Identification of proteins that specifically recognize and bind protofibrillar aggregates of amyloid-β

Protofibrils of the 42 amino acids long amyloid-β peptide are transient pre-fibrillar intermediates in the process of peptide aggregation into amyloid plaques and are thought to play a critical role in the pathology of Alzheimer’s disease. Hence, there is a need for research reagents and potential diagnostic reagents for detection and imaging of such aggregates. Here we describe an in vitro selection of Affibody molecules that bind to protofibrils of Aβ(42)cc, which is a stable engineered mimic of wild type Aβ(42) protofibrils. Several binders were identified that bind Aβ(42)cc protofibrils with low nanomolar affinities, and which also recognize wild type Aβ(42) protofibrils. Dimeric head-to-tail fusion proteins with subnanomolar binding affinities, and very slow dissociation off-rates, were also constructed. A mapping of the chemical properties of the side chains onto the Affibody scaffold surface reveals three distinct adjacent surface areas of positively charged surface, nonpolar surface and a polar surface, which presumably match a corresponding surface epitope on the protofibrils. The results demonstrate that the engineered Aβ(42)cc is a suitable antigen for directed evolution of affinity reagents with specificity for wild type Aβ(42) protofibrils.