Production of monoclonal antibody against human malignant melanoma cell line (HMG) from gingiva
| Project/Area Number |
60480431
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
外科・放射線系歯学
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| Research Institution | Mie University |
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Principal Investigator |
MURATA Mutsuo Mie university, school of medicine, Professor, 医学部, 教授 (80025618)
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| Co-Investigator(Kenkyū-buntansha) |
NOMURA Jouji Mie University hospital, school of medicine, Assistant, 医学部附属病院, 助手 (80172815)
SAITOH Hiroshi Mie University, school of medicine, Assistant, 医学部, 助手 (20135437)
INUI Madoka Mie University hospital, school of medicine, Lecture, 医学部附属病院, 講師 (70159961)
TAGAWA Toshirou Mie University hospital, school of medicine, Lecture, 医学部附属病院, 講師 (30046346)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1986: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1985: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Keywords | Human malignant melanoma / モノクロナール抗体 |
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| Research Abstract |
Monoclonal antibodies(MoAbs) were produced by the cell fusion of mouse splenocytes, which were obtained from the mice immunized with human malignant melanoma cells(HMG) from gingiva, and mouse myeloma cells. In the screening of hybridomas for antibody production, the reactivity of the hybridoma supernatant to HMG cells, which were cultured in a 96-well microplate and fixed by ethanol, was assessed by enzyme immunoassay. Twelve days after the cell fusion, the screening was performed. As a result, positive antibody activities were noted in 54 wells of 576 wells. These hybridomas were immediately subcloned three times by the limiting dilution method using mouse thymocytes. Ultimately, 20 kinds of hybridoma clones which secrete HMG-reactive MoAbs were obtained. The class and subclass of these MoAbs were assessed by Ouchterlony's double immunodiffusion and 4 kinds of IgG1, 1 kind of IgG2a, 6 kinds of IgG3 and 9 kinds of IgM were identified.
Then, the reactivity to HMG tumor, which was formed by inoculation of HMG cells to the nude mouse, was examined immunohistochemically by the ABC method. The reaction products were observed in 14 kinds of MoAbs by the frozen section of various fixation. It seems to be a difference of the antigenic expression between in vitro and in vivo. Moreover, in a formalin-fixed paraffin-embedded section, the reaction products were observed in 11 kinds of MoAbs.
Now we are investigating the specificity of these MoAbs by observation of reactivity of them to various normal and tumor culture cells.
HMG, the antigen in this project, have been growing well for about 9 years above 460 passages and maintain the tumorigenesis and premelanosomes.
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Report
(1 results)
Research Products
(2 results)
