Grant-in-Aid for Developmental Scientific Research (B).
|Research Institution||Gifu Pharmaceutical University|
HAYASHI Kyozo Gifu Pharmaceutical University Pharmacy Professor, 薬学部, 教授 (00029935)
平谷 一 日本ケミカルリサーチ株式会社, 研究所, 取締役
FURUKAWA Yoshiko Gifu Pharmaceutical University Pharmacy, Associate, 薬学部, 助手 (20219108)
HIRANO Kazuyuki Gifu Pharmaceutical University Pharmacy, Professor, 薬学部, 教授 (90057365)
FURUKAWA Shoei Gifu Pharmaceutical University Pharmacy, Associate Professor, 薬学部, 助教授 (90159129)
西谷 裕 国立療養所, 宇多野病院, 院長
NISHITANI Hiroshi Utano National Hospital, President
HIRATANI Hajime JCR Pharmaceutics Co., Ltd. Biotechnology Research Laboratories, Head
|Project Fiscal Year
1990 – 1991
Completed(Fiscal Year 1991)
|Budget Amount *help
¥15,300,000 (Direct Cost : ¥15,300,000)
Fiscal Year 1991 : ¥7,200,000 (Direct Cost : ¥7,200,000)
Fiscal Year 1990 : ¥8,100,000 (Direct Cost : ¥8,100,000)
|Keywords||Alzheimer's disease / cholinergic nerve cells / degeneration / diagnosis / enzyme immunoassay (EIA) / human fibroblast (WS-1) cells / nerve growth factor (NGF) / anti-recombinant human NGF antibody IgG / 老人痴呆 / 神経栄養因子(NTF) / 神経成長因子(NGF) / 塩基性線維芽細胞増殖因子(bFGF) / 酵素免疫測定法(EIA) / 血中レベル / 測定感度 / コリン作動性ニュ-ロン / 酵素免疫測定法 / ヒト線維芽細胞 / 抗ヒトNGF抗体 / ヒト神経成長因子 / 線維芽細胞増殖因子 / 抗ヒト神経成長因子抗体 / 診断 / 開発|
Nerve growth factor(NGF)receptor MRNA has been found to be widely distributed throughout the human central nervous system with the highest levels in the basal f orbrain. This finding suggests that NGF may have function as a retrograde trophic messenger for basal forbrain magnocellular cholinergic nerve cells. The degeneration of the latter constitutes one of the main features of Alzheimer's disease and may be responsible for some of the impairment that characterized this disease. However, little is known about the relationship between Alzheimer's disease and the NGF and other neurotrophic factor levels. This main reason seems to be lack of abundance of these factors in most tissues, and lack of a method to reliably detect the protein levels of these factors including NGF.
Purpose of this research was thus to develop the highly sensitive, specific enzyme immunoassay(EIA)systems for various neurotrophic factors related to the pathogenesis of Alzheimer's disease and to apply these develope
d EIA systems for the diagnosis of the Alzheimer's disease.
Mfajor results are summarized as follows ;
(1)we prepared anti-recombinant human NGF antibody IgG and characterized its property immunologically. This antibody IgG reacted with some animal NGFS, especially with bovine NGF, on immunodiffusion analysis.
(2)Using this antibody IgG, we developed a sensitive two-site EIA system for human NGF, based on the biotin-streptoavidin system. NGF at a concentration as low as 0.02 pg/well(corresponding to 8xlO-19 mol)could be measured with high reproducibility. With this EIA, the detection limit of other, mammalian NGFs was reduced in parallel with the degree of decrease in amino acid seauence homology between them and human NGF.
(3)The NGF in human plasma was found to be 5-10 pg/ml when a 100 mul aliquot of the sample was used.
(4)The specificity of this system was examined using human neurotrophic factor(NT)-3. NT-3 did not show the crossreactivity in this system until the concentration of 10 ng/MI.
(5)Human fibroblast(14S-l)cells were found to synthesize and secrete human NGF in culture and the synthesis/secretion-was regulated in a growth phase-dependent manner.
Preparation of a kit for the measurement of human NGF level in the sera of the patients with Alzheimer's disease. Less