Protocol for quantitative and qualitative analyses of the in vitro aggregation of synthetic β-amyloid -: A method applicable to the identification of substances that may have therapeutic efficacy for Alzheimer's disease

Biochemical studies of beta -amyloid (A beta) aggregation have been hampered by the lack of a simple method to examine simultaneously the extent of aggregation and the structural nature of the aggregates. Consequently, often only the extent of aggregation is analyzed and reported. This means that there is often no knowledge of whether the aggregates consist of fibrils, a structure crucial for their neurotoxicity. Here we describe the development of a protocol for quantitatively: and qualitatively evaluating the in vitro aggregation of synthetic A beta. Specifically, a fluorescein derivatized A beta (1-42) peptide (FITC-A beta (1-42)) was used as the aggregation material. We found that the fluorescent A beta peptide aggregated as efficiently as the native ones and the extent of their aggregation could be determined accurately by fluorescence spectrophotometry. Significantly, our approach is also qualitative because it allows a direct structural examination of A beta fibrils by fluorescence microscopy. In addition. this protocol can also be applied to the identification of substance(s) capable of inhibiting A beta aggregation and further the assessment of the nature of such inhibition. (C) 2000 Elsevier Science B.V. All rights reserved.