000 – All rights reserved

Contents Page
Foreword.iv
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Interferences .4
5 Principle.4
6 Reagents.4
7 Apparatus .5
8 Sampling and sample preparation.5
9 Procedure .6
10 Validity criteria .7
11 Calculation.8
12 Precision.10
13 Test report .10
Annex A (informative) Flowchart and pipetting scheme.11
Annex B (informative) Precision data from an interlaboratory trial .13
Bibliography.14
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 3.
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 International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 15089 was prepared by Technical Committee ISO/TC 147, Water quality,
Subcommittee SC 2, Physical, chemical, biochemical methods.
Annexes A and B of this International Standard are for information only.
iv © ISO 2000 – All rights reserved

INTERNATIONAL STANDARD ISO 15089:2000(E)
Water quality — Guidelines for selective immunoassays for the
determination of plant treatment and pesticide agents
1 Scope
This International Standard specifies a guide for the selective quantitative analysis by immunoassays of
environmental chemicals such as pesticides (including insecticides) or their metabolites in drinking, ground and
surface water.
The application range of the procedure for the analysis of pesticides in drinking water applies to mass
concentrationsW 0,05 �g/l. Therefore, the determination limit should be in this caseu 0,05 �g/l.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 5667-1:1980, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes.
ISO 5667-2:1991, Water quality — Sampling — Part 2: Guidance on sampling techniques.
ISO 5667-3:1994, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples.
ISO/TR 13530:1997, Water quality — Guide to analytical quality control for water analysis.
3 Terms and definitions
For the purposes of this International Standard, the following terms and definitions apply.
3.1
affinity
strength of binding of antibody to analyte
NOTE The strength is defined by the equilibrium constant (K) of the reaction Ab + H = AbH, where Ab = antibody
combining site and H = hapten; K is given by the mass action equation K = c /(c � c ).
AbH Ab H
3.2
analyte
substance to be determined
3.3
antibodies
serum proteins produced in vertebrates in response to immunization and which selectively bind the antigen or
hapten, respectively
NOTE 1 Monoclonal antibodies (mAb) are uniform populations of antibodies which are produced from a single cell clone of
hybridoma cells.
NOTE 2 Polyclonal antibodies (pAb) are a mixed population of antibodies which are produced by several clones of plasma
cells.
NOTE 3 Recombinant antibodies (rAb) are produced using recombinant techniques established in gene technology.
3.4
antibody conjugate
antibody covalently linked to a label such as an enzyme or a fluorochrome
3.5
antigen
substance that stimulates the production of antibodies and reacts with them
3.6
antiserum
immune serum obtained from the blood of immunized vertebrates after removal of cellular components and
coagulation factors
NOTE It usually contains a number of different antibodies which can exhibit different affinities to the antigen/hapten.
3.7
coating conjugate
macromolecule bound to the hapten (also known as a hapten-carrier conjugate), which is immobilized to a solid
phase
NOTE It is used to bind those antibody binding sites which are not occupied by the analyte.
3.8
competitive immunoassay
test which detects the proportion of antibody binding sites which have been occupied by the sample analyte
NOTE This is achieved by adding a tracer which binds to the unoccupied antibody binding sites and produces the
measuring signal after a further reaction.
3.9
cross-reactivity
extent, to which an antibody or an antiserum reacts with a substance which structurally differs from the analyte
NOTE 1 The cross-reactivity of an antibody or an antiserum with this substance is determined by comparing the calibration
curves. The reference curve obtained with the analyte is used as reference quantity (= 100 % cross-reactivity). The cross-
reactivity is usually determined at the IC . The selectivity of an antibody or an antiserum, respectively, is inversely related to the
cross-reactivities. An antibody or an antiserum, respectively, can display different affinities to different substances. With a given
substance, the cross-reactivity of an antiserum can also vary within the measuring range. Usually the structure of the
immunogen essentially determines the selectivity (the so-called specificity) of an antiserum. If cross-reactivities are due to a
mixed population of antibodies as often occurs in an antiserum, the undesired antibodies may be removed by cross-absorption.
NOTE 2 All compounds (present in relevant concentrations) that exhibit cross-reactivity will create false positive results.
3.10
enzyme immunoassay
EIA
immunochemical analysis which detects the occupancy of antibody binding sites by the analyte with the aid of a
tracer (an enzyme-labelled hapten or an enzyme-labelled antibody) and consequently can be used to detect the
analyte concentration in the medium
NOTE The detection procedure is based on the measurement of the enzyme activity of the tracer by means of substrate
conversion.
2 © ISO 2000 – All rights reserved

3.11
enzyme substrate
substance which is converted by the enzyme into a product that can be detected by a measuring device
3.12
excess standard
analyte concentration which, once exceeded, produces no further decrease in the signal measured in the
immunoassay
3.13
fluorescence immunoassay
immunochemical detection procedure which is performed either as an immunoassay with fluorescent substrates or
products, respectively, or as an immunoassay with fluorescence-labelled tracers or antibodies, respectively
3.14
hapten
substance which, because of its small molecular size, does not evoke the production of antibodies unless it is
covalently bound to an immunogen
NOTE Pesticides are examples of haptens.
3.15
heterogeneous immunoassay
test which requires a separation of solid phase-bound and unbound tracers in order to detect the occupancy of
antibody binding sites by the analyte and thus the analyte concentration in the sample
3.16
immunoassay
quantitative assay which is based on the selective analyte/antibody binding and uses tracers for the detection of
the free or occupied antibody binding sites, respectively
3.17
immunogen
substance which triggers an immune response after injection into a vertebrate
3.18
inhibition concentration
IC
analyte concentration which reduces the measuring signal of the zero standard (= 100 %), in the case of IC to
50 % of the zero standard
3.19
luminescence immunoassay
LIA
immunochemical assay which is either performed as an immunoassay detecting luminescent substrate or product,
respectively, or luminescence-labelled tracer
3.20
solid phase immunoassay
heterogeneous immunoassay which uses (depending on the test type) antibodies or coating conjugates,
immobilized to a solid phase
NOTE Usually, both test types are known as ELISA (enzyme-linked immunosorbent assay) when enzyme-labelled tracers
are used.
3.21
tracer
labelled hapten (or antigen) or labelled antibody which, in the case of competitive immunoassays, is used to detect
the percentage of antibody binding sites not being occupied by the analyte
3.22
zero standard
analyte-free standard (method blank) which is used for calibration
4 Interferences
Interferences are caused by improper sampling, for instance due to the choice of equipment or materials which
adsorb or liberate the substances to be analysed. Assay conditions, for instance pH or sample components such as
metal ions, humic acids, salinity and solvents influencing the test components (for instance matrix effects), can
interfere with the analysis. The influence of matrix effects may be assessed by spiking samples with known
amounts of the analyte.
5Principle
Immunoassays are methods which use antibodies produced against defined analytes or analyte groups as
biochemical sensors for the quantification of analyte concentrations. These assays are particularly useful as
screening assays. All immunoassays for the detection of haptens are based on the principle of the competitive
immunoassay. Assays for pesticides have been reported (see references [1] to [4] in the bibliography). A typical
procedure, as an example of an EIA, is described below.
The solid phase variant requires either immobilized antibodies and dissolved hapten tracers (variant a, direct
immunoassay) or immobilized coating conjugate and dissolved antibody tracers (variant b, indirect immunoassay)
in constant ratios. The antibodies are applied in limiting amounts. Therefore components which are not bound to
the coated solid phase can be removed by washing prior to the final detection. This also includes most of the
interfering matrix effects. The more tracer molecules are bound, the lower is the analyte concentration in the
sample.
The immobilization can be achieved by passive adsorption or by covalent binding to functional groups of the solid
phase.
The calibration is performed with solutions of known analyte concentrations.
6 Reagents
6.1 General
Use reagents and water of high purity (for instance "for residue analysis"). Information on type and origin of
antibodies or antisera, respectively, as well as the cross-reactivities shall be stated in the report. Information on
storage and stability of the used reagents shall be requested from the supplier. The selectivity of the antibodies or
antiserum, respectively, shall guarantee that sample c
...

INTERNATIONAL ISO
STANDARD 15089
First edition
2000-03-15
Water quality — Guidelines for selective
immunoassays for the determination of
plant treatment and pesticide agents
Qualité de l'eau — Lignes directrices relatives aux dosages
immunologiques sélectifs pour la détermination des agents de traitement
des plantes et des pesticides
Reference number
©
ISO 2000
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not
be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this
file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this
area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters
were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event
that a problem relating to it is found, please inform the Central Secretariat at the address given below.
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body
in the country of the requester.
ISO copyright office
Case postale 56 � CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 734 10 79
E-mail [e-mail]
Web www.iso.ch
Printed in Switzerland
ii © ISO 2000 – All rights reserved

Contents Page
Foreword.iv
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Interferences .4
5 Principle.4
6 Reagents.4
7 Apparatus .5
8 Sampling and sample preparation.5
9 Procedure .6
10 Validity criteria .7
11 Calculation.8
12 Precision.10
13 Test report .10
Annex A (informative) Flowchart and pipetting scheme.11
Annex B (informative) Precision data from an interlaboratory trial .13
Bibliography.14
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 3.
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 International Standard may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 15089 was prepared by Technical Committee ISO/TC 147, Water quality,
Subcommittee SC 2, Physical, chemical, biochemical methods.
Annexes A and B of this International Standard are for information only.
iv © ISO 2000 – All rights reserved

INTERNATIONAL STANDARD ISO 15089:2000(E)
Water quality — Guidelines for selective immunoassays for the
determination of plant treatment and pesticide agents
1 Scope
This International Standard specifies a guide for the selective quantitative analysis by immunoassays of
environmental chemicals such as pesticides (including insecticides) or their metabolites in drinking, ground and
surface water.
The application range of the procedure for the analysis of pesticides in drinking water applies to mass
concentrationsW 0,05 �g/l. Therefore, the determination limit should be in this caseu 0,05 �g/l.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 5667-1:1980, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes.
ISO 5667-2:1991, Water quality — Sampling — Part 2: Guidance on sampling techniques.
ISO 5667-3:1994, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples.
ISO/TR 13530:1997, Water quality — Guide to analytical quality control for water analysis.
3 Terms and definitions
For the purposes of this International Standard, the following terms and definitions apply.
3.1
affinity
strength of binding of antibody to analyte
NOTE The strength is defined by the equilibrium constant (K) of the reaction Ab + H = AbH, where Ab = antibody
combining site and H = hapten; K is given by the mass action equation K = c /(c � c ).
AbH Ab H
3.2
analyte
substance to be determined
3.3
antibodies
serum proteins produced in vertebrates in response to immunization and which selectively bind the antigen or
hapten, respectively
NOTE 1 Monoclonal antibodies (mAb) are uniform populations of antibodies which are produced from a single cell clone of
hybridoma cells.
NOTE 2 Polyclonal antibodies (pAb) are a mixed population of antibodies which are produced by several clones of plasma
cells.
NOTE 3 Recombinant antibodies (rAb) are produced using recombinant techniques established in gene technology.
3.4
antibody conjugate
antibody covalently linked to a label such as an enzyme or a fluorochrome
3.5
antigen
substance that stimulates the production of antibodies and reacts with them
3.6
antiserum
immune serum obtained from the blood of immunized vertebrates after removal of cellular components and
coagulation factors
NOTE It usually contains a number of different antibodies which can exhibit different affinities to the antigen/hapten.
3.7
coating conjugate
macromolecule bound to the hapten (also known as a hapten-carrier conjugate), which is immobilized to a solid
phase
NOTE It is used to bind those antibody binding sites which are not occupied by the analyte.
3.8
competitive immunoassay
test which detects the proportion of antibody binding sites which have been occupied by the sample analyte
NOTE This is achieved by adding a tracer which binds to the unoccupied antibody binding sites and produces the
measuring signal after a further reaction.
3.9
cross-reactivity
extent, to which an antibody or an antiserum reacts with a substance which structurally differs from the analyte
NOTE 1 The cross-reactivity of an antibody or an antiserum with this substance is determined by comparing the calibration
curves. The reference curve obtained with the analyte is used as reference quantity (= 100 % cross-reactivity). The cross-
reactivity is usually determined at the IC . The selectivity of an antibody or an antiserum, respectively, is inversely related to the
cross-reactivities. An antibody or an antiserum, respectively, can display different affinities to different substances. With a given
substance, the cross-reactivity of an antiserum can also vary within the measuring range. Usually the structure of the
immunogen essentially determines the selectivity (the so-called specificity) of an antiserum. If cross-reactivities are due to a
mixed population of antibodies as often occurs in an antiserum, the undesired antibodies may be removed by cross-absorption.
NOTE 2 All compounds (present in relevant concentrations) that exhibit cross-reactivity will create false positive results.
3.10
enzyme immunoassay
EIA
immunochemical analysis which detects the occupancy of antibody binding sites by the analyte with the aid of a
tracer (an enzyme-labelled hapten or an enzyme-labelled antibody) and consequently can be used to detect the
analyte concentration in the medium
NOTE The detection procedure is based on the measurement of the enzyme activity of the tracer by means of substrate
conversion.
2 © ISO 2000 – All rights reserved

3.11
enzyme substrate
substance which is converted by the enzyme into a product that can be detected by a measuring device
3.12
excess standard
analyte concentration which, once exceeded, produces no further decrease in the signal measured in the
immunoassay
3.13
fluorescence immunoassay
immunochemical detection procedure which is performed either as an immunoassay with fluorescent substrates or
products, respectively, or as an immunoassay with fluorescence-labelled tracers or antibodies, respectively
3.14
hapten
substance which, because of its small molecular size, does not evoke the production of antibodies unless it is
covalently bound to an immunogen
NOTE Pesticides are examples of haptens.
3.15
heterogeneous immunoassay
test which requires a separation of solid phase-bound and unbound tracers in order to detect the occupancy of
antibody binding sites by the analyte and thus the analyte concentration in the sample
3.16
immunoassay
quantitative assay which is based on the selective analyte/antibody binding and uses tracers for the detection of
the free or occupied antibody binding sites, respectively
3.17
immunogen
substance which triggers an immune response after injection into a vertebrate
3.18
inhibition concentration
IC
analyte concentration which reduces the measuring signal of the zero standard (= 100 %), in the case of IC to
50 % of the zero standard
3.19
luminescence immunoassay
LIA
immunochemical assay which is either performed as an immunoassay detecting luminescent substrate or product,
respectively, or luminescence-labelled tracer
3.20
solid phase immunoassay
heterogeneous immunoassay which uses (depending on the test type) antibodies or coating conjugates,
immobilized to a solid phase
NOTE Usually, both test types are known as ELISA (enzyme-linked immunosorbent assay) when enzyme-labelled tracers
are used.
3.21
tracer
labelled hapten (or antigen) or labelled antibody which, in the case of competitive immunoassays, is used to detect
the percentage of antibody binding sites not being occupied by the analyte
3.22
zero standard
analyte-free standard (method blank) which is used for calibration
4 Interferences
Interferences are caused by improper sampling, for instance due to the choice of equipment or materials which
adsorb or liberate the substances to be analysed. Assay conditions, for instance pH or sample components such as
metal ions, humic acids, salinity and solvents influencing the test components (for instance matrix effects), can
interfere with the analysis. The influence of matrix effects may be assessed by spiking samples with known
amounts of the analyte.
5Principle
Immunoassays are methods which use antibodies produced against defined analytes or analyte groups as
biochemical sensors for the quantification of analyte concentrations. These assays are particularly useful as
screening assays. All immunoassays for the detection of haptens are based on the principle of the competitive
immunoassay. Assays for pesticides have been reported (see references [1] to [4] in the bibliography). A typical
procedure, as an example of an EIA, is described below.
The solid phase variant requires either immobilized antibodies and dissolved hapten tracers (variant a, direct
immunoassay) or immobilized coating conjugate and dissolved antibody tracers (variant b, indirect immunoassay)
in constant ratios. The antibodies are applied in limiting amounts. Therefore components which are not bound to
the coated solid phase can be removed by washing prior to the final detection. This also includes most of the
interfering matrix effects. The more tracer molecules are bound, the lower is the analyte concentration in the
sample.
The immobilization can be achieved by passive adsorption or by covalent binding to functional groups of the solid
phase.
The calibration is performed with solutions of known analyte concentrations.
6 Reagents
6.1 General
Use reagents and water of high purity (for instance "for residue analysis"). Information on type and origin of
antibodies or antisera, respectively, as well as the cross-reactivities shall be stated in the report. Information on
storage and stability of the used reagents shall be requested from the supplier. The selectivity of the antibodies or
antiserum, respectively, shall guarantee that sample concentrations deduced from a calibration curve do not
deviate by more than � 10 % from the actual analyte concentrations in these samples. If the specificities of the
antibodies or antiserum, respectively, are low, the interfering analyte shall be removed from the samples by
suitable chemical-physical procedures. Cross-reacting antibodies of an antiserum can be removed by cross-
absorption.
6.2 Buffered washing solution, used for washing for instance phosphate buffered saline (PBS; pH 7,
...

NORME ISO
INTERNATIONALE 15089
Première édition
2000-03-15
Qualité de l'eau — Lignes directrices
relatives aux dosages immunologiques
sélectifs pour la détermination des agents
de traitement des plantes et des pesticides
Water quality — Guidelines for selective immunoassays for the
determination of plant treatment and pesticide agents
Numéro de référence
©
ISO 2000
PDF – Exonération de responsabilité
Le présent fichier PDF peut contenir des polices de caractères intégrées. Conformément aux conditions de licence d'Adobe, ce fichier peut
être imprimé ou visualisé, mais ne doit pas être modifié à moins que l'ordinateur employé à cet effet ne bénéficie d'une licence autorisant
l'utilisation de ces polices et que celles-ci y soient installées. Lors du téléchargement de ce fichier, les parties concernées acceptent de fait la
responsabilité de ne pas enfreindre les conditions de licence d'Adobe. Le Secrétariat central de l'ISO décline toute responsabilité en la
matière.
Adobe est une marque déposée d'Adobe Systems Incorporated.
Les détails relatifs aux produits logiciels utilisés pour la création du présent fichier PDF sont disponibles dans la rubrique General Info du
fichier; les paramètres de création PDF ont été optimisés pour l'impression. Toutes les mesures ont été prises pour garantir l'exploitation de
ce fichier par les comités membres de l'ISO. Dans le cas peu probable où surviendrait un problème d'utilisation, veuillez en informer le
Secrétariat central à l'adresse donnée ci-dessous.
Droits de reproduction réservés. Sauf prescription différente, aucune partie de cette publication ne peut être reproduite ni utilisée sous quelque
forme que ce soit et par aucun procédé, électronique ou mécanique, y compris la photocopie et les microfilms, sans l'accord écrit de l’ISO à
l’adresse ci-après ou du comité membre de l’ISO dans le pays du demandeur.
ISO copyright office
Case postale 56 � CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax. + 41 22 734 10 79
E-mail [e-mail]
Web www.iso.ch
ImpriméenSuisse
ii © ISO 2000 – Tous droits réservés

Sommaire Page
Avant-propos.iv
1 Domaine d'application.1
2 Références normatives .1
3 Termes et définitions.1
4 Interférences .4
5 Principe.4
6 Réactifs .4
7 Appareillage .5
8 Échantillonnage et préparation des échantillons.5
9 Mode opératoire.6
10 Critères de validité.8
11 Évaluation et expression des résultats .8
12 Fidélité .9
13 Rapport d'essai .9
Annexe A (informative) Organigramme et plan de pipettage.11
Annexe B (informative) Résultats d'un essai interlaboratoires.13
Bibliographie .14
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de
normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée aux
comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du comité
technique créé à cet effet. Les organisations internationales, gouvernementales et non gouvernementales, en
liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec la Commission
électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.
Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI, Partie 3.
Les projets de Normes internationales adoptés par les comités techniques sont soumis aux comités membres pour
vote. Leur publication comme Normes internationales requiert l'approbation de 75 % au moins des comités
membres votants.
L’attention est appelée sur le fait que certains des éléments de la présente Norme internationale peuvent faire
l’objet de droits de propriété intellectuelle ou de droits analogues. L’ISO ne saurait être tenue pour responsable de
ne pas avoir identifié de tels droits de propriété et averti de leur existence.
La Norme internationale ISO 15089 a été élaborée par le comité technique ISO/TC 147, Qualité de l'eau,
sous-comité SC 2, Méthodes physiques, chimiques et biochimiques.
Les annexes A et B de la présente Norme internationale sont données uniquement à titre d’information.
iv © ISO 2000 – Tous droits réservés

NORME INTERNATIONALE ISO 15089:2000(F)
Qualité de l'eau — Lignes directrices relatives aux dosages
immunologiques sélectifs pour la détermination des agents de
traitement des plantes et des pesticides
1 Domaine d'application
La présente Norme internationale spécifie des lignes directrices pour l'analyse quantitative sélective, par des
dosages immunologiques, des produits chimiques utilisés dans l'environnement, tels que les pesticides (y compris
les insecticides) ou leurs métabolites présents dans l'eau potable, l'eau souterraine et l'eau de surface.
Le présent mode opératoire pour l’analyse des pesticides dans l'eau potable est applicable à des concentrations en
masseW 0,05 μg/l. En conséquence, il convient que, dans ce cas, la limite de détermination soitu 0,05 μg/l.
2 Références normatives
Les documents normatifs suivants contiennent des dispositions qui, par suite de la référence qui y est faite,
constituent des dispositions valables pour la présente Norme internationale. Pour les références datées, les
amendements ultérieurs ou les révisions de ces publications ne s’appliquent pas. Toutefois, les parties prenantes
aux accords fondés sur la présente Norme internationale sont invitées à rechercher la possibilité d'appliquer les
éditions les plus récentes des documents normatifs indiqués ci-après. Pour les références non datées, la dernière
édition du document normatif en référence s’applique. Les membres de l'ISO et de la CEI possèdent le registre des
Normes internationales en vigueur.
ISO 5667-1:1980, Qualité de l’eau — Échantillonnage — Partie 1: Guide général pour l’établissement des
programmes d’échantillonnage.
ISO 5667-2:1991, Qualité de l’eau — Échantillonnage — Partie 2: Guide général sur les techniques
d’échantillonnage.
ISO 5667-3:1994, Qualité de l’eau — Échantillonnage — Partie 3: Guide général pour la conservation et la
manipulation des échantillons.
ISO/TR 13530, Qualité de l’eau — Guide de contrôle qualité analytique pour l’analyse de l’eau.
3 Termes et définitions
Pour les besoins de la présente Norme internationale, les termes et définitions suivants s'appliquent.
3.1
affinité
degré de liaison de l'anticorps à l'analyte
NOTE Le degré est défini par la constante d'équilibre (K) de la réaction Ab + H = AbH, où Ab est le site récepteur de
l'anticorps et H = haptène; K est donné par l'équation de l'action massique K = c /(c � c ).
AbH Ab H
3.2
analyte
substance à déterminer
3.3
anticorps
protéines de sérum sécrétées par les vertébrés après introduction d'un antigène dans l'organisme réagissant à
l'agression et qui fixent l'antigène ou l’haptène, respectivement, de façon sélective
NOTE 1 Les anticorps monoclonaux (mAb) sont des populations uniformes d'anticorps issues du clone d'une cellule unique
de cellules hybridomes.
NOTE 2 Les anticorps polyclonaux (pAb) sont une population mixte d'anticorps sécrétée par plusieurs clones de cellules de
plasma.
NOTE 3 Les anticorps recombinés (rAb) sont produits grâce aux techniques de recombinaison en vigueur dans le génie
génétique.
3.4
anticorps conjugué
anticorps lié par covalence à un marqueur tel qu'une enzyme ou un fluorochrome
3.5
antigène
substance qui stimule la production d'anticorps et qui réagit à leur contact
3.6
immunosérum
sérum sanguin de vertébrés immunisés, obtenu après élimination des composés cellulaires et des facteurs de
coagulation
NOTE Il contient généralement un certain nombre d'anticorps différents qui peuvent présenter diverses affinités par rapport
à l'antigène/haptène.
3.7
conjugué sur support sensibilisé
macromolécule liée à l'haptène (également appelée conjugué support d'haptène) et immobilisée sur une phase
solide
NOTE Il est utilisé pour lier les sites anticorps non occupés par l'analyte.
3.8
dosage immunologique par compétition
essai qui permet de détecter la proportion de sites anticorps ayant été occupés par l'analyte de l’échantillon
NOTE Pour cela, un traceur est ajouté qui va occuper les sites disponibles de l'anticorps, et après une nouvelle réaction
générera le signal à mesurer.
3.9
réaction croisée
réaction d'un anticorps ou d'un immunosérum avec une substance dont la structure diffère de l'analyte
NOTE 1 La réaction croisée d'un anticorps ou d'un immunosérum avec cette substance est déterminée en comparant les
courbes d'étalonnage. La courbe de référence obtenue avec l'analyte est utilisée comme grandeur de référence (= réaction
croisée à 100 %). La réaction croisée est généralement déterminée à une concentration inhibitrice CI . La sélectivité d'un
anticorps ou d'un immunosérum est inversement proportionnelle aux réactions croisées. Un anticorps ou un immunosérum peut
respectivement présenter diverses affinités avec différentes substances. Avec une substance donnée, la réaction croisée d'un
immunosérum peut également varier au sein de l'étendue de mesure. Généralement, la sélectivité d'un immunosérum (appelée
spécificité) est principalement déterminée par la structure de l'immunogène. Lorsque les réactions croisées sont dues à une
population mixte d'anticorps, ce qui est fréquent pour un immunosérum, il est admis d'éliminer les anticorps indésirables par
absorbance croisée.
2 © ISO 2000 – Tous droits réservés

NOTE 2 Tous les composés (présents à des concentrations importantes) qui présentent une réaction croisée donneront lieu
à des résultats faux positifs.
3.10
dosage immunoenzymatique
EIA
analyse immunochimique qui détecte, à l'aide d'un traceur (un haptène à marqueur enzymatique ou un anticorps à
marqueur enzymatique), l'occupation de sites anticorps par l'analyte et qui peut par conséquent être utilisée pour
détecter la concentration de l’analyte dans le milieu
NOTE La procédure de détection est fondée sur la mesure de l'activité enzymatique du traceur par conversion du substrat.
3.11
substrat enzymatique
substance transformée par l'enzyme en produit détectable par un système de mesure
3.12
étalon par excès
étalon caractérisé par une valeur limite de concentration de l’analyte, au-delà de laquelle il ne se produit plus de
réduction du signal mesuré au cours du dosage immunologique
3.13
dosage immunologique par fluorescence
procédure de détection immunochimique appliquée soit en tant que dosage immunologique avec des substrats ou
des produits fluorescents, ou en tant que dosage immunologique avec des traceurs ou des anticorps à marqueur
fluorescent
3.14
haptène
substance qui, du fait de son faible poids moléculaire, ne peut susciter la formation d'anticorps à moins d'être liée
par covalence à un immunogène
NOTE Les pesticides sont un exemple d'haptènes.
3.15
dosage immunologique hétérogène
essai nécessitant la séparation des traceurs liés et non liés à la phase solide afin de détecter les sites de
l'anticorps occupés par l'analyte et par voie de conséquence la concentration de l’analyte dans l'échantillon
3.16
dosage immunologique
dosage quantitatif fondé sur «l'attraction» de l'analyte/anticorps et qui utilise des traceurs pour la détection des
sites de l’anticorps, respectivement libres ou occupés
3.17
immunogène
substance qui, après injection, déclenche chez un vertébré une réaction immunitaire
3.18
concentration inhibitrice
CI
concentration de l'analyte qui réduit le signal de mesure de l'étalon zéro (= 100 %) dans le cas de CI à50% de
sa valeur
3.19
dosage immunologique par luminescence
LIA
dosage immunochimique réalisé soit en tant que dosage immunologique détecteur de substrats ou de produits
luminescents, soit en tant que dosage immunologique de traceurs à marqueur luminescent
3.20
immunoessai en phase solide
dosage immunologique hétérogène qui utilise (selon le type d'essai) des anticorps ou des conjugués sur support
sensibilisés immobilisés sur une phase solide
NOTE Les deux types d'essai, lorsqu'ils utilisent des traceurs à marqueur enzymatique, sont plus généralement connus
sous le nom de technique ELISA (enzyme-linked immunosorbent assay).
3.21
traceur
haptène (ou antigène) marqué ou anticorps marqué qui, dans le cas de dosage immunologique par compétition,
est utilisé pour détecter le pourcentage de sites anticorps non occupés par l'analyte
3.22
étalon zéro
étalon sans analyte (blanc de méthode) utilisé pour l'étalonnage
4 Interférences
Les interférences ont pour origine un échantillonnage incorrect, dû par exemple au choix des équipements ou des
matériaux qui adsorbent ou libèrent les substances à analyser. Les conditions de dosage, par exemple le pH ou les
composés de l'échantillon tels que les ions métalliques, les acides humiques, la salinité et les solvants, qui
provoquent des interférences avec les composés de l'essai (par exemple des effets de matrice), peuvent affecter
l'analyse. L’influence des effets de matrice peut être évaluée en dopant l’échantillon avec des quantités connues
de l’analyte.
5Principe
Les dosages immunologiques sont des méthodes qui utilisent des anticorps produits par rapport à des analytes
donnés ou des groupes d'analytes définis comme des sondes biochimiques pour la quantification des
concentrations d'analytes. Ces dosages sont particulièrement utiles comme essai de «screening». Tous les
dosages immunologiques pour la détection des haptènes sont fondés sur le principe du dosage immunologique par
compétition. Les dosages concernant les pesticides ont fait l'objet de rapports (voir les références [1] à [4] dans la
bibliographie). Une procédure type, à titre d’exemple d’EIA, est décrite ci-après.
La variante en phase solide requiert soit des anticorps immobilisés et des traceurs haptènes dissous [variante a),
dosage immunologique direct], soit un conjugué sur support sensibilisé et des traceurs anticorps dissous [variante
b), dosage immunologique indirect] en rapport constant. Les anticorps sont appliqués en quantité limitée. Par
conséquent, les composants non liés à la phase solide sensibilisée peuvent être éliminés par lavage avant la
détection finale. Ceci comprend également la plupart des effets de matrice interférents. Plus les molécules traceurs
sont liées, plus la concentration de l'analyte dans l'échantillon est faible.
L'immobilisation peut être obtenue par adsorption passive ou par fixation par liaison covalente aux groupes
fonctionnels de la phase solide.
L'étalonnage est réalisé avec des solutions de concentrations d'analyte connues.
6 Réactifs
6.1 Généralités
Utiliser des réactifs et de l'eau de haute pureté (par exemple «pour analyse des résidus»). Des informations
relatives, respectivement, au type et à l'origine des anticorps ou des immunosérums, ainsi qu'aux réactions
croisées doivent être consignées dans le rapport d’essai. Des informations relatives au stockage et à la stabilité
des réactifs utilisés doivent être demandées au fournisseur. La sélectivité des anticorps ou respectivement des
4 © ISO 2000 – Tous droits réservés

immunosérums doit garantir que les concentrations d'échantillon, déduites d'une courbe d'étalonnage, ne
s'écartent pas de plus de � 10 % des concentrations réelles de l'analyte dans ces échantillons. Lorsque les
spécificités, respectivement, des anticorps ou respectivement des immunosérums sont faibles, l'analyte interférent
doit être éliminé des échantillons selon des procédures chimiques et physiques appropriées. Les anticorps croisés
d'un immunosérum peuvent être éliminés par absorbance croisée.
6.2 Solution
...