2002 Fiscal Year Annual Research Report

新世代抗体を活用する内分泌撹乱物質のハイスループットスクリーニングシステム

Research Project

Project/Area Number	14042204
Research Institution	Tohoku University
Principal Investigator	後藤順一東北大学, 医学部附属病院, 教授 (80006337)
Co-Investigator(Kenkyū-buntansha)	丹羽俊文東北大学, 医療技術短期大学部, 助教授 (90218248) 小林典裕神戸薬科大学, 教授 (90205477)
Keywords	内分泌撹乱物質 / ハイスループット / スクリーニング / エストラジオール / ハプテン / モノクローナル抗体 / ELISA / 遺伝子クローニング
Research Abstract	河川及び下水中の女性ホルモン様物質による生物への影響を精査するために、エストラジオール(E_2)の簡便で信頼度の高いハイスループットスクリーニング法の確立が切望されている。ELISA法は操作が簡単で検体処理能力が大きいため、新しい日常分析法として応用が試みられている。しかし、従来の抗体は必ずしも十分な特異性を示さない。E_2のようなハプテンに対する特異抗体を得るためには、その分子構造を特徴づける官能基から離れた部位にキャリアタンパクを結合させた免疫原を用いることが重要である。そこで、ステロイド核B環(6,7位)又はC環(11位)上にキャリア結合部位を有するハプテン誘導体を用いて各種系統のマウスを免疫し、抗E_2特異モノクローナル抗体の調製を試みた。その結果、7-carboxyethylthioestradiol(7-CET)のウシ血清アルブミン結合体で免疫したBALB/cマウスの脾細胞に由来する2種の抗体産生クローン(#4-4,#17-5;いずれもγ1,κ)を樹立することができた。これら抗体は、ペルオキシダーゼ標識7-CETを用いるELISAにおいて、およそ10〜1000pgのE_2を定量可能な用量作用曲線を与えた。交差反応性はエストロン(0.21%、0.26%)、エストリオール(2.2%、2.6%)、E_2 17-グルクロニド(<0.005%)、E_2 3-サルフェート(<0.05%)で、実用的な特異性を有していた。また、これら抗体の可変部(V_H及びV_L)遺伝子をクローニングして塩基配列を決定し、サブグループ[V_H;III(D)、 V_L;V]と相補性決定部(CDR)のアミノ酸配列を特定した。これらの知見は、CDRへのランダム変異の導入によりaffinity maturationを試みるうえで極めて有用である。

Research Products

(14 results)

All Other

All Publications (14 results)

[Publications] Nariyasu Mano: "Characterization of rat liver bile acid acyl glucuronosyltransferase"Steroids. 67. 257-262 (2002)
[Publications] Takashi Iida: "Potential bile acid metabolites. 24.An efficient synthesis of carboxyl-linked glucosides"Lipids. 37. 101-110 (2002)
[Publications] Norihiro Kobayashi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"Steroids. 67. 827-833 (2002)
[Publications] Nariyasu Mano: "Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture"Journal of Chromatography B. 776. 125-131 (2002)
[Publications] Norihiro Kobayashi: "Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody : Affinity, specificity, and idiotype analysis"Steroids. 67. 733-742 (2002)
[Publications] Sookie La: "Enantioseparation of chiral aromatic amino acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes"Electrophoresis. 23. 4123-4131 (2002)
[Publications] Takashi Iida: "Potential bile acid metabolites.25.Synthesis and chemical property of steueoisomeric 3α,7α,16-and 3α,7α,15-trihydroxy-5β-cholan-24-oic acids"Chemical and Pharmaceutical Bulletin. 50. 1335-1340 (2002)
[Publications] Takashi Iida: "Direct capillary gas chromatographic analysis and thermal stability of carboxy-linked glucosides of bile acids"Chromatographia. 56. 489-494 (2002)
[Publications] Nariyasu Mano: "Separation and determination of diastereomeric flurbiprofen acyl glucuronides in human urine by LC/ESI-MS with a simple column-switching technique"Drug Metabolism and Pharmacokinetics. 17. 142-149 (2002)
[Publications] Norihiro Kobayashi: "Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate : Application to enzyme-linked immunosorbent assay for urinary and serum cortisol"Analytical Sciences. 18. 1309-1314 (2002)
[Publications] Norihiro Kobayashi: "Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity"Journal of Immunological Methods. 272. 1-10 (2003)
[Publications] Norihiro Kobayashi: "Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay"Journal of Immunological Methods. 274. 63-75 (2003)
[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α,6-and 3α,24-dihydroxy-5β-cholan derivatives by dimetyldioxirane"Lipids. (in press). (2003)
[Publications] Takashi Iida: "^1H and ^13C signal assignments of carboxyl-linked glucosides of bile acids"Magnetic Resonance in Chemistry. (in press). (2003)

2002 Fiscal Year Annual Research Report

新世代抗体を活用する内分泌撹乱物質のハイスループットスクリーニングシステム

Principal Investigator

後藤 順一 東北大学, 医学部附属病院, 教授 (80006337)

Research Products

[Publications] Nariyasu Mano: "Characterization of rat liver bile acid acyl glucuronosyltransferase"Steroids. 67. 257-262 (2002)

[Publications] Takashi Iida: "Potential bile acid metabolites. 24.An efficient synthesis of carboxyl-linked glucosides"Lipids. 37. 101-110 (2002)

[Publications] Norihiro Kobayashi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"Steroids. 67. 827-833 (2002)

[Publications] Nariyasu Mano: "Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture"Journal of Chromatography B. 776. 125-131 (2002)

[Publications] Norihiro Kobayashi: "Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody : Affinity, specificity, and idiotype analysis"Steroids. 67. 733-742 (2002)

[Publications] Sookie La: "Enantioseparation of chiral aromatic amino acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes"Electrophoresis. 23. 4123-4131 (2002)

[Publications] Takashi Iida: "Potential bile acid metabolites.25.Synthesis and chemical property of steueoisomeric 3α,7α,16-and 3α,7α,15-trihydroxy-5β-cholan-24-oic acids"Chemical and Pharmaceutical Bulletin. 50. 1335-1340 (2002)

[Publications] Takashi Iida: "Direct capillary gas chromatographic analysis and thermal stability of carboxy-linked glucosides of bile acids"Chromatographia. 56. 489-494 (2002)

[Publications] Nariyasu Mano: "Separation and determination of diastereomeric flurbiprofen acyl glucuronides in human urine by LC/ESI-MS with a simple column-switching technique"Drug Metabolism and Pharmacokinetics. 17. 142-149 (2002)

[Publications] Norihiro Kobayashi: "Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate : Application to enzyme-linked immunosorbent assay for urinary and serum cortisol"Analytical Sciences. 18. 1309-1314 (2002)

[Publications] Norihiro Kobayashi: "Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity"Journal of Immunological Methods. 272. 1-10 (2003)

[Publications] Norihiro Kobayashi: "Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay"Journal of Immunological Methods. 274. 63-75 (2003)

[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α,6-and 3α,24-dihydroxy-5β-cholan derivatives by dimetyldioxirane"Lipids. (in press). (2003)

[Publications] Takashi Iida: "^1H and ^13C signal assignments of carboxyl-linked glucosides of bile acids"Magnetic Resonance in Chemistry. (in press). (2003)

後藤順一東北大学, 医学部附属病院, 教授 (80006337)