Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout ( Oncorhynchus mykiss ): Establishment and Validation of an ELISA
Identifieur interne : 000C89 ( Istex/Corpus ); précédent : 000C88; suivant : 000C90Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout ( Oncorhynchus mykiss ): Establishment and Validation of an ELISA
Auteurs : E. Bon ; U. Barbe ; J. Nu Ez Rodriguez ; B. Cuisset ; C. Pelissero ; J. P Sumpter ; F. Le MennSource :
- Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology [ 1096-4959 ] ; 1997.
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Abstract
Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric form of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20–320 ng/ml (80–20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.
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DOI: 10.1016/S0305-0491(96)00252-0
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Nu Ez" last="Rodriguez">J. Nu Ez Rodriguez</name><affiliation><mods:affiliation>ORSTOM, Centre de Recherches Océanologiques (CRO), BP V18, Abidjan, Côte d'ivoire</mods:affiliation></affiliation></author><author><name sortKey="Cuisset, B" sort="Cuisset, B" uniqKey="Cuisset B" first="B" last="Cuisset">B. Cuisset</name><affiliation><mods:affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Pelissero, C" sort="Pelissero, C" uniqKey="Pelissero C" first="C" last="Pelissero">C. Pelissero</name><affiliation><mods:affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Sumpter, J P" sort="Sumpter, J P" uniqKey="Sumpter J" first="J. P" last="Sumpter">J. 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Le Menn</name><affiliation><mods:affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:941BA0C804C416A015013F99C10A95DE34D15685</idno><date when="1997" year="1997">1997</date><idno type="doi">10.1016/S0305-0491(96)00252-0</idno><idno type="url">https://api.istex.fr/document/941BA0C804C416A015013F99C10A95DE34D15685/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">000C89</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000C89</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout ( Oncorhynchus mykiss ): Establishment and Validation of an ELISA</title><author><name sortKey="Bon, E" sort="Bon, E" uniqKey="Bon E" first="E" last="Bon">E. Bon</name><affiliation><mods:affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Barbe, U" sort="Barbe, U" uniqKey="Barbe U" first="U" last="Barbe">U. Barbe</name><affiliation><mods:affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Rodriguez, J Nu Ez" sort="Rodriguez, J Nu Ez" uniqKey="Rodriguez J" first="J. Nu Ez" last="Rodriguez">J. Nu Ez Rodriguez</name><affiliation><mods:affiliation>ORSTOM, Centre de Recherches Océanologiques (CRO), BP V18, Abidjan, Côte d'ivoire</mods:affiliation></affiliation></author><author><name sortKey="Cuisset, B" sort="Cuisset, B" uniqKey="Cuisset B" first="B" last="Cuisset">B. Cuisset</name><affiliation><mods:affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Pelissero, C" sort="Pelissero, C" uniqKey="Pelissero C" first="C" last="Pelissero">C. Pelissero</name><affiliation><mods:affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</mods:affiliation></affiliation></author><author><name sortKey="Sumpter, J P" sort="Sumpter, J P" uniqKey="Sumpter J" first="J. P" last="Sumpter">J. P Sumpter</name><affiliation><mods:affiliation>Department of Biology and Biochemistry, University of Brunel, Uxbridge Middlesex UB8 3PH, United Kingdom</mods:affiliation></affiliation></author><author><name sortKey="Le Menn, F" sort="Le Menn, F" uniqKey="Le Menn F" first="F" last="Le Menn">F. Le Menn</name><affiliation><mods:affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</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">CBBOLD</title><idno type="ISSN">1096-4959</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1997">1997</date><biblScope unit="volume">117</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="75">75</biblScope><biblScope unit="page" to="84">84</biblScope></imprint><idno type="ISSN">1096-4959</idno></series><idno type="istex">941BA0C804C416A015013F99C10A95DE34D15685</idno><idno type="DOI">10.1016/S0305-0491(96)00252-0</idno><idno type="PII">S0305-0491(96)00252-0</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1096-4959</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Enzyme-linked immunosorbent assay (ELISA)</term><term>fish</term><term>rainbow trout</term><term>reproductive cycle</term><term>vitellogenesis</term><term>vitellogenin</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric form of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20–320 ng/ml (80–20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>E Bon</name><affiliations><json:string>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</json:string></affiliations></json:item><json:item><name>U Barbe</name><affiliations><json:string>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</json:string></affiliations></json:item><json:item><name>J.Nuñez Rodriguez</name><affiliations><json:string>ORSTOM, Centre de Recherches Océanologiques (CRO), BP V18, Abidjan, Côte d'ivoire</json:string></affiliations></json:item><json:item><name>B Cuisset</name><affiliations><json:string>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</json:string></affiliations></json:item><json:item><name>C Pelissero</name><affiliations><json:string>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</json:string></affiliations></json:item><json:item><name>J.P Sumpter</name><affiliations><json:string>Department of Biology and Biochemistry, University of Brunel, Uxbridge Middlesex UB8 3PH, United Kingdom</json:string></affiliations></json:item><json:item><name>F Le Menn</name><affiliations><json:string>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Enzyme-linked immunosorbent assay (ELISA)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>fish</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>rainbow trout</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>reproductive cycle</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>vitellogenin</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>vitellogenesis</value></json:item></subject><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric form of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20–320 ng/ml (80–20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.</abstract><qualityIndicators><score>6.968</score><pdfVersion>1.2</pdfVersion><pdfPageSize>594 x 792 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>6</keywordCount><abstractCharCount>1080</abstractCharCount><pdfWordCount>6175</pdfWordCount><pdfCharCount>37342</pdfCharCount><pdfPageCount>10</pdfPageCount><abstractWordCount>164</abstractWordCount></qualityIndicators><title>Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout ( Oncorhynchus mykiss ): Establishment and Validation of an ELISA</title><pii><json:string>S0305-0491(96)00252-0</json:string></pii><genre><json:string>research-article</json:string></genre><host><volume>117</volume><pii><json:string>S0305-0491(00)X0024-7</json:string></pii><pages><last>84</last><first>75</first></pages><issn><json:string>1096-4959</json:string></issn><issue>1</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>1997</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>1997</publicationDate><copyrightDate>1997</copyrightDate><doi><json:string>10.1016/S0305-0491(96)00252-0</json:string></doi><id>941BA0C804C416A015013F99C10A95DE34D15685</id><score>0.039663434</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/941BA0C804C416A015013F99C10A95DE34D15685/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/941BA0C804C416A015013F99C10A95DE34D15685/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/941BA0C804C416A015013F99C10A95DE34D15685/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout ( Oncorhynchus mykiss ): Establishment and Validation of an ELISA</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>©1997 Elsevier Science Inc.</p></availability><date>1997</date></publicationStmt><notesStmt><note type="content">Section title: General Papers</note><note type="content">Fig. 1: Purification of rainbow trout Vtg. (a) Direct anion-exchange chromatography of E2-treated-male plasma compared to plasma from an untreated-male. The Vtg was eluted with a linear NaCl gradient from 0 to 200 mM in which the Vtg eluted at a salt concentration of 75 mM. (b) The non-competitive ELISA performed using the antibody directed against Vtg showed, in E2-treated male eluted fractions, the appearance of a symmetrical vitellogenic peak B, eluting at the 47th fraction. The absence of immunodetection of Vtg in untreated male eluted fractions demonstrated that the purified molecule was inducible by E2. (c) The non-competitive ELISA performed using the antibody directed against vitelline envelope proteins (VEP) showed, in E2-treated male eluted fractions, that VEP eluted on both sides of the vitellogenic peak (peak B).</note><note type="content">Fig. 2: Specificity of the anti-Vtg antibody. Immunodiffusion was performed in 1.5% agarose gel. The anti-Vtg was located in the central well (0) surrounded by wells containing untreated-male plasma [1], immature-female plasma [2], purified Vtg [3], vitellogenic-female plasma [4], E2-treated-male plasma [5], and egg yolk extract [6]. Samples were allowed to react for up to 24 hr at 4°C.</note><note type="content">Fig. 3: Vtg identification on native-PAGE. (a) Native PAGE of purified Vtg (lane 1), E2-treated-male plasma (lane 2) and untreated-male plasma (lane 3), stained with Coomassie blue. The arrowheads indicate the Vtg, which appears as a single focused band. (b) Corresponding Western blot using the antibody against rainbow trout Vtg (1:600). Vtg was present in the purified Vtg solution (lane 1′) and in E2-treated male plasma (lane 2′), but was clearly absent from untreated-male plasma (lane 3′).</note><note type="content">Fig. 4: Molecular weight determination of Vtg subunits. (a) SDS-PAGE (0.1% SDS) in 7.5% gel, of untreated male plasma without β-mercaptoethanol (lane 1), E2-treated-male plasma without β-merc. (lane 4) and with β-merc. (lane 7), as well as of the electroeluted 176 kDa (protein II) and 390 kDa (protein I) Vtg bands without β-mer. (respectively, lanes 2 and 3), and with β-merc. (respectively, lanes 5 and 6). (b) Corresponding Western blot using the antibody directed against rainbow trout Vtg. The lanes 1′ to 7′ are identified as in Fig. 4a. The molecular weight of the standard proteins is indicated on the left: myosin (200 kDa), β-galactosidase (116.25 kDa), phosphorylase-β (97.4 kDa), BSA (66.2 kDa) and ovalbumin (45 kDa).</note><note type="content">Fig. 5: Competition curves. Competition curves [Log (dose) = f [(Bi-NSB)/(Bo-NSB)] × 100, %] obtained with serial dilutions of various antigens were compared to the competition curve obtained with serial dilutions of the standard Vtg. No competition curve was observed with the untreated male plasma nor the pig serum whereas a great parallelism was observed in most biological samples tested compared to the standard curve (except for egg yolk).</note><note type="content">Fig. 6: Determination of Vtg levels in rainbow trout at various physiological stages. The Vtg plasma levels (μg/ml) varied from nearly undetectable to high. The following values were measured: 1- control males (0.38, n = 19), 2- neomales (1.45, n = 5), 3- triploid females (5.24, n = 11), 4- immature females (64.8, n = 45), 5- juvenile females (423, n = 7), 6- previtellogenic females (1440, n = 7). The highest Vtg levels were measured in 7- vitellogenic females (59600, n = 7), and in 8- E2-treated males (81600, n = 3).</note><note type="content">Fig. 7: Seasonal profile of Vtg in 2-year-old female rainbow trout. (a) Seasonal variations of the gonadosomatic index (GSI, Gonads weight/Body weight × 100, %) and the hepatosomatic index (HSI, liver weight/body weight × 100, %). The spawning period is indicated by an “S.” The abbreviations e. and l. before the calendar months mean, respectively, early and late in the month. (b) Visualization on a Coomassie Blue–stained SDS-PAGE (7.5%) of the seasonal appearance in plasma, of the Vtg. Blood samples were regularly collected from the same marked female throughout the reproductive cycle. The same volume of plasma sample (20 μl) at the same dilution (1:30) was loaded on the gel in all wells. On the right, arrowheads indicate the position of the dimeric form (protein I, 390 kDa) and the monomeric form (protein II, 176 kDa) of the rainbow trout Vtg. (c) Seasonal profile of Vtg plasma levels and seasonal variations of the oocyte diameter. The spawning period is indicated by an “S.”</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout ( Oncorhynchus mykiss ): Establishment and Validation of an ELISA</title><author xml:id="author-1"><persName><forename type="first">E</forename><surname>Bon</surname></persName><note type="biography">E. Bon, Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des Facultés, 33405 Talence Cedex, France</note><affiliation>E. Bon, Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des Facultés, 33405 Talence Cedex, France</affiliation><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation></author><author xml:id="author-2"><persName><forename type="first">U</forename><surname>Barbe</surname></persName><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation></author><author xml:id="author-3"><persName><forename type="first">J.Nuñez</forename><surname>Rodriguez</surname></persName><affiliation>ORSTOM, Centre de Recherches Océanologiques (CRO), BP V18, Abidjan, Côte d'ivoire</affiliation></author><author xml:id="author-4"><persName><forename type="first">B</forename><surname>Cuisset</surname></persName><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation></author><author xml:id="author-5"><persName><forename type="first">C</forename><surname>Pelissero</surname></persName><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation></author><author xml:id="author-6"><persName><forename type="first">J.P</forename><surname>Sumpter</surname></persName><affiliation>Department of Biology and Biochemistry, University of Brunel, Uxbridge Middlesex UB8 3PH, United Kingdom</affiliation></author><author xml:id="author-7"><persName><forename type="first">F</forename><surname>Le Menn</surname></persName><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation></author></analytic><monogr><title level="j">Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology</title><title level="j" type="abbrev">CBBOLD</title><idno type="pISSN">1096-4959</idno><idno type="PII">S0305-0491(00)X0024-7</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1997"></date><biblScope unit="volume">117</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="75">75</biblScope><biblScope unit="page" to="84">84</biblScope></imprint></monogr><idno type="istex">941BA0C804C416A015013F99C10A95DE34D15685</idno><idno type="DOI">10.1016/S0305-0491(96)00252-0</idno><idno type="PII">S0305-0491(96)00252-0</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1997</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric form of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20–320 ng/ml (80–20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>Enzyme-linked immunosorbent assay (ELISA)</term></item><item><term>fish</term></item><item><term>rainbow trout</term></item><item><term>reproductive cycle</term></item><item><term>vitellogenin</term></item><item><term>vitellogenesis</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1997">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/document/941BA0C804C416A015013F99C10A95DE34D15685/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:entity SYSTEM="gr7" NDATA="IMAGE" name="gr7"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>CBB</jid><aid>2283</aid><ce:pii>S0305-0491(96)00252-0</ce:pii><ce:doi>10.1016/S0305-0491(96)00252-0</ce:doi><ce:copyright year="1997" type="full-transfer">Elsevier Science Inc.</ce:copyright></item-info><head><ce:dochead><ce:textfn>General Papers</ce:textfn></ce:dochead><ce:title>Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout (<ce:italic>Oncorhynchus mykiss</ce:italic>): Establishment and Validation of an ELISA</ce:title><ce:author-group><ce:author><ce:indexed-name>Bon</ce:indexed-name><ce:given-name>E</ce:given-name><ce:surname>Bon</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref><ce:cross-ref refid="AFF2">b</ce:cross-ref><ce:cross-ref refid="CORR1">*</ce:cross-ref></ce:author><ce:author><ce:indexed-name>Barbe</ce:indexed-name><ce:given-name>U</ce:given-name><ce:surname>Barbe</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:author><ce:indexed-name>Rodriguez</ce:indexed-name><ce:given-name>J.Nuñez</ce:given-name><ce:surname>Rodriguez</ce:surname><ce:cross-ref refid="AFF3">c</ce:cross-ref></ce:author><ce:author><ce:indexed-name>Cuisset</ce:indexed-name><ce:given-name>B</ce:given-name><ce:surname>Cuisset</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:author><ce:indexed-name>Pelissero</ce:indexed-name><ce:given-name>C</ce:given-name><ce:surname>Pelissero</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:author><ce:indexed-name>Sumpter</ce:indexed-name><ce:given-name>J.P</ce:given-name><ce:surname>Sumpter</ce:surname><ce:cross-ref refid="AFF4">d</ce:cross-ref></ce:author><ce:author><ce:indexed-name>Le Menn</ce:indexed-name><ce:given-name>F</ce:given-name><ce:surname>Le Menn</ce:surname><ce:cross-ref refid="AFF1">a</ce:cross-ref></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>b</ce:label><ce:textfn>Salmonidés d'Aquitaine, Route de Taller, 40260 Castets, France</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>c</ce:label><ce:textfn>ORSTOM, Centre de Recherches Océanologiques (CRO), BP V18, Abidjan, Côte d'ivoire</ce:textfn></ce:affiliation><ce:affiliation id="AFF4"><ce:label>d</ce:label><ce:textfn>Department of Biology and Biochemistry, University of Brunel, Uxbridge Middlesex UB8 3PH, United Kingdom</ce:textfn></ce:affiliation><ce:correspondence id="CORR1"><ce:label>*</ce:label><ce:text>E. Bon, Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des Facultés, 33405 Talence Cedex, France</ce:text></ce:correspondence></ce:author-group><ce:date-received day="8" month="3" year="1996"></ce:date-received><ce:date-accepted day="6" month="9" year="1996"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Rainbow trout, <ce:italic>Oncorhynchus mykiss</ce:italic>, vitellogenin (Vtg) was purified from plasma of E<ce:inf>2</ce:inf>-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric form of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20–320 ng/ml (80–20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Enzyme-linked immunosorbent assay (ELISA)</ce:text></ce:keyword><ce:keyword><ce:text>fish</ce:text></ce:keyword><ce:keyword><ce:text>rainbow trout</ce:text></ce:keyword><ce:keyword><ce:text>reproductive cycle</ce:text></ce:keyword><ce:keyword><ce:text>vitellogenin</ce:text></ce:keyword><ce:keyword><ce:text>vitellogenesis</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout ( Oncorhynchus mykiss ): Establishment and Validation of an ELISA</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout (</title></titleInfo><name type="personal"><namePart type="given">E</namePart><namePart type="family">Bon</namePart><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation><description>E. Bon, Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des Facultés, 33405 Talence Cedex, France</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">U</namePart><namePart type="family">Barbe</namePart><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">J.Nuñez</namePart><namePart type="family">Rodriguez</namePart><affiliation>ORSTOM, Centre de Recherches Océanologiques (CRO), BP V18, Abidjan, Côte d'ivoire</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">B</namePart><namePart type="family">Cuisset</namePart><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">C</namePart><namePart type="family">Pelissero</namePart><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">J.P</namePart><namePart type="family">Sumpter</namePart><affiliation>Department of Biology and Biochemistry, University of Brunel, Uxbridge Middlesex UB8 3PH, United Kingdom</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">F</namePart><namePart type="family">Le Menn</namePart><affiliation>Laboratoire de Biologie de la Reproduction des Poissons, U.A INRA, Université Bordeaux I, Avenue des facultés, 33405 Talence Cedex, France</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">1997</dateIssued><copyrightDate encoding="w3cdtf">1997</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">Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric form of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20–320 ng/ml (80–20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.</abstract><note type="content">Section title: General Papers</note><note type="content">Fig. 1: Purification of rainbow trout Vtg. (a) Direct anion-exchange chromatography of E2-treated-male plasma compared to plasma from an untreated-male. The Vtg was eluted with a linear NaCl gradient from 0 to 200 mM in which the Vtg eluted at a salt concentration of 75 mM. (b) The non-competitive ELISA performed using the antibody directed against Vtg showed, in E2-treated male eluted fractions, the appearance of a symmetrical vitellogenic peak B, eluting at the 47th fraction. The absence of immunodetection of Vtg in untreated male eluted fractions demonstrated that the purified molecule was inducible by E2. (c) The non-competitive ELISA performed using the antibody directed against vitelline envelope proteins (VEP) showed, in E2-treated male eluted fractions, that VEP eluted on both sides of the vitellogenic peak (peak B).</note><note type="content">Fig. 2: Specificity of the anti-Vtg antibody. Immunodiffusion was performed in 1.5% agarose gel. The anti-Vtg was located in the central well (0) surrounded by wells containing untreated-male plasma [1], immature-female plasma [2], purified Vtg [3], vitellogenic-female plasma [4], E2-treated-male plasma [5], and egg yolk extract [6]. Samples were allowed to react for up to 24 hr at 4°C.</note><note type="content">Fig. 3: Vtg identification on native-PAGE. (a) Native PAGE of purified Vtg (lane 1), E2-treated-male plasma (lane 2) and untreated-male plasma (lane 3), stained with Coomassie blue. The arrowheads indicate the Vtg, which appears as a single focused band. (b) Corresponding Western blot using the antibody against rainbow trout Vtg (1:600). Vtg was present in the purified Vtg solution (lane 1′) and in E2-treated male plasma (lane 2′), but was clearly absent from untreated-male plasma (lane 3′).</note><note type="content">Fig. 4: Molecular weight determination of Vtg subunits. (a) SDS-PAGE (0.1% SDS) in 7.5% gel, of untreated male plasma without β-mercaptoethanol (lane 1), E2-treated-male plasma without β-merc. (lane 4) and with β-merc. (lane 7), as well as of the electroeluted 176 kDa (protein II) and 390 kDa (protein I) Vtg bands without β-mer. (respectively, lanes 2 and 3), and with β-merc. (respectively, lanes 5 and 6). (b) Corresponding Western blot using the antibody directed against rainbow trout Vtg. The lanes 1′ to 7′ are identified as in Fig. 4a. The molecular weight of the standard proteins is indicated on the left: myosin (200 kDa), β-galactosidase (116.25 kDa), phosphorylase-β (97.4 kDa), BSA (66.2 kDa) and ovalbumin (45 kDa).</note><note type="content">Fig. 5: Competition curves. Competition curves [Log (dose) = f [(Bi-NSB)/(Bo-NSB)] × 100, %] obtained with serial dilutions of various antigens were compared to the competition curve obtained with serial dilutions of the standard Vtg. No competition curve was observed with the untreated male plasma nor the pig serum whereas a great parallelism was observed in most biological samples tested compared to the standard curve (except for egg yolk).</note><note type="content">Fig. 6: Determination of Vtg levels in rainbow trout at various physiological stages. The Vtg plasma levels (μg/ml) varied from nearly undetectable to high. The following values were measured: 1- control males (0.38, n = 19), 2- neomales (1.45, n = 5), 3- triploid females (5.24, n = 11), 4- immature females (64.8, n = 45), 5- juvenile females (423, n = 7), 6- previtellogenic females (1440, n = 7). The highest Vtg levels were measured in 7- vitellogenic females (59600, n = 7), and in 8- E2-treated males (81600, n = 3).</note><note type="content">Fig. 7: Seasonal profile of Vtg in 2-year-old female rainbow trout. (a) Seasonal variations of the gonadosomatic index (GSI, Gonads weight/Body weight × 100, %) and the hepatosomatic index (HSI, liver weight/body weight × 100, %). The spawning period is indicated by an “S.” The abbreviations e. and l. before the calendar months mean, respectively, early and late in the month. (b) Visualization on a Coomassie Blue–stained SDS-PAGE (7.5%) of the seasonal appearance in plasma, of the Vtg. Blood samples were regularly collected from the same marked female throughout the reproductive cycle. The same volume of plasma sample (20 μl) at the same dilution (1:30) was loaded on the gel in all wells. On the right, arrowheads indicate the position of the dimeric form (protein I, 390 kDa) and the monomeric form (protein II, 176 kDa) of the rainbow trout Vtg. (c) Seasonal profile of Vtg plasma levels and seasonal variations of the oocyte diameter. The spawning period is indicated by an “S.”</note><subject lang="en"><genre>Keywords</genre><topic>Enzyme-linked immunosorbent assay (ELISA)</topic><topic>fish</topic><topic>rainbow trout</topic><topic>reproductive cycle</topic><topic>vitellogenin</topic><topic>vitellogenesis</topic></subject><relatedItem type="host"><titleInfo><title>Comparative Biochemistry and Physiology, Part B: Biochemistry and Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>CBBOLD</title></titleInfo><genre type="journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">199705</dateIssued></originInfo><identifier type="ISSN">1096-4959</identifier><identifier type="PII">S0305-0491(00)X0024-7</identifier><part><date>199705</date><detail type="volume"><number>117</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue pages"><start>1</start><end>158</end></extent><extent unit="pages"><start>75</start><end>84</end></extent></part></relatedItem><identifier type="istex">941BA0C804C416A015013F99C10A95DE34D15685</identifier><identifier type="DOI">10.1016/S0305-0491(96)00252-0</identifier><identifier type="PII">S0305-0491(96)00252-0</identifier><accessCondition type="use and reproduction" contentType="copyright">©1997 Elsevier Science Inc.</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>Elsevier Science Inc., ©1997</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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