A Newly Established ELISA Showing the Effect of the Androgenic Gland on Secondary-Vitellogenic-Specific Protein in the Hemolymph of the Crayfish Cherax quadricarinatus
A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to monitor the onset of secondary vitellogenesis in Cherax quadricarinatus females and in intersex individuals (having both male and female reproductive systems) after removal of the androgenic gland (AG). As a prerequisite for t...
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Veröffentlicht in: | General and comparative endocrinology 1999-07, Vol.115 (1), p.37-45 |
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description | A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to monitor the onset of secondary vitellogenesis in Cherax quadricarinatus females and in intersex individuals (having both male and female reproductive systems) after removal of the androgenic gland (AG). As a prerequisite for the assay, the 106-kDa polypeptide was separated from newly laid C. quadricarinatus eggs by SDS–PAGE, and anti-106-kDa antibody was raised in rabbit. The specificity of the anti-106-kDa polypeptide for proteins specific for the hemolymph of secondary-vitellogenic females was confirmed by double immunodiffusion and immunoblot cross-reactivity tests. A characteristic standard ELISA curve, using egg high-density lipoprotein (HDL), showed linearity between 16 and 500 ng (r=0.953) and was sensitive for amounts as low as 8 ng. The inter- and intraassay coefficients of variance were 14.8 and 7.2%, respectively. Only traces of egg HDL equivalents were detected in the hemolymph of male and primary-vitellogenic females (11 to 110 μg/ml), confirming the specificity of the assay, whereas high levels of such a protein (8–35 mg/ml) were detected in the hemolymph of secondary-vitellogenic females. Removal of the AG from intersex individuals leads to a significant increase in the concentration of vitellogenic-specific protein in the hemolymph (up to 2 mg/ml). Moreover, a significantly lower concentration was found in females subjected to AG transplant (79.3 μg/ml). The ELISA thus provided an accurate and sensitive tool to investigate the influence of the AG on the expression of a vitellogenic-specific protein in female and intersex C. quadricarinatus, confirming the central role of this gland in tuning sexual plasticity in this species. |
doi_str_mv | 10.1006/gcen.1999.7277 |
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As a prerequisite for the assay, the 106-kDa polypeptide was separated from newly laid C. quadricarinatus eggs by SDS–PAGE, and anti-106-kDa antibody was raised in rabbit. The specificity of the anti-106-kDa polypeptide for proteins specific for the hemolymph of secondary-vitellogenic females was confirmed by double immunodiffusion and immunoblot cross-reactivity tests. A characteristic standard ELISA curve, using egg high-density lipoprotein (HDL), showed linearity between 16 and 500 ng (r=0.953) and was sensitive for amounts as low as 8 ng. The inter- and intraassay coefficients of variance were 14.8 and 7.2%, respectively. Only traces of egg HDL equivalents were detected in the hemolymph of male and primary-vitellogenic females (11 to 110 μg/ml), confirming the specificity of the assay, whereas high levels of such a protein (8–35 mg/ml) were detected in the hemolymph of secondary-vitellogenic females. Removal of the AG from intersex individuals leads to a significant increase in the concentration of vitellogenic-specific protein in the hemolymph (up to 2 mg/ml). Moreover, a significantly lower concentration was found in females subjected to AG transplant (79.3 μg/ml). The ELISA thus provided an accurate and sensitive tool to investigate the influence of the AG on the expression of a vitellogenic-specific protein in female and intersex C. quadricarinatus, confirming the central role of this gland in tuning sexual plasticity in this species.</description><identifier>ISSN: 0016-6480</identifier><identifier>EISSN: 1095-6840</identifier><identifier>DOI: 10.1006/gcen.1999.7277</identifier><identifier>PMID: 10375462</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Astacoidea - metabolism ; Cherax quadricarinatus ; Egg Proteins - blood ; Electrophoresis, Polyacrylamide Gel ; Endocrine Glands - physiology ; Enzyme-Linked Immunosorbent Assay - methods ; Female ; Freshwater ; Hemolymph - chemistry ; Immunoblotting ; Immunodiffusion ; Lipoproteins, HDL - blood ; Male ; Ovum - chemistry ; Sensitivity and Specificity ; Vitellogenesis</subject><ispartof>General and comparative endocrinology, 1999-07, Vol.115 (1), p.37-45</ispartof><rights>1999 Academic Press</rights><rights>Copyright 1999 Academic Press.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c381t-16918363afd006d69cd831e41cf3e384cf9bdf90970974dcd735f0545c5b8cbc3</citedby><cites>FETCH-LOGICAL-c381t-16918363afd006d69cd831e41cf3e384cf9bdf90970974dcd735f0545c5b8cbc3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/gcen.1999.7277$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10375462$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sagi, Amir</creatorcontrib><creatorcontrib>Khalaila, Isam</creatorcontrib><creatorcontrib>Abdu, Uri</creatorcontrib><creatorcontrib>Shoukrun, Rami</creatorcontrib><creatorcontrib>Weil, Simy</creatorcontrib><title>A Newly Established ELISA Showing the Effect of the Androgenic Gland on Secondary-Vitellogenic-Specific Protein in the Hemolymph of the Crayfish Cherax quadricarinatus</title><title>General and comparative endocrinology</title><addtitle>Gen Comp Endocrinol</addtitle><description>A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to monitor the onset of secondary vitellogenesis in Cherax quadricarinatus females and in intersex individuals (having both male and female reproductive systems) after removal of the androgenic gland (AG). As a prerequisite for the assay, the 106-kDa polypeptide was separated from newly laid C. quadricarinatus eggs by SDS–PAGE, and anti-106-kDa antibody was raised in rabbit. The specificity of the anti-106-kDa polypeptide for proteins specific for the hemolymph of secondary-vitellogenic females was confirmed by double immunodiffusion and immunoblot cross-reactivity tests. A characteristic standard ELISA curve, using egg high-density lipoprotein (HDL), showed linearity between 16 and 500 ng (r=0.953) and was sensitive for amounts as low as 8 ng. The inter- and intraassay coefficients of variance were 14.8 and 7.2%, respectively. Only traces of egg HDL equivalents were detected in the hemolymph of male and primary-vitellogenic females (11 to 110 μg/ml), confirming the specificity of the assay, whereas high levels of such a protein (8–35 mg/ml) were detected in the hemolymph of secondary-vitellogenic females. Removal of the AG from intersex individuals leads to a significant increase in the concentration of vitellogenic-specific protein in the hemolymph (up to 2 mg/ml). Moreover, a significantly lower concentration was found in females subjected to AG transplant (79.3 μg/ml). The ELISA thus provided an accurate and sensitive tool to investigate the influence of the AG on the expression of a vitellogenic-specific protein in female and intersex C. quadricarinatus, confirming the central role of this gland in tuning sexual plasticity in this species.</description><subject>Animals</subject><subject>Astacoidea - metabolism</subject><subject>Cherax quadricarinatus</subject><subject>Egg Proteins - blood</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Endocrine Glands - physiology</subject><subject>Enzyme-Linked Immunosorbent Assay - methods</subject><subject>Female</subject><subject>Freshwater</subject><subject>Hemolymph - chemistry</subject><subject>Immunoblotting</subject><subject>Immunodiffusion</subject><subject>Lipoproteins, HDL - blood</subject><subject>Male</subject><subject>Ovum - chemistry</subject><subject>Sensitivity and Specificity</subject><subject>Vitellogenesis</subject><issn>0016-6480</issn><issn>1095-6840</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUGP1CAYhonRuOPq1aPh5K0jDC0tx8lk3N1koiajXgmFjymmhVloXecX-Teldk28GJMvIYSHl_A-CL2mZE0J4e9OGvyaCiHW9aaun6AVJaIqeFOSp2hFCOUFLxtyhV6k9I0QUjFOn6MrSlhdlXyzQj-3-AM89Be8T6Nqe5c6MHh_uDtu8bELD86f8NgB3lsLesTB_t5tvYnhBN5pfNMrb3Dw-Ag6eKPipfjqRuj75bw4nkE7m8FPMYzgPM4zR9zCEPrLcO7-ZO6iutj8PN51ENUPfD8pE51W0Xk1TuklemZVn-DV43qNvrzff97dFoePN3e77aHQrKFjQbmgDeNMWZPbMVxo0zAKJdWWAWtKbUVrrCCizlMabWpWWVKVla7aRreaXaO3S-45hvsJ0igHl3T-j_IQpiS5aFhdCvFfkNabkjFaZXC9gDqGlCJYeY5uyEVJSuTsUM4O5exQzg7zhTePyVM7gPkLX6RloFkAyEV8dxBl0g68BuNitiRNcP_K_gUvra06</recordid><startdate>199907</startdate><enddate>199907</enddate><creator>Sagi, Amir</creator><creator>Khalaila, Isam</creator><creator>Abdu, Uri</creator><creator>Shoukrun, Rami</creator><creator>Weil, Simy</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>F1W</scope><scope>H95</scope><scope>L.G</scope><scope>7X8</scope></search><sort><creationdate>199907</creationdate><title>A Newly Established ELISA Showing the Effect of the Androgenic Gland on Secondary-Vitellogenic-Specific Protein in the Hemolymph of the Crayfish Cherax quadricarinatus</title><author>Sagi, Amir ; Khalaila, Isam ; Abdu, Uri ; Shoukrun, Rami ; Weil, Simy</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c381t-16918363afd006d69cd831e41cf3e384cf9bdf90970974dcd735f0545c5b8cbc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Animals</topic><topic>Astacoidea - metabolism</topic><topic>Cherax quadricarinatus</topic><topic>Egg Proteins - blood</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Endocrine Glands - physiology</topic><topic>Enzyme-Linked Immunosorbent Assay - methods</topic><topic>Female</topic><topic>Freshwater</topic><topic>Hemolymph - chemistry</topic><topic>Immunoblotting</topic><topic>Immunodiffusion</topic><topic>Lipoproteins, HDL - blood</topic><topic>Male</topic><topic>Ovum - chemistry</topic><topic>Sensitivity and Specificity</topic><topic>Vitellogenesis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sagi, Amir</creatorcontrib><creatorcontrib>Khalaila, Isam</creatorcontrib><creatorcontrib>Abdu, Uri</creatorcontrib><creatorcontrib>Shoukrun, Rami</creatorcontrib><creatorcontrib>Weil, Simy</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>MEDLINE - Academic</collection><jtitle>General and comparative endocrinology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sagi, Amir</au><au>Khalaila, Isam</au><au>Abdu, Uri</au><au>Shoukrun, Rami</au><au>Weil, Simy</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Newly Established ELISA Showing the Effect of the Androgenic Gland on Secondary-Vitellogenic-Specific Protein in the Hemolymph of the Crayfish Cherax quadricarinatus</atitle><jtitle>General and comparative endocrinology</jtitle><addtitle>Gen Comp Endocrinol</addtitle><date>1999-07</date><risdate>1999</risdate><volume>115</volume><issue>1</issue><spage>37</spage><epage>45</epage><pages>37-45</pages><issn>0016-6480</issn><eissn>1095-6840</eissn><abstract>A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to monitor the onset of secondary vitellogenesis in Cherax quadricarinatus females and in intersex individuals (having both male and female reproductive systems) after removal of the androgenic gland (AG). As a prerequisite for the assay, the 106-kDa polypeptide was separated from newly laid C. quadricarinatus eggs by SDS–PAGE, and anti-106-kDa antibody was raised in rabbit. The specificity of the anti-106-kDa polypeptide for proteins specific for the hemolymph of secondary-vitellogenic females was confirmed by double immunodiffusion and immunoblot cross-reactivity tests. A characteristic standard ELISA curve, using egg high-density lipoprotein (HDL), showed linearity between 16 and 500 ng (r=0.953) and was sensitive for amounts as low as 8 ng. The inter- and intraassay coefficients of variance were 14.8 and 7.2%, respectively. Only traces of egg HDL equivalents were detected in the hemolymph of male and primary-vitellogenic females (11 to 110 μg/ml), confirming the specificity of the assay, whereas high levels of such a protein (8–35 mg/ml) were detected in the hemolymph of secondary-vitellogenic females. Removal of the AG from intersex individuals leads to a significant increase in the concentration of vitellogenic-specific protein in the hemolymph (up to 2 mg/ml). Moreover, a significantly lower concentration was found in females subjected to AG transplant (79.3 μg/ml). The ELISA thus provided an accurate and sensitive tool to investigate the influence of the AG on the expression of a vitellogenic-specific protein in female and intersex C. quadricarinatus, confirming the central role of this gland in tuning sexual plasticity in this species.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>10375462</pmid><doi>10.1006/gcen.1999.7277</doi><tpages>9</tpages></addata></record> |
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subjects | Animals Astacoidea - metabolism Cherax quadricarinatus Egg Proteins - blood Electrophoresis, Polyacrylamide Gel Endocrine Glands - physiology Enzyme-Linked Immunosorbent Assay - methods Female Freshwater Hemolymph - chemistry Immunoblotting Immunodiffusion Lipoproteins, HDL - blood Male Ovum - chemistry Sensitivity and Specificity Vitellogenesis |
title | A Newly Established ELISA Showing the Effect of the Androgenic Gland on Secondary-Vitellogenic-Specific Protein in the Hemolymph of the Crayfish Cherax quadricarinatus |
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