Analysis of anticoagulants for blood-based quantitation of amyloid β oligomers in the sFIDA assay

Abstract

Early diagnostics at the preclinical stage of Alzheimer's disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aβ oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aβ oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aβ particles in blood plasma. For this purpose, EDTA-, citrate- and heparin-treated blood plasma samples from five individual donors were spiked with Aβ coated silica nanoparticles (Aβ-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aβ oligomers in sFIDA assay with a limit of detection of 16 fM.

Aβ oligomer concentration in mouse and human brain and its drug-induced reduction ex vivo

“…It achieves single aggregate particle sensitivity by combining the biochemical principle of a sandwich-ELISA with the readout of fluorescence intensity per pixel as obtained from fluorescence microscopy. Originally developed for the detection of prion protein aggregates, 22 sFIDA has been adapted for the quantitation of Aβ oligomers in cerebrospinal fluid (CSF) 23 and blood 24 and is in further development as a general tool for quantitating all possible protein aggregates. 25 sFIDA is specific for aggregates by using capture and detection antibodies that recognize overlapping or identical epitopes of the aggregated protein of interest.…”

Aβ Oligomer Elimination Restores Cognition in Transgenic Alzheimer’s Mice with Full-blown Pathology

“…To develop an experiment that allows the investigation of target engagement in vivo, we decided to use brain homogenates without enrichment steps for human Aβ, as this could potentially lead to the destruction of native Aβ oligomers and also to the formation of artificial Aβ aggregates formed during Aβ precipitation steps. Because the brain homogenates contain not only Aβ, but also all other brain-derived components, we used the ultra-sensitive and specific sFIDA assay [26, 29–31] for the quantification of Aβ oligomers in the DGC-fractionated brain homogenates ex vivo. The most significant reduction in Aβ containing particles by RD2 treatment was observed in fraction 10, which corresponds to particles with a molecular weight of larger than 400 kDa [14].…”

“…In contrast to conventional ELISAs, however, the readout is obtained by taking the fluorescence micrographs from the glass surface using total internal reflection (TIRF) microscopy, which then yields single-particle sensitivity. The absolute Aβ oligomer concentrations are then calculated on the basis of a suitable calibration standard [26–31].…”

Quantitative detection of α-Synuclein and Tau oligomers and other aggregates by digital single particle counting

“…The biochemical principle of the sFIDA assay was previously described by Kravchenko et al, and Herrmann et al 11 , 49 . In the present study, we used Nunc MicroWell 384-Well plates (Thermo Fisher Scientific, Waltham, USA) functionalized with 211 and Tau5 antibodies as captures, each at 5 µg/mL in 1 x PBS buffer.…”