Lomax D P, Roubal W T, Moore J D, Johnson L L
Environmental Conservation Division, Northwest Fisheries Science Center, National Oceanic and Atmospheric Administration, Seattle, WA 98112, USA.
Comp Biochem Physiol B Biochem Mol Biol. 1998 Dec;121(4):425-36. doi: 10.1016/s0305-0491(98)10125-6.
Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E2) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E2-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen-antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3',5,5'-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10-450 ng ml-1 (85-20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E2-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.
卵黄蛋白原（Vtg）是卵生动物肝脏在雌激素作用下产生的一种卵黄蛋白。卵黄生成通常仅在性成熟雌性动物中观察到，但通过暴露于外源性雌二醇（E2）或模拟雌激素的物质，雄性和幼年动物也可诱导产生。因此，雄性动物Vtg的异常产生可作为接触外源性雌激素的生物学指标。在本研究中，开发并验证了一种用于测量星斑川鲽（Pleuronectes vetulus）中Vtg的酶联免疫吸附测定（ELISA）方法。使用DEAE离子交换和琼脂糖凝胶排阻色谱从注射E₂的雄性星斑川鲽中纯化血浆Vtg，并在兔体内产生针对纯化Vtg蛋白的多克隆抗体。在该测定中，包被在微量滴定板孔上的Vtg与游离Vtg之间建立了对Vtg抗体的竞争。使用辣根过氧化物酶偶联的抗兔二抗检测吸附的抗原 - 抗体复合物，其酶活性用3,3',5,5'-四甲基联苯胺（TMB）底物显示。测定条件提供了稀释样品中10 - 450 ng ml⁻¹（结合率85 - 20%）的可检测Vtg范围。卵黄生成雌性和E₂处理雄性星斑川鲽的血浆稀释曲线与标准稀释曲线平行。我们目前正在进行野外和实验室研究，以调查接触污染物导致的雌激素和抗雌激素活性。
