1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS ISO 23893-3:2013Water quality Biochemicaland physiologicalmeasurements on fishPart 3: Determination of vitellogeninBS ISO 23893-3:2013 BRITISH STANDARDNational forewordThis Br
2、itish Standard is the UK implementation of ISO 23893-3:2013.The UK participation in its preparation was entrusted to TechnicalCommittee EH/3/5, Biological Methods.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to inc
3、lude all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2013. Published by BSI StandardsLimited 2013ISBN 978 0 580 67754 0ICS 13.060.70Compliance with a British Standard cannot confer immunity fromlegal obligations.This Brit
4、ish Standard was published under the authority of theStandards Policy and Strategy Committee on 30 April 2013.Amendments issued since publicationDate Text affectedBS ISO 23893-3:2013 ISO 2013Water quality Biochemical and physiological measurements on fish Part 3: Determination of vitellogeninQualit
5、de leau Mesurages biochimiques et physiologiques sur poisson Partie 3: Dosage de la vitellognineINTERNATIONAL STANDARDISO23893-3First edition2013-04-01Reference numberISO 23893-3:2013(E)BS ISO 23893-3:2013ISO 23893-3:2013(E)ii ISO 2013 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2013All righ
6、ts 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 ei
7、ther ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCase postale 56 CH-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 749 09 47E-mail copyrightiso.orgWeb www.iso.orgPublished in SwitzerlandBS ISO 23893-3:2013ISO 23893-3:2013(E) ISO 2013 All rights r
8、eserved iiiContents PageForeword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Minimum performance criteria 26 Test environment . 27 Reagents 38 Apparatus . 39 Sampling procedure 49.1 Sampling of fish 49.2 Sampling of blood plasma . 49.3 Storage of bloo
9、d plasma samples 510 Analytical procedure 510.1 Preparation of the samples 510.2 Determination of vitellogenin 511 Test report 10Annex A (informative) Examples of results: Fathead minnow sandwich ELISA .12Bibliography .19BS ISO 23893-3:2013ISO 23893-3:2013(E)ForewordISO (the International Organizati
10、on 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 ha
11、s 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.Inter
12、national 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. Publicati
13、on 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
14、.ISO 23893-3 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.ISO 23893 consists of the following parts, under the general title Water quality Biochemical and physiological measurements on fish: Part 1: Sampling of fish, handling and preservation o
15、f samples Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) Technical Specification Part 3: Determination of vitellogeniniv ISO 2013 All rights reservedBS ISO 23893-3:2013ISO 23893-3:2013(E)IntroductionVitellogenin (Vtg) is a large phospholipoglycoprotein produced as the yolk protein prec
16、ursor in the liver of oviparous vertebrates, such as fish. Vtg is secreted from hepatocytes through the secretory pathway, enters the circulation and is taken up by the growing oocyte. Plasma Vtg concentrations are normally an indication of the maturational status of the female fish, for reviews see
17、 References 218. More than a decade ago, several studies demonstrated that male fish caught in rivers and streams had high concentrations of plasma Vtg (e.g. References 1423), caused by chemicals acting like oestrogens present in the environment. Since then, numerous studies have shown the fish Vtg
18、to be a highly responsive biomarker for oestrogenic compounds in both in vitro hepatocyte cell cultures, in vivo aquaria studies, and field studies, for reviews see References 121013162026. Hence, Vtg in fish has become an accepted biomarker of xenooestrogenic and antioestrogenic exposure of chemica
19、ls, effluents, and discharges, and has been proposed in chemical testing, as well as environmental monitoring programmes, e.g. Reference 13.However, recent genetic and immunological analyses have demonstrated a general multiplicity of Vtg forms in fish, References 910. The concentrations of circulat
20、ing Vtg proteins (or Vtg gene transcripts) during oogenesis and their degree of induction by oestrogens appear to vary among species and among different types of Vtg within a species. The kinetics of induction of distinct types of Vtg by oestrogens in fish appears to depend on environmental factors
21、(e.g. water temperature and photoperiod), life history stage, sex, and the concentration and type of oestrogenic compound. Based on these findings, it is important that the Vtg targets in a bioassay for oestrogens in a specific species be demonstrated to be an oestrogen-responsive form, and that the
22、 assay be validated with the species in question before embarking on a monitoring programme, Reference 10.The scientific literature contains a multitude of publications on procedures for determining Vtg in fish samples, using immunoassays. Whereas radioimmunoassays (RIA) were a predominant method in
23、 the 1980s and early 1990s, e.g. References 429, enzyme-linked immunosorbent assays (ELISAs) are the dominating principle today. Both the sandwich and competitive ELISA principles provide sensitive results without the use of radioactive isotopes, and have been successfully applied to determine Vtg l
24、evels in several fish species, e.g. References 368121517192122242527283132.This part of ISO 23893 presents a generalized protocol for both the sandwich and competitive ELISA for use in quantification of Vtg in fish blood plasma samples. The application of standardized methods is strongly advised wit
25、hin monitoring programmes. ISO 2013 All rights reserved vBS ISO 23893-3:2013BS ISO 23893-3:2013Water quality Biochemical and physiological measurements on fish Part 3: Determination of vitellogeninWARNING Persons using this document should be familiar with normal laboratory practice. This document d
26、oes not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions.IMPORTANT It is absolutely essential that tests conducted
27、 in accordance with this document be carried out by suitably qualified staff.1 ScopeThis part of ISO 23893 specifies a method for measuring vitellogenin (Vtg) concentrations in a fish plasma sample employing an enzyme-linked immunosorbent assay (ELISA) method.It applies to fish that are sampled in t
28、he environment (fresh, estuarine or salt water) and to fish exposed to substances or effluents in a laboratory. The method is quantitative when using Vtg antibodies and a Vtg standard well characterized with the species of choice.2 Normative referencesThe following documents, in whole or in part, ar
29、e 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 23893-1, Water quality Biochemical and physiological
30、 measurements on fish Part 1: Sampling of fish, handling and preservation of samples3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1limit of detectionLODlowest content that can be measured with reasonable statistical certaintyEXAMPLE The LOD is
31、often expressed as the reagent blank value plus three times its standard deviation.Note 1 to entry: The method LOD is determined by taking the sample dilution factor into the calculation.3.2limit of quantificationLOQcontent equal to or greater than the lowest concentration point in the calibration c
32、urveEXAMPLE The LOQ is often expressed as the reagent blank value plus 10 times its standard deviation (Reference 11).Note 1 to entry: LOQ is also determined by taking the sample dilution factor into the calculation.INTERNATIONAL STANDARD ISO 23893-3:2013(E) ISO 2013 All rights reserved 1BS ISO 2389
33、3-3:2013ISO 23893-3:2013(E)3.3matrix blankrepresentative sample that does not contain detectable levels of analyteNote 1 to entry: For the purposes of this part of ISO 23893, the analyte is vitellogenin.3.4selectivityability to measure accurately the analyte in the presence of components that can be
34、 expected to be present in the matrixNote 1 to entry: For the purposes of this part of ISO 23893, the analyte is vitellogenin and the matrix is plasma.Note 2 to entry: Selectivity is demonstrated by using “matrix blanks”.4 PrincipleSamples of fish blood plasma are collected essentially as specified
35、in ISO 23893-1; however, with addition of a protease inhibitor (see 7.13 and 9.2). Vitellogenin is determined in the sample by an antibody-based immunoassay, using either of two established variants.In the first, so-called sandwich ELISA, the blood plasma sample is allowed to react with a capture an
36、tibody specific for Vtg (from the same or a closely related species), in a microtitre plate well coated with the antibody. An enzyme-labelled detecting Vtg antibody is then used to produce an antibody “sandwich” that can be detected based on a chromogenic substrate for the enzyme label (e.g. horsera
37、dish peroxidase). A secondary enzyme-labelled antibody can also be used to develop the assay, if the detecting antibody is unlabelled. A standard series based on a purified reference material (Vtg protein from the same or closely related species) is used to develop a quantitative relationship betwee
38、n sample signal and standard amount.The second variant is the competitive ELISA technique, where sample Vtg competes with purified Vtg coated to the microtitre plate walls for binding to a (labelled or unlabelled) Vtg antibody in solution. Development of assay signal follows the same principle as in
39、 the sandwich variant, although the standard series produces an inverse relationship with signal intensity.Only Vtg antibodies or assays that have been demonstrated to perform according to specified performance criteria with the fish species studied should be used in this protocol.5 Minimum performa
40、nce criteriaThe criteria listed below should be regarded as the minimal acceptable performance as defined from a user standpoint on the purpose of performing Vtg analysis. Specific performance criteria need to be established for each specific assay to be used in the study based on in-house (within l
41、aboratory) performance.Selectivity: Matrix blank 10-fold, preferably 50-fold to 100-fold to be practi-cal with the dynamic range found in Vtg concentrationsRecovery: 50 %NOTE The characterization of the “matrix effect” is an important challenge in this regard. It can be difficult to ensure that a “m
42、atrix blank” sample is really devoid of any Vtg.6 Test environmentAll handling operations of plasma samples and standards including the measurement shall be carried out at a temperature of (4 2) C or on crushed ice, except where indicated in the test procedure.2 ISO 2013 All rights reservedBS ISO 23
43、893-3:2013ISO 23893-3:2013(E)7 ReagentsUnless otherwise specified, use only reagents of recognized analytical grade.7.1 Sulfuric acid, 0,3 mol/l or 1,5 mol/l, stop solution.7.2 Crushed ice.7.3 Coating buffer, 50 mmol/l carbonatebicarbonate, pH 9,6.7.4 Washing buffer, phosphate-buffered saline (PBS),
44、 pH 7,3, containing 0,5 g/l polysorbate 20 detergent.7.5 Blocking buffer, washing buffer containing 10 g/l bovine serum albumin (BSA).7.6 Dilution buffer, 10 g/l BSA in PBS.7.7 Substrate buffer, phosphatecitric acid buffer, pH 5,0.7.8 Vtg reference sample.1)7.9 Capture antibody, monoclonal or polycl
45、onal anti-Vtg.1)7.10 Detecting antibody, monoclonal or polyclonal anti-Vtg,1)unconjugated or conjugated to horseradish peroxidase, HRP. In the alternative where the detecting antibody is not conjugated, the detecting antibody shall be harvested from a different species than the capture antibody.7.11
46、 Secondary antibody,2)antibody to Fc (Fragment crystallizable) part of detecting antibody, conjugated to HRP.7.12 Peroxidase substrate, tetramethylbenzidine (TMB), or ortho-phenylenediamine (OPD) + H2O2.7.13 Protease inhibitor, such as aprotinin.8 Apparatus8.1 96-Well microtitre plates, clear, flat-
47、bottomed, absorbing.8.2 96-Well microtitre plates, clear, flat-bottomed, non-absorbing, for the competitive ELISA variant.8.3 Microplate sealing film.8.4 Microplate reader, wavelength 450 nm or 490 nm, depending on substrate used.8.5 Pipettes, with disposable tips 5 l to 1 000 l.8.6 Multi-channel pi
48、pette and reagent reservoir. Alternatively, a stepper pipette with disposable tips (100 l) can be used.1) Vtg reference samples, monoclonal or polyclonal antibodies to fish Vtgs, and complete assay kits (Vtg ELISA kits) are available commercially.2) Enzyme-labelled secondary antibodies are available
49、 commercially. ISO 2013 All rights reserved 3BS ISO 23893-3:2013ISO 23893-3:2013(E)8.7 Test tubes, 1 ml to 50 ml.8.8 Microplate washing device. An automatic or manual plate washer is recommended, but a squeeze bottle or a multichannel/stepper pipette can also be used.8.9 Vortexer.9 Sampling procedure9.1 Sampling of fishSampling should be carried out in the natural environment by fishing or in a laboratory on fish exposed to substances or effluents as specified
