Quantification of the Concentration of Aβ42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay

Abstract

The aggregation of the amyloid beta peptide, Aβ42, implicated in Alzheimer's disease, is characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here we exploit the high sensitivity of chemical chain reactions to the reagent composition to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concentration of fibrillar Aβ42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small number of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the number of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quantitative results of the chain reaction assay are confirmed by qualitative transmission electron microscopy analysis. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.

Metal chelation, radical scavenging and inhibition of Aβ42 fibrillation by food constituents in relation to Alzheimer’s disease

“…ThT fluorescence can be used to detect the formation of Aβ fibrils because the fluorescence emission of ThT is enhanced upon binding to amyloid fibrils. accrual curve was observed, in keeping with an initial lag phase of fibril formation mostly via slow primary nucleation and elongation, followed by exponential growth due to an autocatalytic process involving fast fibril-catalysed secondary nucleation and elongation which becomes the main source of new oligomers, and finally an equilibrium plateau phase (Arosio, Cukalevski, Frohm, Knowles, & Linse, 2014). An empirical sigmoidal model was fitted to each of the fluorescence intensity versus time data sets and the amplitude of the sigmoidal curve (Fig.…”

Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication

“…large majority of toxic oligomers (20)(21)(22). Inhibition of secondary nucleation can therefore lead to a dramatic decrease in the total number of oligomers formed during the aggregation process (21)(22)(23)(24).…”

A sensitive and selective electrochemical biosensor for the determination of beta-amyloid oligomer by inhibiting the peptide-triggered in situ assembly of silver nanoparticles

“…22,23 Therefore, the direct detection of Aβ oligomer level would be more reliable for AD diagnosis than assay of its monomer. 24,25 Recently, a few novel biosensors have been developed for the detection of Aβ oligomer, including electrochemistry, [26][27][28][29] surface plasma resonance (SPR), 30 localized surface plasmon resonance (LSPR), 24,31 fluorescence, 32,33 nuclear magnetic resonance, 34 and surface-enhanced Raman spectroscopy. 35 These methods are feasible, but they require the use of special instruments and/or relatively expensive and variable antibodies for the capture and recognition of Aβ oligomer.…”