Fig. 1

Scheme of sFIDA principle. In sFIDA, capture antibodies directed against a linear epitope on Aβ (Nab228, directed against epitope amino acids 1 − 11) are immobilized on a glass surface, and unoccupied surface area is blocked with bovine serum albumin to reduce unspecific binding events. During sample incubation, monomeric and aggregated Aβ species are bound to the capture antibody. (A) Because sFIDA uses the same or overlapping epitopes for capture and detection, only Aβ aggregates are subsequently detected with fluorescence-labeled antibodies IC16-CF633, which is overlapping with the epitope of the capture antibody (directed against epitope amino acids 2 − 8). (B) For monomeric Aβ, this epitope is already masked by the capture antibody and cannot be bound by the detection antibody. Afterward, the assay surface is imaged by fluorescence microscopy, and pixels above a defined cutoff threshold are counted by image-data analysis (called pixel count). Finally, pixel-based readouts are calibrated into molar particle concentrations using silica nanoparticles (SiNaPs) coated with Aβ as calibration standards. Created with BioRender.com