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Development of immunoassay for fish vitellogenin using universal antibody

使用通用抗体开发鱼类卵黄蛋白原免疫测定法

基本信息

批准号：
11556038
负责人：
HARA Akihiko
金额：
$ 4.61万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

In order to establish universal immunoassay for fish vitellogenins as a biomarker of environmental estrogens, we developed sensitive enzyme immunoassay(EIA)and chemiluminescent immunoassay(CLIA)using monoclonal and/or polyclonal antibodies again st purified egg yolk proteins from salmon and carp.1. Generation of antibodies against egg yolk proteins : Salmon egg yolk proteins are composed of three components : lipovitellin, phosvitin and β'-component(beta). Specific antibodies in rabbits and mice against purified masu salmon lipovitellin and beta, as well as against carp lipovitellin were generated. Beta in carp was not detected in this study.2. Amino acid sequence of masu salmon beta : N-terminal amino acid sequence of masu salmon beta was analyzed and 35 residues were determined. The amino acid sequence was in good agreement with that of rainbow trout previously reported.3. Development of vitellogenin immunoassays : The CLIA for carp vitellogenin was completed within foru hours and measured vitellogenin from 2 to 1000 ng/ml. Cross-reactivity was found in cyprinids, but not in salmonids. Also EIA and CLIA for salmon vitellogenin were developed. As for the EIA, sandwich method using polyclonal and monoclonal antibodies against masu salmon lipovitellin and β'-component, respectively, was used. The EIA measures vitellogenin as low as 0.3 ng/ml. The CLIA developed in this study is called one-step CLIA using two types of antibodies. The CLIA was completed within 6 to 9 hours and covered a range of 122pg/ml to 1000 ng/ml. Both EIA and CLIA for salmon vitellogenin cross-reacted with vitellogenins from other salmonids and therefore can be used as universal immunoassays for salmon vitellogenins.4. Development of commercial vitellogenin assay kit : For the primary screening test prior to analysis by EIA and CLIA, single radial immunodiffusion was developed. This plate is able to measure vitellogenin in a range of 25 to 1000 mg/ml and is ready to be on the market.

为了建立环境雌激素生物标志物鱼卵黄蛋白原的通用免疫分析方法，我们利用单克隆和/或多克隆抗体，对鲑鱼和鲤鱼卵黄蛋白进行了纯化，建立了灵敏的酶免疫分析（EIA）和荧光免疫分析（CLIA）.产生抗蛋黄蛋白的抗体：鲑鱼蛋黄蛋白由三种组分组成：卵黄脂蛋白、卵黄蛋白和β ′-组分（β）。在兔和小鼠中产生针对纯化的鲑鱼卵黄脂磷蛋白和β以及针对鲤鱼卵黄脂磷蛋白的特异性抗体。本研究中未检测到鲤鱼体内的β.马苏鲑鱼β的氨基酸序列：分析马苏鲑鱼β的N-末端氨基酸序列，并确定35个残基。氨基酸序列与虹鳟鱼的一致性较好.卵黄蛋白原免疫测定的开发：鲤鱼卵黄蛋白原的CLIA在四小时内完成，测得的卵黄蛋白原浓度为2至1000 ng/ml。在鲤科鱼类中发现交叉反应，但在鲑科鱼类中没有。同时建立了鲑鱼卵黄蛋白原的EIA和CLIA检测方法。对于EIA，使用分别针对鲑鱼卵黄脂磷蛋白和α ′-组分的多克隆和单克隆抗体的夹心法。EIA测量的卵黄蛋白原低至0.3 ng/ml。本研究中开发的CLIA称为使用两种类型抗体的一步CLIA。CLIA在6至9小时内完成，覆盖范围为122 pg/ml至1000 ng/ml。鲑鱼卵黄蛋白原的EIA和CLIA均能与其他鲑科鱼类的卵黄蛋白原发生交叉反应，因此可作为鲑鱼卵黄蛋白原的通用免疫检测方法.卵黄蛋白原检测试剂盒的研制：采用单向放射免疫扩散法进行初筛。该板能够测量25至1000 mg/ml范围内的卵黄蛋白原，并准备上市。