Development and validation of a homologous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals
Development and validation of a homologous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals
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DOI：
10.1016/s1532-0456(01)00194-6
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发表时间：
2001-07-01
影响因子：
3.9
通讯作者：
Segner, H
中科院分区：
文献类型：
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作者：
Fenske, M;van Aerle, R;Segner, H
用阴离子交换层析从17α-乙炔基雌二醇(EE2)诱导的雌性斑马鱼(Danio Rerio)血浆中分离出卵黄蛋白原(VTG)。用聚丙烯酰胺凝胶电泳法(SDS-PAGE)鉴定VTG分离物的纯度。用纯化的VTG在兔体内制备多克隆抗体，用SDS-PAGE分离的血浆蛋白进行Western印迹分析，证实了VTG抗血清的特异性。这些抗体与雌性斑马鱼血浆中的两种蛋白质发生交叉反应，其分子质量分别约为142和171 kDa。未观察到与任何其他血浆蛋白的交叉反应。以斑马鱼VTG(z-VTG)多克隆抗体为配基，以纯化的z-VTG为标准品，建立了斑马鱼VTG竞争酶联免疫吸附试验(EL ISA)。该方法灵敏度高，检出限为2.0~3.0 ng纯化的VTG/ml，工作范围为3~500 ng/ml(结合率30~85%)。该方法精密度高，批内和批间变异系数分别为7.5+/-2.7和4.9+/-1.4%。成年斑马鱼的血浆和幼斑马鱼的全身匀浆与EI-ISA中的z-VTG标准平行稀释，验证了测定这两种组织中z-VTG的方法。成年雄性斑马鱼经水暴露于EE2可诱导VTG的浓度依赖性诱导，其最低观察效应浓度(LOEC)小于或等于1.67 ng EE2/1(21天暴露)。同源的z-VTG-EL ISA为斑马鱼环境雌激素的研究提供了有价值的工具。(C)2001 Elsevier Science Inc.保留所有权利。
Vitellogenin (VTG) was isolated by anion exchange chromatography from plasma of female zebrafish (Danio rerio) induced with 17 alpha -ethinylestradiol (EE2). The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Purified VTG was used to raise polyclonal antibodies in rabbits and the specificity of the antisera for VTG confirmed by Western blot analysis of plasma proteins separated by SDS-PACE. The antibodies cross-reacted with two proteins in the plasma of female zebrafish, with molecular masses of approximately 142 and 171 kDa. No cross-reactivity was observed with any other plasma proteins. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal zebrafish VTG (z-VTG) antibodies and purified z-VTG as ligand and standard, respectively. The z-VTG ELISA was sensitive with a detection limit of between 2.0 and 3.0 ng purified VTG/ml, and a working range between 3 and 500 ng/ml (30-85% binding). The ELISA demonstrated precision, with inter- and intra-assay variations of 7.5 +/- 2.7 and 4.9 +/- 1.4%, respectively. Plasma from adult zebrafish and whole body homogenates from juvenile zebrafish diluted parallel with the z-VTG standard in the EI-ISA, validating the assay for quantifying z-VTG in both of these tissues. Exposure of adult male zebrafish to EE2 via water induced a concentration-dependent induction of VTG with a lowest observed effect concentration (LOEC) less than or equal to 1.67 ng EE2/1 (for a 21-day exposure). The homologous z-VTG ELISA provides a valuable tool for the study of environmental estrogens in zebrafish. (C) 2001 Elsevier Science Inc. All rights reserved.