| Project/Area Number |
09450301
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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 | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
NAGAMUNE Teruyuki Univ.of Tokyo, Dept.Chem.&Biotechnol., Professor, 大学院・工学系研究科, 教授 (20124373)
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| Co-Investigator(Kenkyū-buntansha) |
KITAYAMA Atsushi Univ.of Tokyo, Dept.Chem.&Biotechnol., Research Associate, 大学院・工学系研究科, 助手 (70270882)
UEDA Hiroshi Univ.of Tokyo, Dept.Chem.&Biotechnol., Lecturer, 大学院・工学系研究科, 講師 (60232758)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥14,100,000 (Direct Cost: ¥14,100,000)
Fiscal Year 1998: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1997: ¥10,700,000 (Direct Cost: ¥10,700,000)
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| Keywords | Immunoassay / Open sandwich method / Variable region of antibody / Antibody HyHEL-10 / Anti-insulin antibody / Anti-digoxin antibody / Anti-NP antibody / Alkaline phosphatase / 蛍光共鳴エネルギー移動 / 均相系非競合型免疫測定法 / 抗ジコキシン抗体 |
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| Research Abstract |
We found that separated VII and VL chains from variable region of antibody HyHEL-10 (anti-Hen Egg Lysozyme antibody) could re-associate in the presence of antigen and demonstrated new immunoassay method, named Open Sandwich method, based on the intrachain interaction of separated Vii and VL (Nature Biotechnology, (1996), 14, 1714-1718). We aimed to elucidate whether this method is applicable to other antigen-antibody immunoassay system and to develop a general Open Sandwich ELISA method based on the intrachain interaction of separated VL and chimeric VH-enzyme.
It was shown that re-association of separated VH and VL chains in the presence of antigen could be observed in two of six anti-insulin monoclonal antibodies investigated, anti-digoxin antibody and anti-NP (4-hydroxy 3-nitrophenyl acetic acid) antibody. These results indicate that such intrachain interaction of separated VH and VL driven by antigen exists generally in many antibodies. We also constructed several gene fusions encoding the mono-domain of antibody variable regions and E.coli alkaline phosphatase (PhoA) and produced the fusion proteins with E.coli secretion system. Open Sandwich ELISA was performed using microtiter plate with immobilized VL, and VH-PhoA as detection reagent which was added to each well with samples. As a result, antigen concentration was determined with wide dynamic range of measurement by only one-each step of incubation and washing.
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