| Project/Area Number |
10555285
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
生物・生体工学
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| Research Institution | Tokyo University of Agriculture & Technology |
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Principal Investigator |
MATSUNAGA Tadashi Tokyo University of Agriculture and Technology, Department of Biotechnology, Professor, 工学部, 教授 (10134834)
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| Co-Investigator(Kenkyū-buntansha) |
TAJIMA Shuji Precision System Science Co., Ltd., President, システム・サイエンス(株), 社長(研究職)
YOHDA Masafumi Tokyo University of Agriculture and Technology, Department of Biotechnology, Associate Professor, 工学部, 助教授 (50250105)
TAKEYAMA Haruko Tokyo University of Agriculture and Technology, Department of Biotechnology, Associate Professor, 工学部, 助教授 (60262234)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥13,400,000 (Direct Cost: ¥13,400,000)
Fiscal Year 1999: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 1998: ¥8,600,000 (Direct Cost: ¥8,600,000)
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| Keywords | magnetic bacteria / bacterial magnetic particles / fusion gene / Protein A / miniaturized immunoassay robot / human IgG / 組み替え磁気微粒子 / 組み変え磁気微粒子 / ルシフェラーゼ / イムノアッセイ / マウスIgG |
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| Research Abstract |
Recently, analysis of the genes concerned with magnetic particle synthesis has been undertaken in Magnetospirillum sp. strain AMB-1. The magA gene was isolated from a nonmagnetic mutant generated by transposon mutagenesis from AMB-1, and completely sequenced. MagA can be used as an anchor protein to express various foreign proteins on membrane of bacterial magnetic particle (BMP) without the need for chemical cross-linking. We have cloned a proteinA-magA hybrid gene into magnetic bacterium strain AMB-1, and produced protein A-BMP complexes. In this study, we developed a fully automated chemiluminescence enzyme immunoassay by using a magnetic separation of antibody-protein A-BMP complexes.
The luminescence intensity of protein A-BMP complexes was 20 times higher than that of BMP from wild type AMB-1. Protein A was successfully expressed on the BMP membrane, and has IgG Fc region-binding activity. Human IgG concentration was measured by fully-automated chemiluminescence enzyme immunoassay system using antibody-protein A-BMP complexes and ALP-antibody. Automated immunoassay system contains a reaction station, tip rack and an automated eight pipettor that is able to attach and detach a strong magnet to a tip surface, and correspondent to 96 well microtiter plate. Microtiter plate can be mounted in the reaction station. There is one rack to hold 8 x 3 tips for reaction. Each reagents is applied to 8 lines of microtiter plate. Dose-response curve was obtained between the luminescence intensity and human IgG concentration. Detection limit was 1 pg/ml. In conclusion, chemiluminescence enzyme immunoassay using antibody-protein A-BMP complexes was developed full-automatically. The fully automated immunoassay system allows precise assay of human insulin in serum.
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