文摘
The process of amyloid-尾 (A尾) fibril formation is genetically and pathologically linked to Alzheimer鈥檚 disease (AD). Thus, a selective and sensitive method for quantifying A尾 fibrils in complex biological samples allows a variety of hypotheses to be tested. Herein, we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding-competent A尾 aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate, and in mouse brain homogenate. Sonicated, proteinase K-treated A尾 fibril-containing tissue homogenates or cell culture media were added to an initially monomeric A尾1鈭?0 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 A尾 aggregates present in the biological sample. An ion-exchange resin amyloid isolation strategy from complex biological samples is demonstrated as an alternative for improving the sensitivity and linearity of the kinetic aggregation assay.