Hennies Mark, Wiesmann Marlene, Allner Bernhard, Sauerwein Helga
Institut für Physiologie, Biochemie und Hygiene der Tiere, Katzenburgweg 7-9, 53115 Bonn, Germany.
Sci Total Environ. 2003 Jun 20;309(1-3):93-103. doi: 10.1016/S0048-9697(03)00005-6.
Vitellogenin (VTG), a phospholipoglycoprotein precursor of egg yolk is synthesized and secreted in the liver in response to circulating estrogens in female fish. Thus, the presence of VTG in male fish is a useful biomarker to identify estrogenic activity of natural or anthropogenic substances in sewage effluents. We report the purification of carp (Cyprinus carpio) and perch (Perca fluviatilis) VTG with the subsequent development and characterization of a specific ELISA for VTG measurement. VTG was purified by combination of ion exchange chromatography and size exclusion chromatography. The purified proteins were used as antigen for antibody production, as standard and tracer in the assay. Carp VTG was stable and showed a characteristic double band after Western blotting of the purified protein and in serum samples, respectively. In perch samples, several bands with lower molecular weight were present and appeared to be degradation products of VTG. The development of the carp VTG ELISA led to a sensitive and valid test system with inter-assay coefficients of variation between 3.0 and 12.3%. In contrast to carp, the described ELISA for perch VTG showed a much higher inter-assay variation up to 24%, possibly attributable to fast protein degradation. In conclusion, the described two-step chromatography is a simple purification method for VTG. Immunological and electrophoretical test systems are valid methods to determine VTG in some species like carp, but for other species like perch in which VTG is not stable, these methods are not applicable.
卵黄蛋白原（VTG）是一种卵黄的磷脂糖蛋白前体，在雌性鱼类中，肝脏会响应循环中的雌激素而合成并分泌该物质。因此，雄性鱼类中存在VTG是一种有用的生物标志物，可用于识别污水中天然或人为物质的雌激素活性。我们报告了鲤鱼（Cyprinus carpio）和鲈鱼（Perca fluviatilis）VTG的纯化过程，以及随后开发并表征用于测量VTG的特异性酶联免疫吸附测定（ELISA）。通过离子交换色谱法和尺寸排阻色谱法相结合的方式纯化VTG。纯化后的蛋白质用作产生抗体的抗原、测定中的标准品和示踪剂。纯化后的鲤鱼VTG具有稳定性，在对纯化蛋白和血清样本进行蛋白质免疫印迹分析后分别显示出特征性的双链带。在鲈鱼样本中，存在几条分子量较低的条带，似乎是VTG的降解产物。鲤鱼VTG ELISA的开发形成了一个灵敏且有效的检测系统，批间变异系数在3.0%至12.3%之间。与鲤鱼不同，所描述的鲈鱼VTG ELISA显示出高达24%的更高批间变异，这可能归因于蛋白质的快速降解。总之，所描述的两步色谱法是一种简单的VTG纯化方法。免疫和电泳检测系统是测定某些物种（如鲤鱼）中VTG的有效方法，但对于其他物种（如鲈鱼，其VTG不稳定），这些方法并不适用。
