| Co-Investigator(Kenkyū-buntansha) |
NAGAMUNE Teruyuki The University of Tokyo, School of Engineering, Professor, 大学院・工学系研究科, 教授 (20124373)
UEDA Takuya The University of Tokyo, School of Engineering, Professor, 大学院・工学系研究科, 教授 (80184927)
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| Budget Amount *help |
¥12,000,000 (Direct Cost: ¥12,000,000)
Fiscal Year 2004: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Research Abstract |
1.A novel system to measure protein-protein interaction (split Fv system).
A novel protein-protein interaction measurement system split Fv (spFv) system was constructed which display one protein fragment on the filamentous phage while another protein fragment is secreted in the culture medium. When the system was applied to the measurement of VH/VL interaction of a model antibody, antigen-dependent change in the interaction was successfully measured (Open Sandwich immunoassay, OS-IA). In addition, the system can display the two domains VH/VL at the same time, which enables biopanning with the antigen. In fact, through the construction of the library of the framework region, we clarified the previously unknown relationship between the antigen binding affinity of Fv and the VH/VL interaction strength, and the residue that determines the VH/VL interaction.
2.OS-IA as a noncompetitive immunoassay principle for the low molecular weight substances
Using the above-mentioned SpFv system, we succe
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eded in the gene cloning of, as well as the noncompetitive immunoassay based on OS-IA with various anti-hapten antibodies (bisphenol A, 11-deoxycortisol, phosphotyrosine, gibbellerin A24, osteocalcin peptide etc.). For every antigen, a higher sensitivity and wider concentration range than those of conventional competitive immunoassays, were attained. Also, practical assays could be performed using combinations of the purified fusion proteins of VH -alkaline phosphatase and such as MBP-VL, which had been expressed using the cloned genes.
3.A mammalian-based selection system for the antigen-specific scFv
Using the IL-3 dependent cultured cells expressing the cell-surface chimeric receptor that can transduce antigen-dependent growth signal, a novel mammalian cell-based selection system for antigen-specific antibody fragment was elucidated. The selected cells can be rapidly converted to glycosylated scFv-secreting cells through the forced expression of Cre recombinase, leading to the name of the system (antigen-mediated selection for antibody producers, ASAP) Less
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