1988 Fiscal Year Final Research Report Summary
Studies on stabilization of a human-mouse heterohybridoma producing sperm immobilizing antibody by recombinant DNA technology.
| Project/Area Number |
62570773
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Hyogo College of Medicine |
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Principal Investigator |
ISOJIMA Shinzo Hyogo College of Medicine, 医学部, 教授 (90068403)
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| 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)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Sperm 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 |
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| 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.
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