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PRODUCTION OF RECOMBINANT HUMAN ANTI-HLA ANTIBODIES

Research Project

Project/Area Number 13672432
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Laboratory medicine
Research InstitutionTokai University

Principal Investigator

IHARA Seiji  Tokai University School of Medicine, Associate Professor, 医学部, 助教授 (50096202)

Co-Investigator(Kenkyū-buntansha) TAKEKOSHI Masataka  Tokai University, School of Medicine, Assistant Professor, 医学部, 講師 (80221373)
Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
KeywordsHLA / B51 / DR9 / recombinant human antibody / Fab / ハイブリドーマ
Research Abstract

We are addressing to produce recombinant human antibodies. The aim of this study is the production of human anti-HLA antibodies. In HLA-typing analysis performed in organ transplantation, paternity test, and analysis of diseases, human anti-HLA serum are used, but the cross reaction occurs in some percentage because human sera are not monoclonal. In addition, since the most sera are obtained from multigravida, it is difficult to prepare all sets of sera which show high specificity and affinity characteristics, and also identify rare case of HLA.
In this study, we aimed to produce recombinant anti-HLA class I B51 and class II DR9 antibodies using PCR primers and expression vector constructed for phage display system.
Memods were as follows: Firstly, B cells isolated from peripheral blood which were donated from B51 and DR9 antibody positive individuals were immortalized by EBV infection. Secondly, in the case of B51, immortalized cells were fused with hetero-myeloma cells and established anti-B51 antibody producing cell lines. In the case of DR9, antibody producing cells were isolated from immortalized cells by EBV infection. RNA was extracted from antibody producing cella and Fab regions of antibodies were PCR amplified. Amplified DNA fragments were cloned to expression vector. In these manners, antibody genes were preserved. Finally, antibodies were expressed and specificities of antibodies were analyzed.
DNA sequencing analysis of L and H chains and Western blot analysis of expressed Fab antibody revealed that anti B51antibody was IgG/λα and DR9 was IgG/κ. Recombinant anti B51 Fab antibody was expressed and purified, and used for FACScan and AHG-LCT analyses. B51 antibody reacted specifically for B51 positive cells, but not for negative cells. The cloning, expression and purification of anti DR9 antibody were completed and analysis of its specificities was under investigation.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (15 results)

All Other

All Publications (15 results)

  • [Publications] Takekoshi M, Maeda F, Nagatsuka Y, Aotsuka S, Ono Y, Ihara S.: "Cloning and expression of human anti-tumor necrosis factor-a monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries"J. Biochemistry. 130. 299-303 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Zhou YH, Takekoshi M, Maeda F, Ihara S, Esumi M.: "Rrecombinant antibody Fab against the hypervariable region I of hepatitis C virus Blocks the virus adsorption to susceptible cells in vitro"Antiviral Research. 56. 51-59 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 井原征治: "抗体医薬によるウイルス感染症の予防と治療"BO(バイオベンチャー). 2(4). 75-80 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 井原征治, 城戸勲, 前田史子, 坂本朋昭, 竹腰正隆: "B型肝炎ウイルス表面抗原に対する人工ヒト抗体の作製"人工血液. 10(4). 116-119 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 竹腰正隆: "医学に役立つHP"臨床外科. 57. 1175-1185 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] ZhouYH, Takekoshi M, Maeda F, Ihara S, Esumi M.: "Recombinant antibody Fab against the hypervariable region I of hepatitis C virus. Blocks the virus adsorption to susceptible cells in vitro"Antiviral Research. 56. 51-59 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Takekoshi M, Maeda F, Nagatsuka Y, Aotsuka S, Ono Y, and Ihara S: "Cloning and expression of human anti-tumor necrosis factor-a monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries"J. Biochemistry. 130. 299-303 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ihara S.: "Prophylaxis and Therapy of Virus Infection by Antibody Medicine"BO(Bioventure). 2, No.4. 75-80 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ihara S., Kido K., Maeda F., Sakamoto T., and Takekoshi M.: "The Production of Recombinant Antibodies against Hepatitis B virus Surface Antigen"Artificial Blood. 10, No4. 116-119 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Takekoshi M.: "Useful Homepage for Medical Doctor"Journal of Clinical Surgery. 57. 1175-1185 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Zhou YH, Takekoshi M, Maeda F, Ihara S, Esumi M.: "Rrecombinant antibody Fab against the hypervariable region I of hepatitis C virus. Blocks the virus adsorption to susceptible cells in vitro"Antiviral Research. 56. 51-59 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 井原征治: "抗体医薬によるウイルス感染症の予防と治療"BO(バイオベンチャー). 2巻4号. 75-80 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 井原征治, 城戸勲, 前田史子, 坂本朋昭, 竹腰正隆: "B型肝炎ウイルス表面抗原に対する人工ヒト抗体の作製"人工血液. 10巻・4号. 116-119 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 竹腰正隆: "医学に役立つHP"臨床外科. 57. 1175-1185 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Takekoshi M, Maeda F, Nagatsuka Y, Aotsuka S, Ono Y, Ihara S: "Cloning and expression of human anti-tumor necrosis factor-α monoclonal antibodies from Epstein-Barr virus transformed oligoclonal libraries"J.Biochemistry. 130. 299-303 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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