| Project/Area Number |
63570115
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Tokushima University |
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Principal Investigator |
YOSHIMOTO Tanihiro School of Medicine Department of Biochemistry, Associate Professor, 医学部, 助教授 (60127876)
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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 |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Arachidonic acid / Cyclooxygenase / 12-Lipoxygenase / Thromboxane / Monoclonal antibody / Enzyme immunoassay / シクロオキシゲナーゼ / リポキシゲナーゼ / モノクローン抗体 |
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| Research Abstract |
Monocloral antibodies against fatty acid cyclooxygenase and arachidonate 12-lipoxygenase of human platelets were prepared. Mice were immunized intraperitoneally with the partially purified preparations of these enzymes as antigens. The hybridoma cans producing monoclonal antibodies were cloned and grown in the abdominal cavity of mice, and the antibodies were purified to IgG from the ascetic fluid.
An enzyme immurioassay for cyclooxygenase was established utilizing two species of monoclonal antibody which bound to different epitopes of the enzyme protein. One of the antibodies was digested with pepsin, and its Fab' fragment was conjugated to horseradish peroxidase. The complex of cyclooxygenase and peroxidase-labeled Fab' was precipitated with the other antibody which had been bound to protein A on the surface of S. aureus. The peroxidase activity in the immunoprecipitate was correlated lineally to the amount of cyclooxygenase present in the assay mixture. This enzyme immunoassay for cyclooxygenase was much more sensitive than the enzyme assay as assessed by the conversion of radioactive arachidonic acid to prostaglandin (PG) H2. Essentially the same enzyme immunoassay was developed for arachidonate 12-lipoxygenase of human platelets.
A clinical case of cyclooxygenase abnormality was reported previously. The cyclooxygenase activity (arachidonic acid => PGH_2) in platelets was markedly reduced, whereas thromboxane syntilase activity (PGF_2 => thromboxane A_2) was at normal level. The enzyme immunoassay established as above was applied to this case to determine the platelet cyclooxygenase level, and detected a normal amount of cyclooxygenase in the patient's platelets. The result indicated that the anomalous cyclooxygenase protein was present in patient's platelets, which was catalytically inactive but immunologically indistinguishable from the normal enzyme.
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