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，其中以基底脑中的表达水平最高。这一发现提示，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。后者的退化构成了阿尔茨海默病的主要特征之一，并可能是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。这一主要原因似乎是在大多数组织中缺乏这些因子的丰度，以及缺乏可靠地检测包括NGF在内的这些因子的蛋白水平的方法。因此，本研究的目的是建立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、特异性的酶免疫分析系统，并应用这些所开发的…主要研究结果如下：(1)制备了抗重组人神经生长因子抗体Ig G，并对其免疫学性质进行了表征。免疫扩散分析表明该抗体可与某些动物NGF特别是牛NGF反应。(2)以生物素-链霉亲和素系统为基础，建立了灵敏的人NGF双位点EIA体系。NGF浓度低至0.02pg/孔(相当于8×10~(-19)摩尔)，重复性好。有了这种EIA，其他哺乳动物NGF的检测下限与它们与人NGF氨基酸序列同源性的降低程度同步降低。(3)当使用100等份样品时，人血浆中NGF的浓度为5-10pg/ml。(4)用人神经营养因子(NT)-3检测该系统的特异性。(5)人成纤维细胞(14S-L)在培养过程中能合成和分泌人神经生长因子，且其合成/分泌受生长时相依赖的调节。较少

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