Analysis of heterogeneous beta A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay
Analysis of heterogeneous beta A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay
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DOI：
10.1074/jbc.271.37.22908
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发表时间：
1996-09-13
影响因子：
4.8
通讯作者：
Beyreuther, K
中科院分区：
文献类型：
--
作者：
Ida, N;Hartmann, T;Beyreuther, K
β A4肽是阿尔茨海默病（AD）脑内老年斑的主要成分，在AD患者和正常人的脑脊液（CSF）和血液中均有发现。虽然β A4 1-40是细胞代谢产生的主要形式，但最近的观察表明，长尾β A4 1-42在AD发病机制中起着更重要的作用。在此，我们建立了针对β A4 1-40和1-42的C-末端的新的单克隆抗体，并将它们用于特异性Western印迹检测。在优化测定条件后，这些抗体检测到低皮克量的β A4，并且CSF中的β A4 1-40和1-42水平都可以通过直接上样样品来测定。β A4 1-40和1-42的泛液水平也通过特异性免疫沉淀，随后通过Western印迹检测来确定。我们发现，与非痴呆对照组相比，AD患者的CSF β A4 1-42水平较低，尽管两组之间存在显著重叠。CSF中β A4 1-40、血浆中β A4 1-40和β A4 1-42的水平在AD患者和对照组之间没有差异。除了4-kDa全长β A4条带外，我们还可以在AD患者和对照的CSF和血浆中检测到β A4的几种N-末端变体。两个N-末端截短的β A4物种在3.3和3.7 kDa的位置迁移被发现在CSF中，而3.7和5 kDa的形式被发现在血浆中。这些不同物质的相对丰度在CSF和血浆中显著不同，表明β A4的细胞来源和/或清除率在这两个隔室中不同。
The beta A4 peptide, a major component of senile plaques in Alzheimer's disease (AD) brain, has been found in cerebrospinal fluid (CSF) and blood of both AD patients and normal subjects, Although beta A4 1-40 is the major form produced by cell metabolism and found in CSF, recent observations suggest that the long-tailed beta A4 1-42 plays a more crucial role in AD pathogenesis. Here, we established new monoclonal antibodies against the C-terminal end of beta A4 1-40 and 1-42, and used them for the specific Western blot detection. After optimizing the assay conditions, these antibodies detected low picogram amount of beta A4, and both beta A4 1-40 and 1-42 levels in CSF could be determined by direct loading of the samples. flood levels of beta A4 1-40 and 1-42 were also determined by specific immunoprecipitation followed by Western blot detection. We found that CSF beta A4 1-42 level is lower in AD patients compared with non-demented controls, although there was a significant overlap between the groups. The level of beta A4 1-40 in CSF, and of beta A4 1-40 as well as beta A4 1-42 in plasma, were not different between AD patients and controls. Besides the 4-kDa full-length beta A4 band, we could also detect several N-terminal variants of beta A4 in CSF and plasma of both AD patients and controls. Two N-terminally truncated beta A4 species migrating at the position of 3.3 and 3.7 kDa were found in CSF, while 3.7- and 5-kDa forms were found in plasma. The relative abundance of these various species were considerably different in the CSF and plasma, suggesting that the cellular source and/or clearance of beta A4 is different in these two compartments.