用于检测斑马鱼（Danio rerio）全身匀浆中卵黄蛋白原的酶联免疫吸附测定法的开发。
Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio).
作者信息
Holbech H, Andersen L, Petersen G I, Korsgaard B, Pedersen K L, Bjerregaard P
机构信息
Danish Centre for Environmental Oestrogen Research, Odense University, Odense, Denmark.
出版信息
Comp Biochem Physiol C Toxicol Pharmacol. 2001 Sep;130(1):119-31. doi: 10.1016/s1532-0456(01)00229-0.
The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish (Danio rerio) by gel filtration and anion exchange chromatography. Polyclonal antibodies against Lv were raised in rabbits. Anti-Lv IgG was purified by affinity chromatography. SDS-PAGE followed by Western blotting was performed to analyse the specificity of the antibody and the immunological similarities between Lv and vitellogenin (Vtg). Anti-Lv IgG was used to develop a direct non-competitive sandwich ELISA to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish. The intra- and interassay variabilities were 5.8% and 10.4%, respectively. The sensitivity was 0.2 ng Vtg x ml(-1) and the practical detection limit was 40 ng Vtg x g(-1) fish (wet weight). Adult male zebrafish were exposed to a nominal water concentration of 10 ng x l(-1) of ethinylestradiol (EE2) in a semi-static exposure system for 7 days. Compared with the control group, exposure to 10 ng EE2 x l(-1) induced a 200-fold increase in Vtg levels.
通过凝胶过滤和阴离子交换色谱法从成熟雌性斑马鱼（Danio rerio）的卵巢中纯化卵黄蛋白——卵黄磷蛋白（Lv）。用兔制备抗Lv的多克隆抗体。通过亲和色谱法纯化抗Lv IgG。进行SDS-PAGE后接着进行蛋白质印迹分析，以分析抗体的特异性以及Lv与卵黄蛋白原（Vtg）之间的免疫相似性。使用抗Lv IgG建立直接非竞争性夹心ELISA，以测量斑马鱼全身匀浆（WBH）中的Vtg浓度。批内和批间变异率分别为5.8%和10.4%。灵敏度为0.2 ng Vtg·ml⁻¹，实际检测限为40 ng Vtg·g⁻¹鱼（湿重）。成年雄性斑马鱼在半静态暴露系统中暴露于名义水浓度为10 ng·l⁻¹的乙炔雌二醇（EE2）7天。与对照组相比，暴露于10 ng EE2·l⁻¹导致Vtg水平增加200倍。
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