1994 Fiscal Year Final Research Report Summary
Development of immunoaffinity purification and immunoassay of thromboxane and prostacyclin metabolites
| Project/Area Number |
05557013
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Tokushima University |
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Principal Investigator |
YAMAMOTO Shozo Department of Biochemistry, Tokushima University School of Medicine Professor, 医学部, 教授 (50025607)
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| Co-Investigator(Kenkyū-buntansha) |
YAMASHITA Kowa Nipponn Kayaku Company Research Laboratories Chief Researcher, 主任研究員
HAMANAKA Nobuyuki Ono Pharmaceutical Company Institute for Drug Development Director, 創薬研究所, 所長
HIRAMATSU Makoto Ono Pharmaceutical Company Minase Research Institute Researcher, 試薬研究室, 研究員
SAWADA Masahumi Ono Pharmaceutical Company Minase Research Institute Chief Researcher, 水無瀬研究所, 主任研究員
HAYASHI Yoko Department of Biochemistry, Tokushima University School of Medicine Research Ass, 医学部, 助手 (60035441)
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| Project Period (FY) |
1993 – 1994
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| Keywords | arachidonic acid / 6-keto-prostaglandin F_<1alpha> / prostaglandin E_2 / thromboxane B_2 / leukotriene C_4 / enzyme immunoassay / monoclonal antibody / 2,3-dinor-6-keto-prostaglandin F_<1alpha> |
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| Research Abstract |
Among the bioactive compounds derived from arachidonic acid, thromboxane (TX) A_2, which is synthesized in platelets, aggregates platelets and constricts blood vessels.Prostacyclin, which is produced predominantly in vascular endothelial cells, inhibits platelet aggregation and relaxs blood vessels. Ateempts have been made for investigation of the in vivo metabolism of these two bioactive compounds, their relevance to various cardiovascular diseases, and validation of their pharmaceutical efficacies.The purposes of this research project are the developments of a simple immunoaffinity purification method and a sensitive immunoassay for 11-dehydro-TXB_2,2,3-dinor-TXB_2 and 2,3-dinor-6-keto-PGF_<1alpha> with metabolic half lives longer than TXB_2 and 6-keto-PGF_<1alpha> which are stable degradation products of very labile TXA_2 and prostacyclin. In the first academic year, an omega-carboxy derivative of 2,3-dinor-6-keto-PGF_<1alpha> was prepared, and its omega-carboxy group was bound to a protein for preparation of an antigen. The conjugate was used as an antigen for preparation of a monoclonal antibody with a higher selectivity in terms of the sidechain length. By the use of this antibody we set up a radioimmunoassay and an enzyme immunoassay of 2,3-dinor-6-keto-PGF_<1alpha>. Cross-reactivity of the antibody with 6-keto-PGF_<1alpha> was less than 0.01%. The peroxidase-linked immunoassay allowed the determination of 2,3-dinor-6-keto-PGF_<1alpha> in a range of 14-1200 pmol. Its IC_<50> value was 120 pmol. In the second academic year, the monoclonal anti-2,3-dinor -6-keto-PGF_<1alpha> antibody was prepared in a large amount, and a solid phase column was prepared for immunoaffinitychromatographic isolation of the urinary metabolite. This technique was also applied to prostaglandin E_2 and leukotriene C_4 contained in synovial fluid of arthritis patients.
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