Abstract
A cell-based assay using homogeneous time-resolved fluorescence has been developed for high throughput screening of putative β-amyloid (Aβ)production inhibitors. In this assay, total Aβ is detected by simply adding two commercially available antibody complexes. The first was a biotinylated monoclonal antibody (4G8), specifically recognizing an epitope comprising the residues 17-24 of the Aβ peptide, complexed with europium cryptate-streptavidin conjugate. The second was a polyclonal antibody (BioS-N), raised against the N-terminus of the Aβ peptide, complexed with an allophycocyanin-anti rabbit antibody conjugate. Binding of the two complexes to the Aβ peptide brought europium cryptate (fluorescence donor) and allophycocyanin (fluorescence acceptor) into close proximity, consequently a fluorescent resonance energy transfer signal was produced upon excitation at 337 nm. The resulting fluorescence signal (665 nm) was then detected using a Discovery™ or a ViewLux™ reader. Detection of Aβ by the proposed method is possible at concentrations of approximately 1 nM. The method was employed for the detection of Aβ secreted from a stable transfected human neuroglioma cell line (H4) overexpressing a mutated form of the human amyloid precursor protein (APP695NL) and developed for robotic automation. At optimized conditions, signal-to-background ratios exceeding 5 and Z factors around 0.7 were achieved in a 384-well format. High throughput screening of 56,913 potential Aβ production inhibitors led to identification of new non-cytotoxic and cell permeable compounds with potencies in the submicromolar range.
Keywords: amyloid inhibitors, high throughput screening, homogenous time-resolved fluorescence
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