An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds

R.S. Boshuizen, J.P.M. Langeveld, M. Salmona, A. Williams, R.H. Meloen, J.P. Langendijk

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    23 Citations (Scopus)


    Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2 mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Aß(1¿42), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10¿400 ¿M. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106¿126
    Original languageEnglish
    Pages (from-to)372-380
    JournalAnalytical Biochemistry
    Issue number2
    Publication statusPublished - 2004


    • stained amyloid fibrils
    • congo red
    • thioflavine-t
    • branched polyamines
    • alzheimers-disease
    • molecular-cloning
    • dextran sulfate
    • cultured-cells
    • scrapie
    • prp


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