Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, School of Food and Environment, Dalian University of Technology, Panjin 124221, China.
State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361102, China.
Chemosphere. 2018 Apr;197:477-484. doi: 10.1016/j.chemosphere.2018.01.086.
A lipovitellin (Lv) based sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in marine medaka (Oryzias melastigma). Lv and Vtg were purified from the unfertilized eggs and the whole body homogenates (WBH) of estradiol (E)-exposed fish. The purified Lv sample appeared as three clear bands (118, 112 and 100 kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and was identified as an Lvs mixture from VtgAa and VtgAb by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. Polyclonal antibody against marine medaka VtgAa was also raised. Compared with Vtg, Lv was more stable to heat stress (37 °C for 8 h or 4 °C for a week) and repeated freeze/thaw stress. In addition, western blot analysis revealed that marine medaka Vtg and Lv had similar immunogenicity. Therefore, in this study, Lv was applied instead of Vtg as the standard to establish an ELISA. The Lv standard curve was parallel to serial WBH dilutions of E-exposed fish, and the absorbance values were very low in control male samples, suggesting the specificity and feasibility of the method for Vtg quantification. The developed assay was sensitive with the detection limit of 3.1 ng/mL and had a working range between 15.6 and 500 ng/mL. The intra- and inter-assay coefficients of variation were both below 5%. Moreover, the standard curves of Lv antigen treated under different stresses were almost identical, indicating high robustness of the assay. Overall, our study provides an important methodology reference for quantification of marine medaka Vtg.
建立了一种基于卵黄脂磷蛋白（Lv）的夹心酶联免疫吸附测定法（ELISA），用于定量海洋泥鳅（Oryzias melastigma）的卵黄蛋白原（Vtg）。Lv 和 Vtg 从雌二醇（E）暴露鱼的未受精卵和全鱼匀浆（WBH）中纯化。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中，纯化的 Lv 样品显示为三个清晰的条带（118、112 和 100 kDa），并通过液相色谱-串联质谱（LC-MS/MS）分析鉴定为 VtgAa 和 VtgAb 的 Lvs 混合物。还制备了针对海洋泥鳅 VtgAa 的多克隆抗体。与 Vtg 相比，Lv 对热应激（37°C 8 h 或 4°C 1 周）和反复冻融应激更稳定。此外，Western blot 分析表明，海洋泥鳅 Vtg 和 Lv 具有相似的免疫原性。因此，在本研究中，Lv 被用作标准物代替 Vtg 建立 ELISA。Lv 标准曲线与 E 暴露鱼的 WBH 系列稀释液平行，而对照雄性样本的吸光度值非常低，表明该方法用于 Vtg 定量具有特异性和可行性。所开发的测定法具有灵敏度，检测限为 3.1ng/mL，工作范围在 15.6 和 500ng/mL 之间。内和间测定的变异系数均低于 5%。此外，在不同应激下处理的 Lv 抗原的标准曲线几乎相同，表明该测定法具有很高的稳健性。总体而言，本研究为海洋泥鳅 Vtg 的定量提供了重要的方法学参考。
