| Project/Area Number |
08672506
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Mie University |
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Principal Investigator |
TOMITA Masahiro Mie Univ, Fac.of Eng.Associate Prof., 工学部, 助教授 (20183494)
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| 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)
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1998: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1997: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1996: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | monoclonal antibody / pulsed electric field / avidin / biotin / B lymphocyte / myeloma cell / hybridoma / 生体外免疫法 / モノクローカル抗体 / 電気融合法 / B細胞選択 / クロスリンカー |
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| 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.
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