Development of immunoassay for fish vitellogenin using universal antibody

点击翻译按钮获取中文摘要

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.