Project/Area Number |
12650782
|
Research Category |
Grant-in-Aid for Scientific Research (C)
|
Allocation Type | Single-year Grants |
Section | 一般 |
Research Field |
生物・生体工学
|
Research Institution | The University of Tokyo |
Principal Investigator |
UEDA Hirosi Graduate School of Frontier Sciences, The University of Tokyo, Associate Professor, 大学院・新領域創成科学研究科, 助教授 (60232758)
|
Co-Investigator(Kenkyū-buntansha) |
NAGAMUNE Teruyuki Graduate School of Engineering, The University of Tokyo, Professor, 大学院・工学系研究科, 教授 (20124373)
|
Project Period (FY) |
2000 – 2001
|
Project Status |
Completed (Fiscal Year 2001)
|
Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
Keywords | Antibody Engineering / Open Sandwich Assay / Phage Display / Combinatorial / Immunoassay / FV Fragment / Lysozime / Variable Region / ELISA / スクリーニング / リゾチーム |
Research Abstract |
The objective of the study was to elucidate a rapid selection system to obtain a Fv that is suitable to the novel immunoassay based on the stabilization of Fv by bound antigen (Open Sandwich ELISA). To this end, a novel filamentous phage display system termed 'spFv system' in which antigen binding ability of Fv as well as VH/VL interaction can be easily evaluated. Based on a model experiment using HyHEL-10, which is a known antibody to be suitable to Open Sandwich ELISA, the principle was shown to work by simply changing the host strain used. Also, a model selection of HyHEL-10 against another nonbonding antibody was shown to work. Along with this, the analysis of important amino acid residue(s) in the determination of VH/VL interaction strength was performed by phage display of human antibody model. As a result, H95 of CDR H3 was shown to be a key determinant of VH/VL interaction. To attain higher sensitivity of homogeneous Open Sandwich immunoassays, novel methods utilizing the energy transfer from a luciferase to a fluorescent protein, as well as enzymatic complementation of two enzyme fragments were engineered. As a result, 10 to thousand folds improvement in sensitivity was attained compared with previous homogeneous assays.
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