Abstract
BackgroundBeside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer’s disease (AD) being composed of aggregated fibrils of β-amyloid (Aβ). During the underlying fibrillogenic pathway, starting from a surplus of soluble Aβ and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of β-amyloid and to have tools at hand to monitor the distribution of aggregates in the brain or even act as disease modulators, it is essential to develop highly sensitive antibodies that can discriminate between diverse aggregates of Aβ.ResultsHere we report the generation and characterization of a variety of amyloid-β specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from Aβ42 peptides. These antigens were used for the generation of two Aβ42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naïve human phage display libraries resulted in multiple unique binders specific for amyloid-β. Three of the obtained antibodies target the N-terminal part of Aβ42 although with varying epitopes, while another scFv binds to the α-helical central region of the peptide. The affinities of the antibodies to various Aβ42 aggregates as well as their ability to interfere with fibril formation and disaggregation of preformed fibrils were determined. Most significantly, one of the scFv is fibril-specific and can discriminate between two different fibril forms resulting from variations in the acidity of the milieu during fibrillogenesis.ConclusionWe demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti β-amyloid scFvs that are capable of accurately discriminating between minute conformational differences.
Highlights
IntroductionAmyloid plaques are the major histological hallmarks of Alzheimer’s disease (AD) being composed of aggregated fibrils of β-amyloid (Aβ)
Beside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer’s disease (AD) being composed of aggregated fibrils of β-amyloid (Aβ)
Antigen preparation (Aβ42) Fractions of Aβ42 monomers, protofibrils and mature fibrils were prepared from synthetic Aβ42 peptide to serve as antigens
Summary
Amyloid plaques are the major histological hallmarks of Alzheimer’s disease (AD) being composed of aggregated fibrils of β-amyloid (Aβ). Histological hallmarks of AD are neurofibrillary tangles, comprised of hyperphosphorylated tau protein [2,3], and amyloid plaques that are composed of aggregated amyloid-β peptides [4,5,6]. Amyloid-β is regarded as the main culprit causing the neuropathology in AD and is released from the amyloid precursor protein by sequential cleavage with β- and γ-secretases. This processing results in peptides of various amino acids (aa) in length with the majority being 40 aa (90%) and 42 aa (10%) long [7], . Oligomers and protofibrils are widely regarded as the main toxic species in AD the exact nature of the toxic entity - if such a form even exists [18] - has yet to be elucidated [19,20,21,22,23,24,25]
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