Aβ Conformation Dependent Antibodies and Alzheimer's Disease

Sammanfattning: Soluble intermediates of the amyloid-β (Aβ) aggregation process are suggested to play a central role in the pathogenesis of Alzheimer’s disease (AD) by causing synaptic dysfunction and neuronal loss. In this thesis, soluble Aβ aggregates have been studied with a particular focus on the Aβ protofibril, which has served as the antigen for developing conformation dependent monoclonal antibodies.Antibodies generated from mice immunized with Aβ protofibrils were characterized regarding Aβ binding properties and the amino acid sequences of their antigen binding sites. A conformation dependent IgG antibody, mAb158, was further characterized and found to bind to Aβ protofibrils with a 200-fold higher affinity than to monomeric Aβ without affinity for soluble amyloid-β precursor protein (AβPP) or other amyloidogenic proteins. A sandwich enzyme-linked immunosorbent assay (ELISA) based on mAb158 was used to measure soluble Aβ protofibrils in brain extracts from AβPP-transgenic mice. Low levels of protofibrils could also be detected in human AD brain. However, positive signals generated from measurements in AD and control CSF samples were attributed to interference from heterophilic antibodies (HA), generating false positive signals by cross-binding the assay antibodies; consequently, a study on HA interference in Aβ oligomer ELISAs was initiated. A large set of plasma and CSF samples from AD and non-AD subjects were analyzed with and without measures taken to block HA interference, revealing that virtually all signals above the assay limit of detection were false and generated by HA interference.Many types of soluble Aβ aggregates have been described and suggested to impair neuron and synapse function. To investigate the soluble Aβ pool, synthetic Aβ and brain extracts from AβPP-transgenic mice and AD patients were ultracentrifuged on a density gradient to separate Aβ by size under native conditions. Four distinct gradient fractions were defined based on the appearance of synthetic Aβ in atomic force microscopy (AFM) and immunoreactivity in our protofibril specific sandwich ELISA. Interestingly, most Aβ from AD patients and AβPP-transgenic mice separated in the same fraction as toxic synthetic protofibrils.