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Protein based infections such as prion diseases have lately attracted a large amount of interest, primarily due to the Mad Cow Epidemic in Great Britain, and the increase of Alzheimer's disease and related diseases in the ageing Western society. Infective proteins are very stable and almost untraceable prior to infection making them ideal as biological weapons. Particularly if the used agent is of human origin, the immunoresponse can be avoided, leaving no trace of the infectious agent. The transient nature of infectious oligomeric intermediates of misfolded proteins or peptide fragments that later matures into fibrillar aggregates makes them hard to study, and methods to detect and study these species are sparse. There exist a number of fluorescent probes that bind specifically to protein amyloidic structures. Thioflavins (ThT, ThS), Congo and Nile red, 4-(dicyanovinyl)-julolidine (DCVJ), as well as derivatives amino-8-naphtalene sulphonate (ANS, Bis-ANS) which are known to bind to the fibrillar or pre-fibrillar states with dissociation constants of typically 1 - 20 μM. Here, transthyretin (TTR), insulin and lysozyme were used as model proteins to detect different amyloid precursor states for diseases such as senile systemic amyloidosis, familial amyloidotic polyneuropathy (FAP) and iatrogenic amyloidosis. Specifically, the probes were employed in static assays to characterize protofibrillar and mature amyloid fibrillar states using steady state and time-resolved fluorescence techniques. Particularly, we investigate and report on the possibility to detect protofibrillar states at low concentration levels using modern fluorescence array detector systems in conjunction with lasers operating in the blue or ultraviolett wavelengths as excitation source. Results of ANS, ThT and a ThT analogue (abbreviated ThC) are discussed.
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