Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease

Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A beta peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A beta protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results: For specific detection of A beta protofibrils we have used a monoclonal antibody, mAb158, selective for A beta protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Ab aggregates in solid- phase reactions, allowing detection of just 0.1 pg/ml A beta protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Ab protofibril detection by up to 25- fold. The assay was used to measure soluble Ab aggregates in brain homogenates from mice transgenic for a human allele predisposing to A beta aggregation. Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of A beta aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.