Li Chun-Ri, Jung Kwun-Soon, Lee Sang-Hoon, Kim Andre, Park Jang-Su
Department of Chemistry and Centre for Innovative Bio-physio Sensor Technology, Pusan National University, Busan 609-735, South Korea.
Chemosphere. 2006 Jun;63(9):1485-90. doi: 10.1016/j.chemosphere.2005.09.041. Epub 2005 Nov 14.
A sandwich enzyme-linked immunosorbant assay (ELISA) was developed to quantitatively detect Bullfrog (Rana catesbeiana) vitellogenin (VTG) levels. This procedure involved a sandwich ELISA using monoclonal antibodies of BVmA1 and BVmA2 against Bullfrog-VTG, and BVmA1 conjugated to horseradish peroxidase as the detection antibody. The assay range was between 9.4 ng/ml and 1200 ng/ml and the recovery of the VTG added to Bullfrog control male serum was 92.0-108.8%. Male Bullfrog was induced by injection 17beta-estradiol (E2) for four weeks and Bullfrog-VTG levels were measured each week. Histological analysis was performed for investigating the correlation of the effect to male reproduction and Bullfrog-VTG level variation depending on E2 dose. After two weeks of E2 exposure, the induced Bullfrog-VTG level was significantly higher than Bullfrog control female (p<0.05). After four weeks of E2 exposure, the rupture and fusion of seminiferous tubule in the testes of male Bullfrog were shown and provided direct evidence that the reproduction of male Bullfrog was affected by estrogenic compounds. Bullfrog-VTG bioassay, using the sandwich ELISA, could be a sensitive and useful tool for quantification of estrogenic principles in aquatic environments.
开发了一种夹心酶联免疫吸附测定法（ELISA）来定量检测牛蛙（Rana catesbeiana）卵黄蛋白原（VTG）水平。该程序涉及使用针对牛蛙VTG的BVmA1和BVmA2单克隆抗体的夹心ELISA，以及与辣根过氧化物酶偶联的BVmA1作为检测抗体。测定范围为9.4 ng/ml至1200 ng/ml，添加到牛蛙对照雄性血清中的VTG回收率为92.0 - 108.8%。对雄性牛蛙注射17β-雌二醇（E2）诱导四周，每周测量牛蛙VTG水平。进行组织学分析以研究该效应与雄性繁殖的相关性以及取决于E2剂量的牛蛙VTG水平变化。E2暴露两周后，诱导的牛蛙VTG水平显著高于牛蛙对照雌性（p<0.05）。E2暴露四周后，雄性牛蛙睾丸中的生精小管出现破裂和融合，这为雄性牛蛙的繁殖受到雌激素化合物影响提供了直接证据。使用夹心ELISA的牛蛙VTG生物测定法可能是定量水生环境中雌激素成分的一种灵敏且有用的工具。
