鲫鱼(Carassius auratus)卵黄蛋白原的ELISA检测

采用Sephadex G-200柱层析从鲫鱼(Carassius auratus)成熟鱼卵中提取卵黄脂磷蛋白并制备抗卵黄脂磷蛋白抗血清.用DEAE-cellulose阴离子交换层析和Sephacryl S-300分子筛凝胶层析相结合的两步层析从经17b-雌二醇诱导的雌鱼血浆中提纯卵黄蛋白原.免疫印迹反应显示卵黄脂磷蛋白和卵黄蛋白原都与抗卵黄脂磷蛋白抗血清有特异的反应.以抗卵黄脂磷蛋白抗血清作为抗体,卵黄蛋白原作为竞争抗原建立了间接竞争法酶联免疫吸附反应(ELISA)方法来检测雄鱼血液中卵黄蛋白原含量.该方法可测的最低浓度为7.8ng/mL,工作范围为0.39~25mg/mL,批内和批间误差分别为6.5%和7.5%.

Development of ELISA for detecting Carassius auratus vitellogenin

Sephadex G-200 chromatography was used to extract lipovitellin from mature fish egg; and the antiserum which resists lipovitellin was prepared. Vtg was purified by DEAEcellulose anion-exchange and Sephacryl size-exclusion combined two step chromatography from 17b-estradiol (E2)-injected female Carassius auratus blood. An indirect competitive enzyme-linked immunosorbent assay (ELISA) for detecting Vtg content in the blood of male Carassius auratus was developed with antiserum of lipovitellin resistance as antibody and vitellogen in as competition antigen. Of this technique, the lowest detectable concentration was 7.8ng/mL; and the working range was 0.39~25mg/mL; the intra- and the inter-assay coefficients of variation were 6.5% and 7.5% respectively.