2000 Fiscal Year Final Research Report Summary
Production of monoclonal antibodies reactive to brain pathologies of Alzheimer's disease by Immunizing-menbraine preparation from the human entorhinal cortex.
| Project/Area Number |
11470060
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Experimental pathology
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| Research Institution | Kyoto Prefectural University of Medicine |
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Principal Investigator |
FUKUYAMA Ryuichi Kyoto Prefectural University of Medicine, Assistant Professor, 医学部, 講師 (60199271)
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| Co-Investigator(Kenkyū-buntansha) |
FUSHIKI Shinji Kyoto Prefectural University of Medicine, Professor, 医学部, 教授 (80150572)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Alzheimer's disease / neuronal cell death / monoclonal antibody / entorhinal cortex / electro cell fusion / immunohistochemistry / myeloma / human brain |
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| Research Abstract |
Applying monoclonal hybridoma techniques, we attempted to isolate monoclonal antibodies (mAbs) which label pathological changes in brains of Alzheimer's disease patients (AD). Crude membrane fractions from human entorhinal and visual cortical areas were used as a combination of desiredand undesiredantigen. Three protocols employed in this study were SOFISTIC (combination of in vivo immunosuppression and in vitro immunization protocols), pSOFISTIC (primedSOFISTIC) and conventional in vitro immunization procedures(cIV). PAI myeloma cells and lymphocytes were fused with polyethylene glycol and in some experiments by electrofusion apparatus (LF101, Gen System Inc.). Paraffin embeddedand formalin fixed human brain sections and freshly frozen rat brain sections were stained immunohistochemically for screening hybridomas. Isotypes of Ig was also determined
In total, more than 4000 hybridoma clones were producedby three protocols. Efficacy in producing hybridomas and their Ig types were all sim
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ilar. Numbers of wells whose culture media positively stained human brain sections were higher in SOFISTIC and pSOFISTIC than in cIV, while those which negatively stained human but positively stained rat brain sections were higher in cIV than in other 2 protocols, indicating that cyclophosphamide preferentially suppresses subsets of lymphocytes activated with common antigen species. From these results, SOFISTIC is to be the best among three protocols. Culture media of hybridomas stained neurons, astroglia, intracellular organellae and neural tracts as well as pathological changes such as corpora amylacea, neurofibrillary tangles, senile plaques and neurons with granulovascular degeneration. One culture medium did not stain CA1 neurons of AD hippocampus. These results indicate that our goal to isolate mAbs that label pathological changes of AD brains appears successful. Currently, we are undertaking subcloning of hybridomas that detectedpathological changes and other interesting morphologies. Less
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