| Project/Area Number |
15590494
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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 |
Laboratory medicine
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| Research Institution | Okayama Prefectural University |
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Principal Investigator |
KIMOTO Masumi Okayama Prefectural University, Fac.Health and Welfare Science, Professor, 保健福祉学部, 教授 (40108866)
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| Co-Investigator(Kenkyū-buntansha) |
TSUJI Hideaki Okayama Prefectural University, Fac.Health and Welfare Science, Professor, 保健福祉学部, 教授 (20093875)
YAMASHITA Hiromi Okayama Prefectural University, Fac.Health and Welfare Science, Assistant Professor, 保健福祉学部, 講師 (70254563)
HIEMORI Miki Okayama Prefectural University, Fac.Health and Welfare Science, Assistant Researcher, 保健福祉学部, 助手 (80326412)
MATSUOKA Hidehiro Kurume University School of Medicine, Department of Internal medicine, Assistant Professor, 医学部, 講師 (80248393)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Nitric oxide / asymmetric dimethylarginine / N^G,N^G-dimethylarginine dimethylaminohydrolase / monoclonal antibody / atherosclerosis / inhibition ELISA / risk factor for cardiovascular disease / 心臓血管動脈硬化症 / 組み換え型ヒトDDAH / ハプテン抗原 / 組換え型ヒトDDAH |
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| Research Abstract |
Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Elevated ADMA plasma levels have been reported in connection with diseases associated with an impaired endothelial arginine/NO pathway and endothelial dysfunction, such as atherosclerosis, hypercholesterolemia, chronic heart failure, diabetes mellitus, and hypertension. Moreover, ADMA has recently been shown to be a risk factor for cardiovascular diseases. Therefore, there is increasing interest in measuring ADMA levels in human subjects, experimental animals, and cell culture. So far, the quantitative determination of ADMA by high-performance liquid chromatography (HPLC) analysis has been the most widely applied method. We designed specific two assay systems, the first is a method using enzymatic reaction with N^G,N^G-dimethylarginine dimethylaminohydrolase (E.C. 3.5.3.18 : DDAH) that degrades ADMA to citrulline, and the second an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against ADMA. In general, the analytical performance of ELISA is assessed above all by evaluating its specificity, analytical sensitivity, and facility. Therefore, we investigated the development of a novel ELISA assay for ADMA focusing on the second system described above. First we attempted to prepare a monoclonal antibody (mAb) against ADMA using BALB/c mouse immunized with ADMA-MBS-BSA conjugate. The mAb 3C12 obtained reacted specifically ADMA within a variety of amino acids including arginine derivatives and more sensitively to MBS-acylated ADMA than ADMA. We constructed a standard assay system due to the inhibition ELISA using the mAb. The limit of quantitation was 0.4nM, and the mean recovery was 95% after all treatments for the assay system. We determined ADMA levels in human plasma and found good correlation of the values measured by HPLC (r=0.98,p<0.01).
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