| Project/Area Number |
63480479
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | Osaka Medical College |
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Principal Investigator |
INAI Sinya Osaka Medical College, Clinical Pathol., Prof., 医学部, 教授 (90131317)
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| Co-Investigator(Kenkyū-buntansha) |
HATANAKA Michiyo Osaka Medical College, Clinical, Assistant Prof., 医学部, 助手 (50218484)
TAKUBO Takayuki Osaka Medical College, Clinical Pathol., lecturer, 医学部, 講師 (60163359)
高田 裕子 大阪医科大学, 医学部, 助手 (30197107)
森山 剛 大阪医科大学, 医学部, 助手 (90182279)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1988: ¥6,400,000 (Direct Cost: ¥6,400,000)
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| Keywords | Complement / C9 / MoAb to C9 / C9 Related peptides / モノクロナール抗C9抗体 |
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| Research Abstract |
Complement component C9 is a globular plasma protein which has the ability to insert into membranes containing a preformed C5b-8 complex. C9 proceeds a drastic conformational change upon interaction with C5b-8 complex. Although the entire primary structure has been already demonstrated, the structure and function relationship is not fully understood. Recently, we have obtained monoclonal antibodies against human C9 (P40 and X197) which effectively inhibit hemolytic activity of C9 through preventing C9 binding to C5b-8 complex. The inhibition mechanism suggests that the epitopes to which these antibodies bind lie on the binding site of C9 to C5b-8 complex. In this research, we intended to localize the epitopes recognized by these antibodies in order to clarify the structure and function relationship of C9. C9 was proteolized with either alpha-thrombin or trypsin, or cleaved with BNPS-skatole and the resulting fragments were used in Western blots for epitope mapping. Several peptides within the amino-acid sequence of C9 assumed to be the epitope were synthesized and tested reactivity to the antibodies. Reactivity of Melittin, the major component of bee venom, was also tested, since it shares functional and structural characteristics with C9. The results indicate that both P40 and X197 recognize the sequence region close to the C terminal of C9 molecule designated as domain 5.
We also demonstrated that P40 recognizes native C9, but not inactive C9 or SC5-9 complex formed in serum. Using P40 as a capture antibody, we developed a sensitive ELISA method for C9 detection in human serum.
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