2003 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, 医学部, 助教授 (90218248) 小林典裕神戸薬科大学, 薬学部, 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 / 抗体 / 遺伝子クローニング / 相補性決定部 / CDRシャッフリング
Research Abstract	環境中女性ホルモン様物質の影響を精査するために、エストラジオール(E_2)のハイスループットスクリーニング法が切望され、高親和力で特異的な抗体が求められている。野生型抗体の相補性決定部(CDR)にランダム変異を導入することにより高性能な改変抗体の創製が可能と期待される。そこで、error-prone PCRにより得られる変異CDR遺伝子断片を野生型抗体のフレームワーク(FR)遺伝子にランダムに組み込む"CDRシャッフリング"に基づく変異抗E_2抗体の創製を企てた。先に樹立した抗E_2抗体産生ハイブリドーマ株よりRNAを単離し、RT-PCRによりH鎖及びL鎖可変部(V_H,V_L)遺伝子をクローニングした。これらを連結して一本鎖Fvフラグメント(scFv)の遺伝子を構築し、その産物である野生型scFvを用いて競合ELISAを行った結果、遊離E_2添加量2-200ngにおいて用量作用曲線が得られた。本scFv遺伝子を鋳型として0.5又は1.0mM MnCl_2存在下でerror-prone PCRを行い、変異クローン27種のV_H及びV_L塩基配列を解析したところ、最大13%の高い効率で点変異の導入が認められた。その一方で、V_H、V_L各ドメインに対応する遺伝子の全長をそれぞれ6本の一本鎖合成オリゴDNA(5'側から#1〜#6;およそ60〜120merの範囲)に分解し,これをrecursive PCRに付すことで元のscFv遺伝子が再構成されることを確認した。得られた変異scFv遺伝子群のCDR部分をPCRにより増幅することで、変異CDR遺伝子断片群(#2、3、5に相当)を調製することができる。これらを野生型FR遺伝子断片(#1、#4、#6に相当)と混合してrecursive PCRに付すことで得られるCDRシャッフリングライブラリーから、親和力と特異性に優れる変異抗体の探索が可能と期待される。

Research Products

(23 results)

All Other

All Publications (23 results)

[Publications] Nariyasu Mano: "Inhibition of the rat hepatic microsomal flurbiprofen acyl glucuronidation by bile acids"Journal of Pharmaceutical Sciences. 92. 2098-2108 (2003)
[Publications] Takashi Iida: "^1H and ^<13>C NMR signal assignments of carboxyl-linked glucuronides of bile acids"Magnetic Resonance Chemistry. 41. 250-264 (2003)
[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α,6- and 3α,24-dihydroxy-5β-cholane derivatives by dimethyldioxirane"Lipids. 38. 281-287 (2003)
[Publications] Nariyasu Mano: "Synthesis and characterization of deoxycholyl 2-deoxyglucuronide : A water-soluble affinity labeling reagent"Lipids. 38. 873-879 (2003)
[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] Naoto Suzuki: "Determination of urinary 12(S)-hydroxyeicosatetraenoic acid by liquid chromatography/tandem mass spectrometry with column-switching technique : Sex difference in healthy voluntees and patients with diabetes mellitus"Journal of Chromatography B. 783. 383-389 (2003)
[Publications] Lilian Rumi Tsuruta: "Characterization of 11-dehydro-thromboxane B_2 recombinant antibody obtained by phage display technology"Prostaglandins, Leukotrienes & Essential Fatty Acids. 68. 273-284 (2003)
[Publications] Naoto Nakagawa: "Brain and heart specific alteration of methamphetamine(MAP) distribution in MAP-senstized rat"Biological Pharmaceutical Bulletin. 26. 506-509 (2003)
[Publications] Takehiko Suzuki: "Identification and characterization of novel rat and human gonad specific organic anion transporters"Molecular Endocrinology. 17. 1203-1215 (2003)
[Publications] Takashi Iida: "An improved method for the capillary gas chromatographic derivatization of polyhydroxylated steroids having tert-hydroxyl groups"Analytical Sciences. 19. 1317-1321 (2003)
[Publications] Takashi Iida: "A comparative study of remote-oxyfunctionalization of unactivated carbons in 5β-steroids by dimethyldioxirane and 2,6-dichloropyridine N-oxide/Ruthenium porphyrin/HBr"Arkivoc. 2003. 170-179 (2003)
[Publications] Norihiro Kobayashi: "Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody agains 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay"Journal of Immunological Methods. 274. 63-75 (2003)
[Publications] Sookie La: "Simultaneous chiral discrimination of multiple profens by cyclodextrin-modifeid capillary electrophoesis in normal and reversed polarity modes"Electrophoresis. 24. 2642-2649 (2003)
[Publications] Nariyasu Mano: "Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption mass spectrometry"Analytical Sciences. 19. 1469-1472 (2003)
[Publications] Mami Kida: "Vectorial transport of bile acids in immortalized mouse bile duct cells"Hepatology Research. 27. 151-157 (2003)
[Publications] Nariyasu Mano: "Presence of protein-bound unconjugated bile acids in the cytoplasmic fraction of rat brain"Journal of Lipids Research. 45. 295-300 (2004)
[Publications] Nariyasu Mano: "Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using novel affinity labeling reagent, acyl adenylate"Biochemistry. 43. 2041-2048 (2004)
[Publications] Genta Kakiyama: "Chemical synthesis of (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid and its taurine and glycine conjugates : a major bile acid in the rat"Journal of Lipid Research. 45. 567-573 (2004)
[Publications] Tsuyoshi Mikkaichi: "Isolation and characterization of a novel human kidney-specific digoxin transporter and its rat homologue"Proceedings of National Academy of Sciences in USA. in press. (2004)
[Publications] Shoujiro Ogawa: "Remote-oxyfunction of unactivated carbons in (5β)-3-oxobile acids by 2,6-dichloropyridine N-oxide catalyzed by ruthenium-porphyrin and HBr : direct lactonization at C-20"Organic Biomolecular Chemistry. in press. (2004)
[Publications] Naoto Suzuki: "Quantitative liquid chromatography-tandem mass spectrometric analysis of 11-dehydro TXB_2 in the urine"Prostagrandins & Other Lipid Metabolites. in press. (2004)
[Publications] Hiroki Tsukamoto: "Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production in peroxisome proliferator-activated receptor γ-independent manner"Prostagrandins & Other Lipid Metabolites. in press. (2004)
[Publications] Mitsuobu Okuyama: "Enzyme-linked immunosorbent assay for monitoring toxic digoxin congeners in milk based on a newly generated monoclonal anti-digoxin"Analytical Chemistry. in press. (2004)

2003 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: "Inhibition of the rat hepatic microsomal flurbiprofen acyl glucuronidation by bile acids"Journal of Pharmaceutical Sciences. 92. 2098-2108 (2003)

[Publications] Takashi Iida: "^1H and ^<13>C NMR signal assignments of carboxyl-linked glucuronides of bile acids"Magnetic Resonance Chemistry. 41. 250-264 (2003)

[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α,6- and 3α,24-dihydroxy-5β-cholane derivatives by dimethyldioxirane"Lipids. 38. 281-287 (2003)

[Publications] Nariyasu Mano: "Synthesis and characterization of deoxycholyl 2-deoxyglucuronide : A water-soluble affinity labeling reagent"Lipids. 38. 873-879 (2003)

[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] Naoto Suzuki: "Determination of urinary 12(S)-hydroxyeicosatetraenoic acid by liquid chromatography/tandem mass spectrometry with column-switching technique : Sex difference in healthy voluntees and patients with diabetes mellitus"Journal of Chromatography B. 783. 383-389 (2003)

[Publications] Lilian Rumi Tsuruta: "Characterization of 11-dehydro-thromboxane B_2 recombinant antibody obtained by phage display technology"Prostaglandins, Leukotrienes & Essential Fatty Acids. 68. 273-284 (2003)

[Publications] Naoto Nakagawa: "Brain and heart specific alteration of methamphetamine(MAP) distribution in MAP-senstized rat"Biological Pharmaceutical Bulletin. 26. 506-509 (2003)

[Publications] Takehiko Suzuki: "Identification and characterization of novel rat and human gonad specific organic anion transporters"Molecular Endocrinology. 17. 1203-1215 (2003)

[Publications] Takashi Iida: "An improved method for the capillary gas chromatographic derivatization of polyhydroxylated steroids having tert-hydroxyl groups"Analytical Sciences. 19. 1317-1321 (2003)

[Publications] Takashi Iida: "A comparative study of remote-oxyfunctionalization of unactivated carbons in 5β-steroids by dimethyldioxirane and 2,6-dichloropyridine N-oxide/Ruthenium porphyrin/HBr"Arkivoc. 2003. 170-179 (2003)

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

[Publications] Sookie La: "Simultaneous chiral discrimination of multiple profens by cyclodextrin-modifeid capillary electrophoesis in normal and reversed polarity modes"Electrophoresis. 24. 2642-2649 (2003)

[Publications] Nariyasu Mano: "Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption mass spectrometry"Analytical Sciences. 19. 1469-1472 (2003)

[Publications] Mami Kida: "Vectorial transport of bile acids in immortalized mouse bile duct cells"Hepatology Research. 27. 151-157 (2003)

[Publications] Nariyasu Mano: "Presence of protein-bound unconjugated bile acids in the cytoplasmic fraction of rat brain"Journal of Lipids Research. 45. 295-300 (2004)

[Publications] Nariyasu Mano: "Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using novel affinity labeling reagent, acyl adenylate"Biochemistry. 43. 2041-2048 (2004)

[Publications] Genta Kakiyama: "Chemical synthesis of (22E)-3α,6β,7β-trihydroxy-5β-chol-22-en-24-oic acid and its taurine and glycine conjugates : a major bile acid in the rat"Journal of Lipid Research. 45. 567-573 (2004)

[Publications] Tsuyoshi Mikkaichi: "Isolation and characterization of a novel human kidney-specific digoxin transporter and its rat homologue"Proceedings of National Academy of Sciences in USA. in press. (2004)

[Publications] Shoujiro Ogawa: "Remote-oxyfunction of unactivated carbons in (5β)-3-oxobile acids by 2,6-dichloropyridine N-oxide catalyzed by ruthenium-porphyrin and HBr : direct lactonization at C-20"Organic Biomolecular Chemistry. in press. (2004)

[Publications] Naoto Suzuki: "Quantitative liquid chromatography-tandem mass spectrometric analysis of 11-dehydro TXB_2 in the urine"Prostagrandins & Other Lipid Metabolites. in press. (2004)

[Publications] Hiroki Tsukamoto: "Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production in peroxisome proliferator-activated receptor γ-independent manner"Prostagrandins & Other Lipid Metabolites. in press. (2004)

[Publications] Mitsuobu Okuyama: "Enzyme-linked immunosorbent assay for monitoring toxic digoxin congeners in milk based on a newly generated monoclonal anti-digoxin"Analytical Chemistry. in press. (2004)

後藤順一東北大学, 医学部附属病院, 教授 (80006337)