An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids
Identifieur interne : 001038 ( Istex/Corpus ); précédent : 001037; suivant : 001039An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids
Auteurs : Daniel P. Lomax ; William T. Roubal ; James D. Moore ; Lyndal L. JohnsonSource :
- Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology [ 1096-4959 ] ; 1998.
Abstract
Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E2) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E2-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml−1 (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E2-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.
Url:
DOI: 10.1016/S0305-0491(98)10125-6
Links to Exploration step
ISTEX:0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids</title><author><name sortKey="Lomax, Daniel P" sort="Lomax, Daniel P" uniqKey="Lomax D" first="Daniel P" last="Lomax">Daniel P. Lomax</name><affiliation><mods:affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</mods:affiliation></affiliation></author><author><name sortKey="Roubal, William T" sort="Roubal, William T" uniqKey="Roubal W" first="William T" last="Roubal">William T. Roubal</name><affiliation><mods:affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</mods:affiliation></affiliation></author><author><name sortKey="Moore, James D" sort="Moore, James D" uniqKey="Moore J" first="James D" last="Moore">James D. Moore</name><affiliation><mods:affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</mods:affiliation></affiliation></author><author><name sortKey="Johnson, Lyndal L" sort="Johnson, Lyndal L" uniqKey="Johnson L" first="Lyndal L" last="Johnson">Lyndal L. Johnson</name><affiliation><mods:affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4</idno><date when="1998" year="1998">1998</date><idno type="doi">10.1016/S0305-0491(98)10125-6</idno><idno type="url">https://api.istex.fr/document/0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">001038</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001038</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids</title><author><name sortKey="Lomax, Daniel P" sort="Lomax, Daniel P" uniqKey="Lomax D" first="Daniel P" last="Lomax">Daniel P. Lomax</name><affiliation><mods:affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</mods:affiliation></affiliation></author><author><name sortKey="Roubal, William T" sort="Roubal, William T" uniqKey="Roubal W" first="William T" last="Roubal">William T. Roubal</name><affiliation><mods:affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</mods:affiliation></affiliation></author><author><name sortKey="Moore, James D" sort="Moore, James D" uniqKey="Moore J" first="James D" last="Moore">James D. Moore</name><affiliation><mods:affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</mods:affiliation></affiliation></author><author><name sortKey="Johnson, Lyndal L" sort="Johnson, Lyndal L" uniqKey="Johnson L" first="Lyndal L" last="Johnson">Lyndal L. Johnson</name><affiliation><mods:affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology</title><title level="j" type="abbrev">CBB</title><idno type="ISSN">1096-4959</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998">1998</date><biblScope unit="volume">121</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="425">425</biblScope><biblScope unit="page" to="436">436</biblScope></imprint><idno type="ISSN">1096-4959</idno></series><idno type="istex">0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4</idno><idno type="DOI">10.1016/S0305-0491(98)10125-6</idno><idno type="PII">S0305-0491(98)10125-6</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1096-4959</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E2) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E2-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml−1 (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E2-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>Daniel P Lomax</name><affiliations><json:string>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</json:string></affiliations></json:item><json:item><name>William T Roubal</name><affiliations><json:string>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</json:string></affiliations></json:item><json:item><name>James D Moore</name><affiliations><json:string>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</json:string></affiliations></json:item><json:item><name>Lyndal L Johnson</name><affiliations><json:string>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Biomarker</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ELISA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>English sole</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Environmental estrogens</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Vitellogenin</value></json:item></subject><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E2) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E2-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml−1 (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E2-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.</abstract><qualityIndicators><score>7.568</score><pdfVersion>1.2</pdfVersion><pdfPageSize>552 x 770 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>5</keywordCount><abstractCharCount>1528</abstractCharCount><pdfWordCount>6820</pdfWordCount><pdfCharCount>41228</pdfCharCount><pdfPageCount>12</pdfPageCount><abstractWordCount>214</abstractWordCount></qualityIndicators><title>An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids</title><pii><json:string>S0305-0491(98)10125-6</json:string></pii><genre><json:string>research-article</json:string></genre><host><volume>121</volume><pii><json:string>S0305-0491(00)X0048-X</json:string></pii><pages><last>436</last><first>425</first></pages><issn><json:string>1096-4959</json:string></issn><issue>4</issue><genre><json:string>journal</json:string></genre><language><json:string>unknown</json:string></language><title>Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology</title><publicationDate>1998</publicationDate></host><categories><wos><json:string>science</json:string><json:string>zoology</json:string><json:string>biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>health sciences</json:string><json:string>biomedical research</json:string><json:string>biochemistry & molecular biology</json:string></scienceMetrix></categories><publicationDate>1998</publicationDate><copyrightDate>1998</copyrightDate><doi><json:string>10.1016/S0305-0491(98)10125-6</json:string></doi><id>0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4</id><score>0.029737534</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a">An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>©1998 Elsevier Science Inc.</p></availability><date>1998</date></publicationStmt><notesStmt><note type="content">Fig. 1: Optimal assay concentrations for Vtg and anti-Vtg were determined by coating wells with serial dilutions of purified Vtg and incubating with different antibody concentrations. The chosen routine conditions were 200 ng ml−1 for Vtg coating and 1:100 000 for antibody dilution.</note><note type="content">Fig. 2: (A) Binding displacement curves obtained with Vtg standard (○) and serial dilutions of plasma from vitellogenic female English sole (■) and E2-injected male English sole (•). Control male English sole (+) exhibited no significant cross-reactivity with the Vtg antiserum at dilutions greater than 100-fold. (B) Linearization of binding curves by logit transformation. Parallelism of the regression curves was assessed by F-test on mean squares. No significant differences were seen between the standard preparation (s=−1.49; r2=0.99), vitellogenic female English sole (s=−1.54; r2=0.99) and E2-treated male English sole (s=−1.48; r2=0.97). All points are the mean of triplicate determinations.</note><note type="content">Fig. 3: (A) Binding displacement curves obtained with Vtg standard (○) and serial dilutions of plasma from different species: female rock sole (□), female starry flounder (▴) and female sand sole (♦). (B) Parallelism was observed between the standard and plasma from rock sole (s=−1.39; r2=0.95) and starry flounder (s=−1.40; r2=0.97). Sand sole (s=−0.93; r2=0.96) plasma showed some cross-reactivity with the Vtg antiserum but was not statistically parallel with the standard. All points are the mean of duplicate determinations.</note><note type="content">Fig. 4: Plasma Vtg levels in adult female English sole captured in the field at different stages of ovarian maturation. Values are the mean±S.E.; n in parentheses. ND, none detected.</note><note type="content">Fig. 5: Plasma Vtg levels in juvenile English sole induced with estradiol. Plasma samples were collected 2 days after E2 injections. Vtg was not detected in any of the control groups or the three lowest E2 doses (data not shown). Values are the mean±S.E.; n in parentheses.</note><note type="content">Fig. 6: Vtg levels in two types of hepatocyte media culture at 1, 3 and 5 days following isolation and plating of English sole hepatocytes. Values are the mean of duplicate samples.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids</title><author xml:id="author-1"><persName><forename type="first">Daniel P</forename><surname>Lomax</surname></persName><note type="correspondence"><p>Corresponding author. Tel.: +1 206 8603314; fax: +1 206 8603335; e-mail: dan.lomax@noaa.gov</p></note><affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</affiliation></author><author xml:id="author-2"><persName><forename type="first">William T</forename><surname>Roubal</surname></persName><affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</affiliation></author><author xml:id="author-3"><persName><forename type="first">James D</forename><surname>Moore</surname></persName><affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</affiliation></author><author xml:id="author-4"><persName><forename type="first">Lyndal L</forename><surname>Johnson</surname></persName><affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</affiliation></author></analytic><monogr><title level="j">Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology</title><title level="j" type="abbrev">CBB</title><idno type="pISSN">1096-4959</idno><idno type="PII">S0305-0491(00)X0048-X</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998"></date><biblScope unit="volume">121</biblScope><biblScope unit="issue">4</biblScope><biblScope unit="page" from="425">425</biblScope><biblScope unit="page" to="436">436</biblScope></imprint></monogr><idno type="istex">0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4</idno><idno type="DOI">10.1016/S0305-0491(98)10125-6</idno><idno type="PII">S0305-0491(98)10125-6</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1998</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E2) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E2-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml−1 (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E2-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>Biomarker</term></item><item><term>ELISA</term></item><item><term>English sole</term></item><item><term>Environmental estrogens</term></item><item><term>Vitellogenin</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1998-09-10">Modified</change><change when="1998">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>CBB</jid><aid>6120</aid><ce:pii>S0305-0491(98)10125-6</ce:pii><ce:doi>10.1016/S0305-0491(98)10125-6</ce:doi><ce:copyright year="1998" type="full-transfer">Elsevier Science Inc.</ce:copyright></item-info><head><ce:title>An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole (<ce:italic>Pleuronectes vetulus</ce:italic>): development, validation and cross-reactivity with other pleuronectids</ce:title><ce:author-group><ce:author><ce:given-name>Daniel P</ce:given-name><ce:surname>Lomax</ce:surname><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:author><ce:given-name>William T</ce:given-name><ce:surname>Roubal</ce:surname></ce:author><ce:author><ce:given-name>James D</ce:given-name><ce:surname>Moore</ce:surname></ce:author><ce:author><ce:given-name>Lyndal L</ce:given-name><ce:surname>Johnson</ce:surname></ce:author><ce:affiliation><ce:textfn>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>Corresponding author. Tel.: +1 206 8603314; fax: +1 206 8603335; e-mail: dan.lomax@noaa.gov</ce:text></ce:correspondence></ce:author-group><ce:date-received day="1" month="4" year="1998"></ce:date-received><ce:date-revised day="10" month="9" year="1998"></ce:date-revised><ce:date-accepted day="18" month="9" year="1998"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E<ce:inf>2</ce:inf>) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (<ce:italic>Pleuronectes vetulus</ce:italic>) was developed and validated. Plasmatic Vtg was purified from E<ce:inf>2</ce:inf>-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml<ce:sup>−1</ce:sup> (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E<ce:inf>2</ce:inf>-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords class="keyword"><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Biomarker</ce:text></ce:keyword><ce:keyword><ce:text>ELISA</ce:text></ce:keyword><ce:keyword><ce:text>English sole</ce:text></ce:keyword><ce:keyword><ce:text>Environmental estrogens</ce:text></ce:keyword><ce:keyword><ce:text>Vitellogenin</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole (</title></titleInfo><name type="personal"><namePart type="given">Daniel P</namePart><namePart type="family">Lomax</namePart><affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</affiliation><description>Corresponding author. Tel.: +1 206 8603314; fax: +1 206 8603335; e-mail: dan.lomax@noaa.gov</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">William T</namePart><namePart type="family">Roubal</namePart><affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">James D</namePart><namePart type="family">Moore</namePart><affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Lyndal L</namePart><namePart type="family">Johnson</namePart><affiliation>Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E., Seattle, WA 98112, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1998</dateIssued><dateModified encoding="w3cdtf">1998-09-10</dateModified><copyrightDate encoding="w3cdtf">1998</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract lang="en">Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E2) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E2-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen–antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10–450 ng ml−1 (85–20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E2-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.</abstract><note type="content">Fig. 1: Optimal assay concentrations for Vtg and anti-Vtg were determined by coating wells with serial dilutions of purified Vtg and incubating with different antibody concentrations. The chosen routine conditions were 200 ng ml−1 for Vtg coating and 1:100 000 for antibody dilution.</note><note type="content">Fig. 2: (A) Binding displacement curves obtained with Vtg standard (○) and serial dilutions of plasma from vitellogenic female English sole (■) and E2-injected male English sole (•). Control male English sole (+) exhibited no significant cross-reactivity with the Vtg antiserum at dilutions greater than 100-fold. (B) Linearization of binding curves by logit transformation. Parallelism of the regression curves was assessed by F-test on mean squares. No significant differences were seen between the standard preparation (s=−1.49; r2=0.99), vitellogenic female English sole (s=−1.54; r2=0.99) and E2-treated male English sole (s=−1.48; r2=0.97). All points are the mean of triplicate determinations.</note><note type="content">Fig. 3: (A) Binding displacement curves obtained with Vtg standard (○) and serial dilutions of plasma from different species: female rock sole (□), female starry flounder (▴) and female sand sole (♦). (B) Parallelism was observed between the standard and plasma from rock sole (s=−1.39; r2=0.95) and starry flounder (s=−1.40; r2=0.97). Sand sole (s=−0.93; r2=0.96) plasma showed some cross-reactivity with the Vtg antiserum but was not statistically parallel with the standard. All points are the mean of duplicate determinations.</note><note type="content">Fig. 4: Plasma Vtg levels in adult female English sole captured in the field at different stages of ovarian maturation. Values are the mean±S.E.; n in parentheses. ND, none detected.</note><note type="content">Fig. 5: Plasma Vtg levels in juvenile English sole induced with estradiol. Plasma samples were collected 2 days after E2 injections. Vtg was not detected in any of the control groups or the three lowest E2 doses (data not shown). Values are the mean±S.E.; n in parentheses.</note><note type="content">Fig. 6: Vtg levels in two types of hepatocyte media culture at 1, 3 and 5 days following isolation and plating of English sole hepatocytes. Values are the mean of duplicate samples.</note><subject><genre>Keywords</genre><topic>Biomarker</topic><topic>ELISA</topic><topic>English sole</topic><topic>Environmental estrogens</topic><topic>Vitellogenin</topic></subject><relatedItem type="host"><titleInfo><title>Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>CBB</title></titleInfo><genre type="journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">199812</dateIssued></originInfo><identifier type="ISSN">1096-4959</identifier><identifier type="PII">S0305-0491(00)X0048-X</identifier><part><date>199812</date><detail type="volume"><number>121</number><caption>vol.</caption></detail><detail type="issue"><number>4</number><caption>no.</caption></detail><extent unit="issue pages"><start>363</start><end>470</end></extent><extent unit="pages"><start>425</start><end>436</end></extent></part></relatedItem><identifier type="istex">0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4</identifier><identifier type="DOI">10.1016/S0305-0491(98)10125-6</identifier><identifier type="PII">S0305-0491(98)10125-6</identifier><accessCondition type="use and reproduction" contentType="copyright">©1998 Elsevier Science Inc.</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>Elsevier Science Inc., ©1998</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Eau/explor/EsturgeonV1/Data/Istex/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001038 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Istex/Corpus/biblio.hfd -nk 001038 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Eau
|area= EsturgeonV1
|flux= Istex
|étape= Corpus
|type= RBID
|clé= ISTEX:0AEEC0E3CCFDB14C38478558DCE0D5DD6BA096A4
|texte= An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole ( Pleuronectes vetulus ): development, validation and cross-reactivity with other pleuronectids
}}
| This area was generated with Dilib version V0.6.27. |  |