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1998 Fiscal Year Final Research Report Summary

Selective Production of monoclonal Antibodies Using a Specific Affinity between Avidin and Biotin

Research Project

Project/Area Number 08672506
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biological pharmacy
Research InstitutionMie University

Principal Investigator

TOMITA Masahiro  Mie Univ, Fac.of Eng.Associate Prof., 工学部, 助教授 (20183494)

Co-Investigator(Kenkyū-buntansha) KOBAYASHI Jun  Mie Univ, Fac.of Eng.Research Associate, 工学部, 助手 (70242930)
YOSHIMURA Tetsuro  Mie Univ, Fac.of Eng.Professor, 工学部, 教授 (30035472)
MIYAJIMA Shigetoshi  Mie Univ, Fac.of Eng.Professor, 工学部, 教授 (80239409)
Project Period (FY) 1996 – 1998
Keywordsmonoclonal antibody / pulsed electric field / avidin / biotin / B lymphocyte / myeloma cell / hybridoma
Research Abstract

A new method for the production of monoclonal antibodies were developed. First, an antigen-avidin was prepared to select the B lymphocytes pre-immunized by the antigen. A bifunctional cross-linker, MBS (m-maleimidobenzoyl N-hydroxysuccinimide), was employed for this purpose. Lysine residues in the avidin and cysteine residues in the antigen were covalently linked by use of heterofunctional groups in MBS such as NHS (N-hydroxysuccinimide) and maleimide, respectively. On the one hand, myeloma cells were biotinylated by NHS-biotin. Finally, B lymphocyte-antigen-avidin and biotin-myeloma cell were combined to produce B lymphocyte-antigen-avidin-biotin-mycloma cell complex. This B lymphocyte-myeloma cell complex was then selectively fused by a pulsed electric field (PEF) with a few kVcm^<-1> for a duration in 10mus range. The fusion efficiency of this new method resulted in 20 to 30-fold higher than that obtained by poly(ethylene glycol) method, which is commonly used for the production of monoclonal antibodies.
In the next step, a method for in vitro immunization was employed to further develop this new method. The advantage of the usage of in vitro system is that (I) it can markedly reduce the period for the immunization and the amount of an antigen, and (II) it enables to use the antigens which make adverse effects on in vivo system. After B lymphocytes immunized in vitro system were selected by antigen-biotin, they were combined with biotin-myeloma cells by use of a specific affinity between avidin and biotin. The B lymphocyte-antigen-biotin- streptavidin-biotin-mycloma cell complex was then fused by an electric field. In consequence, we could succeed in obtaining hybridoma cells which can produce monoclonal antibodies against the given antigen using not only in vivo system but also in vitro system with a newly developed PEF method.

  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] Masahiro Tomita: "Selective Production of Monoclonal Antibodies Mediated by B Lymphocyte Receptors" Protein Engineering. 9(9). 819 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masahiro Tomita: "A New Method for the Production of Monoclonal Antibodies" New Technology Japan. 24(3). 15 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masahiro Tomita: "Serological Relationship between Inclusion Body Proteins and a Virus Enhancing Factor of an Entomopoxvirus" Applied Entomology and Zoology. 33(2). 277-280 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masahiro Tomita: "Selective Production of Monoclonal Antibodies Mediated by B Lymphocyte Receptors" Protein Engineering. 9 (9). 819 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masahiro Tomita: "A New Method for the Production of Monoclonal Antibodies" New Technology Japan. 24 (3). 15 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masahiro Tomita: "Serological Relationship between Inclusion Body Proteins and a Virus Enhancing Factor of an Entomopoxvirus" Applied Entomology and Zoology. 33 (2). 277-280 (1998)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-12-08  

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