人脑
固相萃取
抗体
啮齿动物
生物
色谱法
化学
分子生物学
萃取(化学)
免疫学
生态学
神经科学
作者
Thomas A. Lanz,Joel B. Schachter
标识
DOI:10.1016/j.jneumeth.2006.03.023
摘要
In the process of developing species-specific, immunosorbent assays for brain amyloid-β (Aβ) in non-transgenic animals, we have demonstrated an artifact that impedes accurate quantitation of Aβ in this assay format. Using synthetic peptides, cerebrospinal fluid (CSF), or plasma samples, no nonspecific binding or cross-species immunoreactivity was detected in human or rodent Aβ assays. However, extracts of guinea pig brain (human Aβ sequence) or rat brain (rodent Aβ sequence) demonstrated immunoreactivity regardless of which capture antibody, detection antibody, or reporter method (colorimetric or fluorescent) was used. This immunoreactivity remained even in the absence of a capture antibody. Various blocking conditions failed to resolve the nonspecific binding of detection antibodies in the presence of brain extracts. Fractionation of DEA-extracted guinea pig brain over Sephadex G-50 demonstrated the feasibility of separating specific from nonspecific binding components in the brain extracts. Thus, a solid phase extraction method, compatible with multiple extraction buffers, has been developed to isolate and concentrate Aβ from brain extracts. This isolation method eliminates non-specific binding components from brain extracts and allows for accurate quantitation and robust detection of multiple Aβ peptides in extracts from wild-type animals.
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