Ammonium hydroxide treatment of A beta produces an aggregate free solution suitable for biophysical and cell culture characterization

Alzheimer's disease is the leading cause of dementia in the elderly. Pathologically it is characterized by the presence of amyloid plaques and neuronal loss within the brain tissue of affected individuals. It is now widely hypothesised that fibrillar structures represent an inert structure. Biophysical and toxicity assays attempting to characterize the formation of both the fibrillar and the intermediate oligomeric structures of Aβ typically involves preparing samples which are largely monomeric; the most common method by which this is achieved is to use the fluorinated organic solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Recent evidence has suggested that this method is not 100% effective in producing an aggregate free solution. We show, using dynamic light scattering, size exclusion chromatography and small angle X-ray scattering that this is indeed the case, with HFIP pretreated Aβ peptide solutions displaying an increased proportion of oligomeric and aggregated material and an increased propensity to aggregate. Furthermore we show that an alternative technique, involving treatment with strong alkali results in a much more homogenous solution that is largely monomeric. These techniques for solubilising and controlling the oligomeric state of Aβ are valuable starting points for future biophysical and toxicity assays

Central amyloid-beta-specific single chain variable fragment ameliorates A-beta aggregation and neurotoxicity

Anti-amyloid-β immunotherapies are a promising therapeutic approach for the treatment and prevention of Alzheimer's disease (AD). Single chain antibody fragments (scFv) are an attractive alternative to whole antibodies due to their small size, single polypeptide format and inability to stimulate potentially undesirable Fc-mediated immune effector functions. We have generated the scFv derivative of anti-Aβ monoclonal antibody, 1E8, known to target residues 17-22 of Aβ. Here we show that the soluble 1E8 scFv binds to the central region of Aβ with an affinity of ∼55 nM and significantly reduces fibril formation of Aβ. Furthermore, 1E8 scFv ameliorates Aβ-mediated toxicity in the PC12 cell line and murine primary neuronal cultures. This ability to both target the central region of Aβ and prevent Aβ neurotoxicity in vitro makes it a promising therapeutic antibody building block for further functionalization, toward the treatment of AD