Journal
JOURNAL OF NEUROSCIENCE METHODS
Volume 157, Issue 1, Pages 71-81Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jneumeth.2006.03.023
Keywords
abeta; amyloid-beta; Alzheimer's disease; brain homogenate; ELISA; nonspecific binding; solid-phase extraction
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In the process of developing species-specific, immunosorbent assays for brain amyloid-beta (A beta) in non-transgenic animals, we have demonstrated an artifact that impedes accurate quantitation of A beta 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 A beta assays. However, extracts of guinea pig brain (human A beta sequence) or rat brain (rodent A beta 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 A beta from brain extracts. This isolation method eliminates non-specific binding components from brain extracts and allows for accurate quantitation and robust detection of multiple A beta peptides in extracts from wild-type animals. (c) 2006 Elsevier B.V. All rights reserved.
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