A Kinetic Aggregation Assay Allowing Selective and Sensitive Amyloid-尾 Quantification in Cells and Tissues

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• 作者：Deguo Du ; Amber N. Murray ; Ehud Cohen ; Hyun-Eui Kim ; Ryan Simkovsky ; Andrew Dillin ; Jeffery W. Kelly
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：March 15, 2011
• 年：2011
• 卷：50
• 期：10
• 页码：1607-1617
• 全文大小：1127K
• 年卷期：v.50,no.10(March 15, 2011)
• ISSN：1520-4995

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.