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

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 本研究的主要结果如下：（1）制备了抗重组人神经生长因子抗体IgG，并对其免疫学特性进行了鉴定。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

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