title: Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin.
abstract: Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.
authors.affiliation: EnBioTec Laboratories, Co., Ltd., Ishikawa Create Labo 202, 2-13, Asahidai, Tatsunokuchi, Ishikawa 923-1211, Japan. nishi@enbiotec.co.jp
authors.forename: Kazuto
authors.initials: K
authors.lastname: Nishi
authors.forename: Miyuki
authors.initials: M
authors.lastname: Chikae
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Hatano
authors.forename: Haruki
authors.initials: H
authors.lastname: Mizukami
authors.forename: Michiaki
authors.initials: M
authors.lastname: Yamashita
authors.forename: Ryuzo
authors.initials: R
authors.lastname: Sakakibara
authors.forename: Eiichi
authors.initials: E
authors.lastname: Tamiya
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology; D001202:Ascitic Fluid / Q000737:chemistry; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006735:Horseradish Peroxidase / Q000737:chemistry; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D016014:Linear Models; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009990:Oryzias / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014819:Vitellogenins / Q000032:analysis* / Q000096:biosynthesis / Q000097:blood
chemical_list: D000911:Antibodies, Monoclonal; D014819:Vitellogenins; D004958:Estradiol; D006735:Horseradish Peroxidase