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The process of amyloid-β (Aβ) fibril formation is genetically and pathologically linked to
Alzheimer's disease (AD). Thus, a selective and sensitive method for the quantification of Aβ
amyloid fibrils in complex biological samples enables a variety of hypotheses to be tested. Herein
we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding competent
Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate and
in mouse brain homogenate. Sonicated, proteinase K treated Aβ-fibril-containing tissue
homogenates or cell culture media were added to an initially monomeric Aβ1–40 reporter peptide
to seed an in vitro nucleated aggregation reaction. The reduction in the half time (t50) of the
amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present
in the biological sample. An ion exchange resin amyloid isolation strategy from complex
biological samples is demonstrated as an alternative to improve the sensitivity and linearity of the
kinetic aggregation assay.
Alzheimer's disease (AD). Thus, a selective and sensitive method for the quantification of Aβ
amyloid fibrils in complex biological samples enables a variety of hypotheses to be tested. Herein
we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding competent
Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate and
in mouse brain homogenate. Sonicated, proteinase K treated Aβ-fibril-containing tissue
homogenates or cell culture media were added to an initially monomeric Aβ1–40 reporter peptide
to seed an in vitro nucleated aggregation reaction. The reduction in the half time (t50) of the
amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present
in the biological sample. An ion exchange resin amyloid isolation strategy from complex
biological samples is demonstrated as an alternative to improve the sensitivity and linearity of the
kinetic aggregation assay.
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