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Development of quickly diagnosis for silkworm virus by use of monoclonal antibody

单克隆抗体快速诊断家蚕病毒的研究进展

基本信息

批准号：
04454065
负责人：
MIYAJIMA Shigetoshi
金额：
$ 3.84万
依托单位：
MIE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

In order to develop a simple ana quickly diagnosis for silkworm virus we made the monoclonal antibodies for cytoplasmic-polyhedrosis virus (CPV) and dnhancing factor of nuclear-polyhedrosis virus infection (EF) by hybridoma selection method.1.Three-group clones of monoclonal antibody for CPV-I strain were obtained. Thst is, one group was remarkably reacted to CPV-I strain, the second reacted to CPV-H strain, and the third reacted in the intermediate degrees. These results suggested that both strains pessessed the common epitope between CPV-H and CPV-I strains. But some of them showed a little different reaction between two strains.2. There are two types of inclusion body produced by Pseudaletia separata entomopoxvirus. One is called spheroid enclosed virus particles and the other is spindle without virus particle in it. Enhancing factor (EF) is known as the increasing the nuclear-polyhedrosis virus infection.So we mede a monoclonal antibody for the enhancing factor protein. But this mo … More noclonal antibody did not react for spheroidin which was a main constructed protein of spheroid. On the contrary the another inclusion protein, spindle, reacted remarkably to this monoclonal antibody. From the result it was demonstrated that 49.5KDa protein, i.e.main constructed protein of spindle, had common epitope to enhancing factor.We tried to develop the immunosensor constructed by monoclonal antibody and crystal bibrator to detect the cytoplasmic-polyhedrosis virus of the silkworm simply and quickly. At first we purified the IgG from monoclonal antibody for CPV and tested its reaction. The IgG showed the highly titers, i.e.1 x 10^<-5> g IgG could detect 1 ug/ml CPV by ELISA.In addition to this it was clear that this monoclonal antibody reacted both CPV core protein and CPV polyhedrin by western blotting analysis. The IgG conjugated the crystal bibrator smeared with protein A was showed the variation of co-frequency. By remove the non-specific adsorption we might develop the high sensitive immunosensor. Less

为了建立家蚕病毒的简便、快速诊断方法，我们用杂交瘤筛选法制备了抗细胞质多角体病毒(CPV)和核型多角体病毒感染因子(EF)的单抗。1.获得了三组抗CPV-I株的单抗。即，一组对CPV-I株有明显反应，第二组对CPV-H株有反应，第三组对CPV-I株有中等程度的反应。这些结果表明，两株病毒都具有CPV-H株和CPV-I株的共同表位。但它们中的一些在两个品系之间表现出略有不同的反应。油菜昆虫痘病毒产生的包涵体有两种类型。一种是球形包裹型病毒颗粒，另一种是无病毒颗粒的纺锤形颗粒。增强因子(EF)被称为增加核型多角体病毒感染，因此我们研制了一种针对增强因子蛋白的单抗。但是这个mo…更多的非克隆抗体不与球体的主要构建蛋白球蛋白反应。相反，另一种包涵体蛋白--纺锤体与该单抗发生显著反应。结果表明，49.5 KDa蛋白是家蚕纺锤体的主要结构蛋白，具有共同的增强因子表位。本研究试图建立一种利用单抗和晶体生物构建的免疫传感器来快速、简便地检测家蚕细胞质多角体病毒。首先，我们从抗CPV的单抗中提纯了Ig G，并对其反应进行了检测。该单抗具有较高的效价，即1×10~(-5)~(-5)~(-5)~(gt；g)抗体可检测到1ug/mlCPV。此外，Western blotting分析表明，该单抗可与CPV核心蛋白和CPV多角体蛋白反应。与A蛋白涂片的晶体生物膜上的免疫球蛋白呈同频变化。通过去除非特异性吸附，我们可以开发出高灵敏度的免疫传感器。较少