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Preparation of Monoclonal Antibody against Mutagenic Sugar Chain Which Specifically Occurs in Hepatoma Cells and Its Evaluation for Diagnostics

Research Project

Project/Area Number 10556079
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Applied molecular and cellular biology
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

KUMAGAI Hidehiko  Kyoto Univ. Grad. Sch. Biostudies, Professor, 生命科学研究科, 教授 (70027192)

Co-Investigator(Kenkyū-buntansha) ISHIHARA Hideharu  Gifu Univ. Fac. Eng., Professor, 工学部, 教授 (60021599)
KOUKETSU Mamoru  Gifu Univ. Fac. Eng., Research Associate, 工学部, 助手 (50178208)
YAMAMOTO Kenji  Kyoto Univ. Grad. Sch. Biostudies, Professor, 生命科学研究科, 教授 (70109049)
SAWABU Norio  Kanazawa Univ. Canser Inst., Professor, がん研究所, 教授 (90019969)
FUKUMI Hironobu  Fukui Industrial Univ. Fac. Eng., Professor, 工学部, 教授 (90288340)
Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥11,800,000 (Direct Cost: ¥11,800,000)
Fiscal Year 1999: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥8,600,000 (Direct Cost: ¥8,600,000)
Keywordshepatoma / monoclonal antibody / mutagenic sugar chain / α-fetoprotein / alkylglycoside / endoglycosidase / transglycosylation / トランスフェリン / エンドグリコシダーゼ / 腫瘍マーカー / ガン性変異 / 複合型二本鎖糖鎖
Research Abstract

It is known that the structure of oligosaccharide existed in glycoproteins and cell membrane frequently mutate in cancer cells. It was found that the structure of oligosaccharide in glycoprotein α-fetoprotein from serum was varied in hepatoma patients. Therefore, we intended to develop a simple method to prepare the monoclonal antibody specific for the mutagenic oligosaccharide of glycoprotein. In this study, we synthesized the novel glycoconjugate (alkylglycoside) having oligosaccharide of glycoprotein as immunogen using transglycosylation activity of microbial endoglycosidase (endo-β-N-acetylglucosaminidase), and prepared the monoclonal antibody against asparagine-linked bianntenary complex-type of oligosaccharide.
1. We prepared a large amount of glycopeptide containing bianntenary complex-type of oligosaccharide from hen egg yolk as the donor of ligosaccharide for binding to alkylgucoside.
2. We synthesized alkylglycoside as the immunogen of asparagine-linked bianntenary complex-type … More of oligosaccharide using transglycosylation activity of Mucor endoglycosidase (Endo-M). We incubated egg yolk glycopeptide and n-octyl-β-D-glucoside with Endo-M, pH6.0, at 37℃ for 12hr, and isolated the transglycosylation product by HPLC. The product was confirmed as the alkylglycoside having complex-type of oligosaccharide by mass spectrometry.
3. We immunized mice by the alkylglycoside. The immunogen was injected to mice intraperitonealy four times. After last booster, spleen cells were harvested and mixed with myeloma cells, and fused. Three weeks later, hybridomas grew and the screening of hybridomas producing antibody against oligosaccharide was carried out by ELISA method using human serum tranferrin glycopeptide and egg yolk glycopetide, both of which contained bianntenary complex-type of oligosaccharide. As the result, five hybridoma cell lines were obtained as the producer of antibody. Using these hybridomas, cloning was carried out by limiting dilution method. Then, we obtained three hybridoma clones which produced antibody against bianntenary complex-type of oligosaccharide.
4. We also prepared the hybridoma producing asialo-bianntenary complex-type of oligo-saccharide according to the same method described above. Less

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] K.Yamanoto et al.: "Chemoenzymatic synthesis of a novel glycopeptide using a microbial endoglycosidase"Carbohydr. Res.. 305. 415-422 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Haneda et al.: "Chemo-enzymatic synthesis of a calcitonin derivatives containing Nlinked oligosaccharides"Bioorg. Med. Chem. Let.,. 8. 1303-1306 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 山本憲二: "微生物のエンドグリコシダーゼの活性解析とその応用"蛋白質核酸酵素. 43. 866-874 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y.Motoo et al.: "Expression of pancreatitis-associated protein(PAP) mRNA in gastrointestinal cancers"Int. J. Pancreatol,. 25. 11-16 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S.Akiba et al.: "Transglycosylation activity of the Endo-β-1, 4-Glucanase from Aspergillus niger IFO31125 and its application"J. Biosci. Bioeng.,. 87. 576-580 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y.Motoo et al.: "Serum levels of pancreatitis-associated protein in digestive diseases with special reference to gastrointestinal cancers"Dig. Dis. Sci.,. 44. 1142-1147 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 沢武紀雄: "肝臓病学:Basic Science(戸田剛太郎ほか編)"医学書院. 839-848 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Yamamoto et al.: "Chemoenzymatic synthesis of a novel glycopeptide using a microbial endoglycosidase"Carbohydr. Res.. 305. 415-422 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Haneda et al.: "Chemo-enzymatic synthesis of calcitonin derivatives containing N-linked oligosaccharides"Bioorg. Med. Chem. Let.. 8. 1303-1306 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Yamamoto: "Microbial Endoglycosidases and Their Application"Protein, Nucleic acid, Enzyme. 43. 866-874 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Motoo et al.: "Expression of pancreatitis-associated protein (PAP) mRNA in gastrointestinal cancers"Int. J. Pancreatol. 25. 11-16 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S. Akiba et al.: "Transglycosylation activity of the Endo-β-1, 4-Glucanase from Aspergillus niger IFO31125 and Its application"J. Biosci. Bioeng.. 87. 576-580 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Motoo et al.: "Serum levels of pancreatitis-associated protein in digestive diseases with special reference to gastrointestinal cancers"Dig. Dis. Sci.. 44. 1142-1147 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N. Sawabu: "Studies on hepatic disease, Basic Science (ed. by K. Toda et al.)"Igaku-shoin Co.. 839-848 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S.Akiba et al.: "Transglycosylation Activity of the Endo-β-1,4-Glucanase from Aspergillus niger IFO31125 and Its Application"J.Biosci.Bioeng.. 87・5. 576-580 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] H.Ashida et al.: "Characterization of Endo-α-N-acetylgalactosaminidase from Bacillus sp.And Syntheses.of Neo-oligosaccharides Using Its Trans-glycosylation Activity"Arch.Biochem.Biophys.. 373・2. 394-400 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y.Motoo et al.: "Expression of pancreatics-associated protein(PAP)mRNA in gastrointestinal cancers" Int.J.Pancreatol.25(1). 11-16 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Y.Motoo et al.: "Expression of multidrug resistance gene(mdr-1)mRNA in gastric and colorectal cancers" Anticancer Res.18. 1903-1906 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Yamamoto et al.: "Chemoenzymatic synthesis of a novel glycopeptide using a microbial endoglycosidase" Carbohydr.Res.305. 415-422 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Haneda et al.: "Chemo-enzymatic synthesis of calcitonin derivatives containing N-linked oligosaccharides" Bioorg.Med.Chem.Let.8. 1303-1306 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 沢武 紀雄: "肝臓病学:Basic Science(戸田剛太郎ほか編)" 医学書院, 839-848 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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