Develpment of the Enzyme Immunoassay System for the Substances Related to the Pathogenesis of Alzheimer's Disease

阿尔茨海默病发病相关物质酶联免疫分析系统的研制

• 批准号: 02557037
• 负责人: HAYASHI Kyozo
• 金额: $ 9.79万
• 依托单位: Gifu Pharmaceutical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557037/
• 关键词: 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; 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

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 … More 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

神经生长因子（Nerve growth factor，NGF）受体mRNA广泛分布于人的中枢神经系统，以基底脑含量最高。这一发现表明，NGF可能是基底前脑大细胞胆碱能神经细胞的逆行营养信使。后者的变性构成阿尔茨海默病的主要特征之一，并且可能是导致这种疾病的一些特征性损害的原因。然而，关于阿尔茨海默病与NGF和其他神经营养因子水平之间的关系知之甚少。本研究的目的是建立一种高灵敏度、特异性的神经营养因子酶免疫分析（enzyme immunoassay，EIA）系统，并将其应用于阿尔茨海默病（Alzheimer's disease，AD）神经营养因子的检测。 ...更多信息 d用于阿尔茨海默病诊断的EIA系统。Mfajor结果总结如下：（1）我们制备了抗重组人NGF抗体IgG，并对其免疫学性质进行了表征。免疫扩散分析表明，该抗体IgG与某些动物NGFS，特别是牛NGF反应。（2）利用此抗体IgG，我们建立了一种基于生物素-链亲和素系统的敏感的人NGF双位点酶免疫分析系统。可以以高再现性测量低至0.02 μ g/孔（对应于8 × 10 -19 mol）的浓度的NGF。用这种EIA，其他哺乳动物NGF的检测限随着它们与人NGF之间氨基酸序列同源性的降低程度而降低。（3）当使用100穆尔等分试样的样品时，发现人血浆中的NGF为5-10 pg/ml。（4）用人神经营养因子（NT）-3检测该系统的特异性。NT-3在10 ng/ml浓度时才显示交叉反应性。（5）人成纤维细胞（14 S-1）在体外培养条件下可合成和分泌人神经生长因子，其合成/分泌受生长期的调控。少

项目成果

Junko Miyasaka,Kyozo Hayashi: "Bombesin/GRP Stimulates a Protein Kinase in Swiss 3T3 Cells that Phosphorylates Microtuble-Associated Protein Kinase" Journal of Clinical Biochemistry and Nutrition.

Junko Miyasaka、Kyozo Hayashi：“Bombesin/GRP 刺激瑞士 3T3 细胞中的蛋白激酶，使微管相关蛋白激酶磷酸化”《临床生物化学与营养学杂志》。

Takekazu Ohi,Shoei Furukawa,Kyozo Hayashi,Shigeru Matsukura: "Ganglioside Stimulation of Nerve Growth Factor Synthesis in Cultured Rat Schwann Cells" Biochemistry International. 20. 739-746 (1990)

Takekazu Ohi、Shoei Furukawa、Kyozo Hayashi、Shigeru Matsukura：“神经节苷脂刺激培养的大鼠雪旺细胞中神经生长因子的合成”生物化学国际。

Ryosuke Ikegami, Yoshiko Furukawa, Kyosuke Kaechi, Kyozo Hayashi, and Shoei Furukawa: "Effects of catecholamines and 4-methylcatechol on the synthesis and secretion of nerve growth factors by rat sciatic nerve segments in culture." Biomed. Res.11(1). 61-6

Ryosuke Ikegami、Yoshiko Furukawa、Kyosuke Kaechi、Kyozo Hayashi 和 Shoei Furukawa：“儿茶酚胺和 4-甲基儿茶酚对培养中大鼠坐骨神经节段合成和分泌神经生长因子的影响。”

Tetsuo Adachi, Kazuyuki Hirano, Kyozo Hayashi, Yasutoshi Muto, and Fumitaka Okuno: "A one-step enzyme immunoassay for human manganese superoxide dismutase with monoclonal antibodies." Free Radical Biol. Med.8(1). 25 (1990)

Tetsuo Adachi、Kazuyuki Hirano、Kyozo Hayashi、Yasutoshi Muto 和 Fumitaka Okuno：“一种使用单克隆抗体对人锰超氧化物歧化酶进行一步酶联免疫分析。”

Toshitaka Nabeshima,Shinichi Ogawa,Hirohisa Ishimaru,Tsutomu Kameyama,Taneo Fukuta,Rie Takeuchi,Kyozo Hayashi: "Memory Impairment and Morphological Changes in Rats Induced by Active Fragment of Anti-Nerve Growth Factor-Antibody" Biochemical and Biophysica

Toshitaka Nabeshima、Shinichi Okawa、Hirohisa Ishimaru、Tsutomu Kameyama、Taneo Fukuta、Rie Takeuchi、Kyozo Hayashi：“抗神经生长因子抗体活性片段诱导大鼠记忆损伤和形态变化”生化与生物物理学