Structural differences of amyloid-beta fibrils revealed by antibodies from phage display
Background: Beside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer's disease (AD) being composed of aggregated fibrils of beta-amyloid (A beta). During the underlying fibrillogenic pathway, starting from a surplus of soluble A beta and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of beta-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 A beta. Results: Here we report the generation and characterization of a variety of amyloid-beta specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from A beta 42 peptides. These antigens were used for the generation of two A beta 42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naive human phage display libraries resulted in multiple unique binders specific for amyloid-beta. Three of the obtained antibodies target the N-terminal part of A beta 42 although with varying epitopes, while another scFv binds to the a-helical central region of the peptide. The affinities of the antibodies to various A beta 42 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. Conclusion: We demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti beta-amyloid scFvs that are capable of accurately discriminating between minute conformational differences.