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Studies on stabilization of a human-mouse heterohybridoma producing sperm immobilizing antibody by recombinant DNA technology.

Research Project

Project/Area Number 62570773
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Obstetrics and gynecology
Research InstitutionHyogo College of Medicine

Principal Investigator

ISOJIMA Shinzo  Hyogo College of Medicine, 医学部, 教授 (90068403)

Co-Investigator(Kenkyū-buntansha) YAMASAKI Noriyuki  Hyogo College of Medicine, 医学部, 助手 (50174644)
SHIGETA Minoru  Hyogo College of Medicine, 医学部, 講師 (80122315)
KOYAMA Koji  Hyogo College of Medicine, 医学部, 助教授 (00068496)
Project Period (FY) 1987 – 1988
Project Status Completed (Fiscal Year 1988)
Budget Amount *help
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1988: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1987: ¥1,400,000 (Direct Cost: ¥1,400,000)
KeywordsSperm immobilizing antibody / human-mouse heterohybridoma / immunoglobulin gene / recombinant DNA / heavy chain class-switch / キメラ遺伝子 / クラススイッチ / 形質 転換細胞腫 / ハムスター試験 / 受精阻害作用 / ヒトーマウスヘテロハイブリドーマ / ヒト型モノクローナル抗体 / 形質転換細胞腫 / 形質転換細胞株
Research Abstract

For stabilizing the gene coding for sperm immobilizing monoclonal antibody (IgM, lambda from a human-mouse hetero-hybridoma (H6-3C4), the human rearranged immunoglobulin mu-chain genes were cloned from the hybridoma. The cloned V region of the heavy chain (VH) gene was ligated to human immunoglibulin gamma1-heavy chain constant region (C gamma 1) genes. This resulted in the heavy-chain class-switched from mu-chain to gamma 1-chain of H6-3C4 asntibody.The class-switched heavy-chain gene as well as the cloned lambda-chain gene were introduced into mouse myeloma cell line X63Ag8.653 by protoplast fusion and electroporation respectively.Five stable transformants were established. The culture supernatants were electrophoresed on a SDS-PAGE and the proteins were transblotted to the nitrocellulose membranes. Followed by staining with peroxidase conjugated anti-human gamma-chain or lambda-chain antibodies. Under non-reducing condition, all transformants secreted the complete form of human IgG molecules with a molecular weight of 160Kd. Under reduciht condition, the 54Kd band of human gamma-chain and 25Kd band of human lambda-chain were observed. The titers of sperm immobilizing antibody of the culture supernatants of the transformants were in the range of 10-20 units in SI50 titer.The amounts of monoclonal antibodies secreted from the transformants were about 0.2-0.5umg/ml. The SI50 titers and IgM concentrations in the supernatant of H6-3C4 hybridoma were about 5,000 units and 2.0mug/ml, respectively. If we considered the lower complement-dependent sperm immobilizing activity of IgG1 as compared to IgM, the affinity of the recombinant antibody appeared to be the same as that of the original H6-3C4 monoclonal antibody. Thus we succeeded in establishing the stable transformants producing human IgG1 which fully retained the specificity of the original sperm immobilizng human monoclonal IgM.

Report

(3 results)
  • 1988 Annual Research Report   Final Research Report Summary
  • 1987 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Shinzo Isojima,et al.: J.Reprod.Immunol.10. 67-78 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] 山崎則行 他: 産婦進歩. 39. 567 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] Shinji Komori,et al.: Clin.Exp.Immunol. 71. 508-516 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] Shinzo Isojima: Am.J.Reprod .Immunol.Microbiol.17. 150-155 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] Shinzo Isojima, et al.: "Establishment and characterization of a human hybridoma secreting monoclonal antibody with high titers os sperm immobilizing and agglutinating activities against human seminal plasma." J. Reprod. Immunol.10. 67-78 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] Shinji Komori et al.: "Production of heavy-chain class-switch variants of human monolconal antibody by recombinant DNA technology." Clin. Exp. Immunol.71. 508-516 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] Shinzo isojima: "Recent advances in defining human seminal plasma antigens using monoclonal antibodies." Am. J. Reprod. Immunol. Microbiol.17. 150-155 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] Shinji Komori,et al.: Clin.Exp.Immunol.71. 508-516 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Shinzo Isojima: Am.J.Reprod.Immunol.Microbiol.17. 150-155 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Isojima.S.et al: J.Reprod.Immunol. 10. 67-78 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] 山崎 則行,他: 産婦進歩. 39. 567 (1987)

    • Related Report
      1987 Annual Research Report
  • [Publications] Komori,S.et al: Clin.exp.Immunol.

    • Related Report
      1987 Annual Research Report

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Published: 1987-04-01   Modified: 2016-04-21  

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