1990 年度実績報告書

モノクロ-ナル抗体を用いた低分子生理活性ペプチドの超高感度測定法の開発及び実用化

研究課題

研究課題/領域番号	02557112
研究機関	京都大学
研究代表者	中尾一和京都大学, 医学部, 講師 (00172263)
研究分担者	島津章京都大学, 医学部, 助手 (90196494) 井上健塩野義研究所, 部長研究員
キーワード	生理活性ペプチド / ナトリウム利尿ペプチド / エンドセリン / バゾプレッシソ / モノクロ-ナル抗体 / エンザイムノアッセイ / イムノラジオメトリックアッセイ / エピト-プ
研究概要	従来よりホルモンの測定に多用されてきたラジオイムノアッセイ(RIA)より測定感度が100〜1000倍以上高感度である低分子生理活性ペプチドの超高感度測定法の開発と実用化を目的として、ナトリウム利尿ペプチドファミリ-(ANP、BNP、CNP)、エンドセリン(ET)ファミリ-(ETー1、ETー2、ETー3)バゾプレッシン(AVP)等に対するモノクロ-ナル抗体の開発を行なった。特にサンドイッチ酵素抗体法(EIA)やイムノラジオメトリックアッセイ(IRMA)の開発のためには低分子生理活性ペプチドの異なるエピト-プを認識するモノクロ-ナル抗体の開発が必要なので、上記生理活性ペプチドの種々のフラグメントを化学合成して、carrier蛋白であるウシサイログロブリンとconjugateし、Balb/cマウスに免疫した。血清抗体価の上昇を確認したマウスの脾細胞をマウスミエロ-マ細胞X63Ag8.653と細胞融合して、ハイブリド-マを調整し、限界希釈法にてモノクロ-ナル抗体産生ハイブリド-マをクロ-ニングした。本年度までに開発できたモノクロ-ナル抗体はANP8種類、ヒトBNP2種類、ブタBNP2種類、ラットBNP3種類、エンドセリン5種類、AVP5種類であり、これらの結合定数Kaはすべて10^<-9>〜10^<-11>Mと高親和性であり、エピト-プも異なっている。このうち、ANPではEIAとIRMAに利用可能なモノクロ-ナル抗体が確認できており、現時点での最少検出量は、EIAで0.01fmol/tube、IRMAで0.05fmol/tubeまで検出可能になってきており、実用化が期待できる段階にきている。現在、他の生理活性ペプチドのEIA、IRMAについても検討を続けている。

研究成果

(27件)

すべてその他

すべて文献書誌 (27件)

[文献書誌] G.Takemura,H.Fujiwara,H.Yoshida,M.Mukoyama,Y.Saito,K.Nakao,T.Fujiwara,T.Uegaito,H.Imura and C.Kawai: "Identification and distribution of atrial natriuretic polypeptide in ventricular myocardium of humans with myocardical infarction." J.Pathology. 161. 285-292 (1990)
[文献書誌] G.Shirakami,K.Nakao,Y.Saito,T.Magaribuchi,H.Nagata,M.Jougasaki,M.Mukoyama,H.Arai,K.Hosoda,S.Suga,Y.Ogawa,K.Mori,H.Imura.: "Endothelin in human cerebrospinal fluid." Therapeutic Research. 11. 3913-3918 (1990)
[文献書誌] M.Mukoyama,K.Nakao,Y.Saito,Y.Ogawa,K.Hosoda,S.Suga,G.Shirakami,M.Jougasaki,K.Obata,H.Yasue and H.Imura.: "Brain natriuretic peptide is a novel cardiac hormone secreted from the ventricle in humans." Proceedings of the XIth International Congress of Nephrology. (1990)
[文献書誌] T.Fujiwara,H.Fujiwara,G.Takemura,M.Mukoyama,Y.Saito,K.Nakao,H.Imura,M.Nakano,K.Baba.: "Expression and distribution of atrial natriuretic polypeptide in the ventricles of children with myocarditis and/or myocardial infarction secondary to Kawasaki disease: Immunohistochemical study." Am.Heart J.120. 612-618 (1990)
[文献書誌] Y.Kambayashi,K.Nakao,H.Imura,T.Kawabata,M.Nakamura,K.Inouye,N.Yoshida and H.Imura.: "Biological characterization of human brain natriuretic peptide (BNP) and rat BNP: speciesーspecific actions of BNP." Biochem.Biophys.Res.Commun.173. 599-605 (1990)
[文献書誌] K.Obata,H.Yasue,K.Okumura,K.Matsuyama,H.Ogawa,M.Kurose,Y.Saito,K.Nakao,H.Imura and M.Nobuyoshi.: "Atrial natriuretic polypeptide is removed by the lungs and released into the atrium as well as the right atrium in humans." J.Am.Coll.Cardiol.15. 1537-1543 (1990)
[文献書誌] Y.Kambayashi,K.Nakao,M.Mukoyama,Y.Saito,Y.Ogawa,S.Shiono,K.Inouye,N.Yoshida and H.Imura.: "Isolation and sequence determination of humand brain natriuretic peptide in human atrium." FEBS Lett.259. 341-345 (1990)
[文献書誌] K.Nishimura,T.Ban,Y.Saito,K.Nakao,H.Imura.: "Atrial pacing stimulates secretion of atrial natriuretic polypeptide without elevation of atrial pressure in awake dogs with experimental complete atrioventricular block." Circ.Res.66. 115-122 (1990)
[文献書誌] K.Nakao,H.Itoh,Y.Kambayashi,K.Hosoda,Y.Saito,T.Yamada,M.Mukoyama,H.Arai,G.Shirakami,S.Suga,M.Jougasaki,Y.Ogawa,K.Inouye and H.Imura.: "Rat brain natriuretic peptideーisolation from rat heart and tissue distributionー." Hypertension. 15. 774-778 (1990)
[文献書誌] Y.Ogawa,K.Nakao,M.Mukoyama,G.Shirakami,H.Itoh,K.Hosoda,Y.Saito,H.Arai,S.Suga,M.Jougasaki,T.Yamada,Y.Kambayashi,K.Inouye,H.Imura: "Rat brain natriuretic peptideーTissue distribution and molecular formー." Endocrinology. 126. 2225-2227 (1990)
[文献書誌] M.Mukoyama,K.Nakao,Y.Saito,Y.Ogawa,K.Hosoda,S.Suga,G.Shirakami,M.Jougasaki and H.Imura.: "Human brain natriuretic peptide,a novel cardiac hormone." Lancet. 335. 801-802 (1990)
[文献書誌] T.Maeda,M.Niwa,K.Shigematsu,M.Kurihara,Y.Kataoka,K.Nakao,H.Imura,H.Matsuo,H.Tsuchiyama,M.Ozaki.: "Specific [ ^<125>I]brain natriuretic peptideー26 binding sites in rat and pig kidney." Eur.J.Pharmacol.176. 341-350 (1990)
[文献書誌] Y.Saito,K.Nakao,M.Mukoyama and H.Imura.: "Increased plasma endothelin level in patients with essential hypertension." N.Engl.J.Med.322. 205 (1990)
[文献書誌] Y.Saito,K.Nakao,M.Mukoyama,G.Shirakami,H.Itoh,T.Yamada,H.Arai,K.Hosoda,S.Suga,M.Jougasaki,Y.Ogawa,S.Nakajima,M.Ueda,H.Imura: "Application of monoclonal antibodies for endothelin to hypertensive research." Hypertension. 15. 734-738 (1990)
[文献書誌] K.Nishimura,Y.Saito,T.Hidaka,T.Ishihara,K.Nakao,H.Imura,Y.Okamoto and T.Ban.: "Does atrial appendectomy aggravate secretory function of atrial natriuretic polypeptide?" J.Thoracic and Cardiovascular Surgery. (1990)
[文献書誌] M.Jougasaki,H.Yasue,M.Mukoyama,K.Nakao and K.Takahashi.: "Appearance of atrial natriuretic peptide in the ventricles in patients with myocardial infarction." Am.Heart J.119. 92-96 (1990)
[文献書誌] H.Itoh,K.Nakao,M.Mukoyama,Y.Saito,T.Yamada,G.Shirakami,H.Arai,K.Hosoda,S.Suga,I.Yoshida and H.Imura.: "Preparation of monoclonal antibodies against brain natriuretic peptide and their application to radioimmunoassay and passive immunization." Endocrinology. 127. 1292-1300 (1990)
[文献書誌] M.Mukoyama,K.Nakao,Y.Saito,Y.Ogawa,K.Hosoda,S.Suga,G.Shirakami,M.Jougasaki and H.Imura.: "Increased human brain natriuretic peptide in congestive heart failure." N.Engl.J.Med.323. 757-758 (1990)
[文献書誌] H.Itoh,K.Nakao,M.Mukoyama,H.Arai,T.Yamada,Y.Saito,S.Shiono,K.Hosoda,G.Shirakami,S.Suga and H.Imura.: "Pathophysiological role of augmented atrial natriuretic polypeptide (ANP) gene expression in DOCAーsalt hypertensionーEffects of ANP monoclonal antibody." Am.J.Hypertension. (1991)
[文献書誌] M.Mukoyama,K.Nakao,K.Hosoda,S.Suga,Y.Saito,Y.Ogawa,G.Shirakami,M.Jougasaki,K.Obata,H.Yasue,Y.Kambayashi,K.Inouye,H.Imura: "Brain natriuretic peptide (BNP) as a novel cardiac hormone in humansーEvidence for an exquisite dual natriuretic peptide system,atrial natriuretic peptide and brain natriuretic peptide" J.Clin.Invest.(1991)
[文献書誌] K.Hosoda,K.Nakao,M.Mukoyama,Y.Saito,M.Jougasaki,G.Shirakami,S.Suga,Y.Ogawa,H.Yasue and H.Imura.: "Expression of brain natriuretic peptide gene in human heartーProduction in ventricleー." Hypertension. (1991)
[文献書誌] M.Yoshibayashi,K.Nishioka,K.Nakao,Y.Saito,S.Temma,M.Matsumura,T.Ueda,G.Shirakami,H.Imura,H.Mikawa: "Plasma endothelin levels in healthy children: high values in early infancy." J.Cardiovas.Pharmacol.(1991)
[文献書誌] G.Shirakami,K.Nakao,Y.Saito,T.Magaribuchi,M.Jougasaki,M.Mukoyama,H.Arai,K.Hosoda,S.Suga,Y.Ogawa,T.Yamada,K.Mori and H.Imura.: "Acute pulmonary alveolar hypoxia increases lung and plasma endothelinー1 levels in conscious rats." Life Sci.48. 969-976 (1991)
[文献書誌] Y.Saito,K.Nakao,G.Shirakami,M.Mukoyama,H.Arai,K.Hosoda,S.Suga,Y.Ogawa and H.Imura.: "Endothelinー1 (ETー1) in patients with cronic renal failure." J.Cardiovasc.Pharmacol.(1991)
[文献書誌] O.Nakagawa,K.Nakao,Y.Saito,G.Shirakami,M.Jougasaki,M.Mukoyama,K.Hosoda,S.Suga,Y.Ogawa,I.Kishimoto,Y.Komatsu,N.Hama,H.Imura: "Isolation and characterization of porcine endothelinーlike peptide" J.Cardiovasc.Pharmacol.in press. (1991)
[文献書誌] M.Hasegawa,N.Sagawa,Y.Ihara,A.Okagaki,X.M.Li,K.Inamori,H.Itoh,T.Mori,Y.Saito,G.Shirakami,K.Nakao and H.Imura.: "The levels of endothelin in the human amniotic fluid at various stages of pregnancy." J.Cardiovasc.Pharmacol.in press. (1991)
[文献書誌] Y.Ihara,N.Sagawa,M.Hasegawa,A.Okagaki,X.M.Li,K.Inamori,H.Itoh,T.Mori,Y.Saito,G.Shirakami,K.Nakao,H.Imura: "The levels of endothelin in the material blood and cord blood at various stage of pregnancy." J.Cardiovasc.Pharmacol.in press. (1991)

1990 年度 実績報告書

モノクロ-ナル抗体を用いた低分子生理活性ペプチドの超高感度測定法の開発及び実用化

研究代表者

中尾 一和 京都大学, 医学部, 講師 (00172263)

研究成果

[文献書誌] G.Takemura,H.Fujiwara,H.Yoshida,M.Mukoyama,Y.Saito,K.Nakao,T.Fujiwara,T.Uegaito,H.Imura and C.Kawai: "Identification and distribution of atrial natriuretic polypeptide in ventricular myocardium of humans with myocardical infarction." J.Pathology. 161. 285-292 (1990)

[文献書誌] G.Shirakami,K.Nakao,Y.Saito,T.Magaribuchi,H.Nagata,M.Jougasaki,M.Mukoyama,H.Arai,K.Hosoda,S.Suga,Y.Ogawa,K.Mori,H.Imura.: "Endothelin in human cerebrospinal fluid." Therapeutic Research. 11. 3913-3918 (1990)

[文献書誌] M.Mukoyama,K.Nakao,Y.Saito,Y.Ogawa,K.Hosoda,S.Suga,G.Shirakami,M.Jougasaki,K.Obata,H.Yasue and H.Imura.: "Brain natriuretic peptide is a novel cardiac hormone secreted from the ventricle in humans." Proceedings of the XIth International Congress of Nephrology. (1990)

[文献書誌] Y.Kambayashi,K.Nakao,H.Imura,T.Kawabata,M.Nakamura,K.Inouye,N.Yoshida and H.Imura.: "Biological characterization of human brain natriuretic peptide (BNP) and rat BNP: speciesーspecific actions of BNP." Biochem.Biophys.Res.Commun.173. 599-605 (1990)

[文献書誌] K.Obata,H.Yasue,K.Okumura,K.Matsuyama,H.Ogawa,M.Kurose,Y.Saito,K.Nakao,H.Imura and M.Nobuyoshi.: "Atrial natriuretic polypeptide is removed by the lungs and released into the atrium as well as the right atrium in humans." J.Am.Coll.Cardiol.15. 1537-1543 (1990)

[文献書誌] Y.Kambayashi,K.Nakao,M.Mukoyama,Y.Saito,Y.Ogawa,S.Shiono,K.Inouye,N.Yoshida and H.Imura.: "Isolation and sequence determination of humand brain natriuretic peptide in human atrium." FEBS Lett.259. 341-345 (1990)

[文献書誌] K.Nishimura,T.Ban,Y.Saito,K.Nakao,H.Imura.: "Atrial pacing stimulates secretion of atrial natriuretic polypeptide without elevation of atrial pressure in awake dogs with experimental complete atrioventricular block." Circ.Res.66. 115-122 (1990)

[文献書誌] K.Nakao,H.Itoh,Y.Kambayashi,K.Hosoda,Y.Saito,T.Yamada,M.Mukoyama,H.Arai,G.Shirakami,S.Suga,M.Jougasaki,Y.Ogawa,K.Inouye and H.Imura.: "Rat brain natriuretic peptideーisolation from rat heart and tissue distributionー." Hypertension. 15. 774-778 (1990)

[文献書誌] Y.Ogawa,K.Nakao,M.Mukoyama,G.Shirakami,H.Itoh,K.Hosoda,Y.Saito,H.Arai,S.Suga,M.Jougasaki,T.Yamada,Y.Kambayashi,K.Inouye,H.Imura: "Rat brain natriuretic peptideーTissue distribution and molecular formー." Endocrinology. 126. 2225-2227 (1990)

[文献書誌] M.Mukoyama,K.Nakao,Y.Saito,Y.Ogawa,K.Hosoda,S.Suga,G.Shirakami,M.Jougasaki and H.Imura.: "Human brain natriuretic peptide,a novel cardiac hormone." Lancet. 335. 801-802 (1990)

[文献書誌] T.Maeda,M.Niwa,K.Shigematsu,M.Kurihara,Y.Kataoka,K.Nakao,H.Imura,H.Matsuo,H.Tsuchiyama,M.Ozaki.: "Specific [ ^<125>I]brain natriuretic peptideー26 binding sites in rat and pig kidney." Eur.J.Pharmacol.176. 341-350 (1990)

[文献書誌] Y.Saito,K.Nakao,M.Mukoyama and H.Imura.: "Increased plasma endothelin level in patients with essential hypertension." N.Engl.J.Med.322. 205 (1990)

[文献書誌] Y.Saito,K.Nakao,M.Mukoyama,G.Shirakami,H.Itoh,T.Yamada,H.Arai,K.Hosoda,S.Suga,M.Jougasaki,Y.Ogawa,S.Nakajima,M.Ueda,H.Imura: "Application of monoclonal antibodies for endothelin to hypertensive research." Hypertension. 15. 734-738 (1990)

[文献書誌] K.Nishimura,Y.Saito,T.Hidaka,T.Ishihara,K.Nakao,H.Imura,Y.Okamoto and T.Ban.: "Does atrial appendectomy aggravate secretory function of atrial natriuretic polypeptide?" J.Thoracic and Cardiovascular Surgery. (1990)

[文献書誌] M.Jougasaki,H.Yasue,M.Mukoyama,K.Nakao and K.Takahashi.: "Appearance of atrial natriuretic peptide in the ventricles in patients with myocardial infarction." Am.Heart J.119. 92-96 (1990)

[文献書誌] H.Itoh,K.Nakao,M.Mukoyama,Y.Saito,T.Yamada,G.Shirakami,H.Arai,K.Hosoda,S.Suga,I.Yoshida and H.Imura.: "Preparation of monoclonal antibodies against brain natriuretic peptide and their application to radioimmunoassay and passive immunization." Endocrinology. 127. 1292-1300 (1990)

[文献書誌] M.Mukoyama,K.Nakao,Y.Saito,Y.Ogawa,K.Hosoda,S.Suga,G.Shirakami,M.Jougasaki and H.Imura.: "Increased human brain natriuretic peptide in congestive heart failure." N.Engl.J.Med.323. 757-758 (1990)

[文献書誌] H.Itoh,K.Nakao,M.Mukoyama,H.Arai,T.Yamada,Y.Saito,S.Shiono,K.Hosoda,G.Shirakami,S.Suga and H.Imura.: "Pathophysiological role of augmented atrial natriuretic polypeptide (ANP) gene expression in DOCAーsalt hypertensionーEffects of ANP monoclonal antibody." Am.J.Hypertension. (1991)

[文献書誌] K.Hosoda,K.Nakao,M.Mukoyama,Y.Saito,M.Jougasaki,G.Shirakami,S.Suga,Y.Ogawa,H.Yasue and H.Imura.: "Expression of brain natriuretic peptide gene in human heartーProduction in ventricleー." Hypertension. (1991)

[文献書誌] M.Yoshibayashi,K.Nishioka,K.Nakao,Y.Saito,S.Temma,M.Matsumura,T.Ueda,G.Shirakami,H.Imura,H.Mikawa: "Plasma endothelin levels in healthy children: high values in early infancy." J.Cardiovas.Pharmacol.(1991)

[文献書誌] G.Shirakami,K.Nakao,Y.Saito,T.Magaribuchi,M.Jougasaki,M.Mukoyama,H.Arai,K.Hosoda,S.Suga,Y.Ogawa,T.Yamada,K.Mori and H.Imura.: "Acute pulmonary alveolar hypoxia increases lung and plasma endothelinー1 levels in conscious rats." Life Sci.48. 969-976 (1991)

[文献書誌] Y.Saito,K.Nakao,G.Shirakami,M.Mukoyama,H.Arai,K.Hosoda,S.Suga,Y.Ogawa and H.Imura.: "Endothelinー1 (ETー1) in patients with cronic renal failure." J.Cardiovasc.Pharmacol.(1991)

[文献書誌] O.Nakagawa,K.Nakao,Y.Saito,G.Shirakami,M.Jougasaki,M.Mukoyama,K.Hosoda,S.Suga,Y.Ogawa,I.Kishimoto,Y.Komatsu,N.Hama,H.Imura: "Isolation and characterization of porcine endothelinーlike peptide" J.Cardiovasc.Pharmacol.in press. (1991)

[文献書誌] M.Hasegawa,N.Sagawa,Y.Ihara,A.Okagaki,X.M.Li,K.Inamori,H.Itoh,T.Mori,Y.Saito,G.Shirakami,K.Nakao and H.Imura.: "The levels of endothelin in the human amniotic fluid at various stages of pregnancy." J.Cardiovasc.Pharmacol.in press. (1991)

[文献書誌] Y.Ihara,N.Sagawa,M.Hasegawa,A.Okagaki,X.M.Li,K.Inamori,H.Itoh,T.Mori,Y.Saito,G.Shirakami,K.Nakao,H.Imura: "The levels of endothelin in the material blood and cord blood at various stage of pregnancy." J.Cardiovasc.Pharmacol.in press. (1991)

1990 年度実績報告書

中尾一和京都大学, 医学部, 講師 (00172263)