唐鱼卵黄蛋白原的ELISA检测方法的建立

目的探索建立唐鱼卵黄蛋白原的ELISA检测方法。方法以卵黄脂磷蛋白（Lv）抗血清为抗体,以纯化的Lv作为抗原建立间接酶联免疫吸附反应（ELISA）方法检测雄性唐鱼（Tanichthys albonubes）整体匀浆液中的卵黄蛋白原（Vtg）。结果利用ELISA方法测定了经17-β雌二醇（E2）和不同浓度DDTs暴露21 d诱导的雄鱼整体匀浆液中的Vtg含量,可以直接在1块或在不同的酶标板上准确地进行比较。经0.055 mg/mL E2诱导的雄鱼整体匀浆液中Vtg含量为4148.33μg/g;当暴露DDTs浓度为0.0275、0.0137和0.0067 mg/mL时,雄鱼整体匀浆液中Vtg含量分别为1109.43、911.16和1322.79μg/g,与丙酮溶剂对照组462.79μg/g比较差异存在显著性（P〈0.05）。结论建立了唐鱼卵黄蛋白原的ELISA检测方法 ,本方法的检测灵敏度为8.1 ng/mL,批内误差为8.59%,批间误差为6.28%,工作范围为3.26～209.25 ng/mL。在该范围内,标准曲线具有良好的线性和重复性。

Development and Application of an ELISA Technique for Detecting Tanichthys albonubes Vitellogenin

Objective The aim of this study was to set up an indirect enzyme-linked immunosorbent assay（ELISA）for detecting Tanichthys albonubes vitellogenin.Methods ELISA was established for detecting Vtg in the whole body homogenate（WBH） of male T.albonubes.The technique was developed with antiserum against lipovitellin（Lv） as antibody and Lv as antigen.Results Adult male T.albonubes was exposed to nominal water（control group）,to DDTs at concentrations of 0.0275 mg /mL,0.0137 mg /mL and 0.0067 mg /mL（DDTs exposure groups） and to 17-β estradiol at concentration of 0.0550 mg /mL（E2 exposure group） in a static exposure system for 21 days.WBH samples of male fish could be checked in the same one coated well and different coated wells to develop screening of environmental estrogen.Compared with control group（462.79 μg /g）,the Vtg concentrations in DDTs exposure groups（1 109.43 μg /g,911.16 μg/g,and 1 322.79 μg/g,respectively） and E2 exposure group（4 148.33 μg/g） were significantlyly different（P 0.05）.Conclusion An ELISA assay for detecting T.albonubesvitellogenin has been set up.The working range is 3.26 to 209.25 ng /mL,the sensitivity is 8.1 ng /mL,the intra-assay and inter-assay coefficients of variation are 8.59% and 6.28%,respectively.This technique shows a good linearity and repeatability in standard curves.