| Project/Area Number |
08672601
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用薬理学・医療系薬学
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| Research Institution | Akita University |
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Principal Investigator |
ITOH Kunihiko Akita University School of Medicine, Associate Professor., 医学部, 助教授 (90221770)
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| Co-Investigator(Kenkyū-buntansha) |
MIZUGAKI Michinao Tohoku University School of Medicine, Professor., 医学部, 教授 (60004595)
SUZUKI Toshio Akita University School of Medicine, Professor., 医学部, 教授 (20108559)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1996: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Phage display system / Human antibodies / Combinatorial libraries / Rotaviruses / ファージディスプレイシステム |
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| Research Abstract |
The goal of this research project is the establishing of the conventional procedure for making human monoclonal antibodies applicable for the prophylaxis or therapy using phage display system. We have selected rotaviruses as a target since they are recognized as the most important cause of severe viral gastroenteritis in humans and animals. The immunoglobulin Fd and light chain gene segments were amplified from PBL of two healthy individuals (ON and AO) with high serum titer to human rotavirus Wa (HRV Wa) using RT-PCR,then the combinatorial lgG1, k Fab phage display library were constructed. Five rounds of panning of these libraries with rabbit Ab-captured HRV Wayielded an approximately 30-fold enrichment in eluted phage. Eight Fab clones were finally isolated from these libraries by screening in the sandwich ELISA followed by the Bst NI finger printing. All Fab clones reacted with HRV Wa in a concerntration dependent manner with no cross-reactivity to a panel of Ags. The Fab clones from ON library showed the strain cross-reactivity, on the contrary, those from AO library were specific for Wa. Although the immunoblotting analysis revealed that Fabs from two individual libraries react with the identical Ag, the VP6 protein, they would recognize the different epitopes because of their distinct strain specificity and the different VH gene usage. From these results, the phage display system would be powerful tool for making clinically useful human monoclonal antibodies, and the isolated Fabs would be useful for detection or identification of rotaviruses from biological specimen in clinical laboratory testing.
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