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Demonstration of a common artifact in immunosorbent assays of brain extracts: Development of a solid-phase extraction protocol to enable measurement of amyloid-β from wild-type rodent brain
- Source :
- Journal of Neuroscience Methods. 157:71-81
- Publication Year :
- 2006
- Publisher :
- Elsevier BV, 2006.
-
Abstract
- In the process of developing species-specific, immunosorbent assays for brain amyloid-beta (Abeta) in non-transgenic animals, we have demonstrated an artifact that impedes accurate quantitation of Abeta 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 Abeta assays. However, extracts of guinea pig brain (human Abeta sequence) or rat brain (rodent Abeta 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 Abeta from brain extracts. This isolation method eliminates non-specific binding components from brain extracts and allows for accurate quantitation and robust detection of multiple Abeta peptides in extracts from wild-type animals.
- Subjects :
- Male
Amyloid beta
Blotting, Western
Enzyme-Linked Immunosorbent Assay
Rats, Sprague-Dawley
Guinea pig
Cerebrospinal fluid
Species Specificity
Animals
Humans
Solid phase extraction
Binding site
Brain Chemistry
Analysis of Variance
Amyloid beta-Peptides
biology
Tissue Extracts
General Neuroscience
Brain
Dextrans
Molecular biology
Peptide Fragments
Rats
Blot
Sephadex
biology.protein
Binding Sites, Antibody
Antibody
Artifacts
Subjects
Details
- ISSN :
- 01650270
- Volume :
- 157
- Database :
- OpenAIRE
- Journal :
- Journal of Neuroscience Methods
- Accession number :
- edsair.doi.dedup.....8664f76c59447b3f64fc2a2ce3ee48a8