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

神经生长因子（NGF）受体MRNA广泛分布于人类中枢神经系统，在基底脑中水平最高。这一发现提示NGF可能作为基底脑大细胞胆碱能神经细胞的逆行营养信使起作用。后者的退化是阿尔茨海默病的主要特征之一，可能是导致这种疾病特征的一些损害的原因。然而，人们对阿尔茨海默病与神经生长因子和其他神经营养因子水平之间的关系知之甚少。其主要原因似乎是这些因子在大多数组织中缺乏丰度，并且缺乏可靠检测这些因子（包括NGF）蛋白水平的方法。因此，本研究的目的是开发与阿尔茨海默病发病机制相关的各种神经营养因子的高灵敏度、特异性酶免疫测定（EIA）系统，并将这些系统应用于阿尔茨海默病的诊断。主要结果总结如下：(1)制备了抗重组人NGF抗体IgG，并对其性质进行了免疫学鉴定。免疫扩散分析表明，该抗体IgG可与部分动物NGFS，特别是与牛NGF发生反应。(2)利用该抗体IgG，在生物素-亲链亲和素系统的基础上，建立了一个敏感的人NGF的双位点EIA系统。NGF浓度低至0.02 pg/孔（相当于8xlO-19 mol），重现性高。通过此EIA，其他哺乳动物NGF的检出限与它们与人类NGF之间氨基酸序列同源性的降低程度平行降低。(3)人血浆中NGF的含量为5 ~ 10 pg/ml。(4)采用人神经营养因子(NT)-3检测该系统的特异性。NT-3在浓度为10 ng/MI时才表现出交叉反应性。(5)培养的人成纤维细胞（14S-l）可合成和分泌人NGF，且合成/分泌受生长阶段依赖性调节。测定阿尔茨海默病患者血清中人神经生长因子水平试剂盒的制备。少

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