Abstract
BackgroundProtein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ 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 Aβ 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.ResultsFor specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ 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 Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ 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 Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.ConclusionsThe proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.
Highlights
Protein aggregation plays important roles in several neurodegenerative disorders
The SP-proximity ligation assay (PLA) has been previously shown to provide a broader dynamic range and a lower limit of detection for a wide range of proteins compared to standard sandwich Enzyme-linked immunosorbent assay (ELISA) protein assays [14]
Thereafter, the Ab protofibrils were detected using a pair of PLA probes both utilizing the same monoclonal antibody as the capture reagent
Summary
Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Ab peptides are cornerstones in the pathology of Alzheimer’s disease. We have established a proximity ligation assay (PLA) for specific and sensitive detection of Ab protofibrils via simultaneous recognition of three identical determinants present in the aggregates. Cerebrospinal fluid (CSF) is often investigated for levels of Ab42, tau and phoshotau in routine diagnostics of AD [1], where decreased Ab42 and increased tau and/or phospho-tau (Thr181P) in CSF are indicative of the disease. These measures are reasonably good predictors of future conversion to AD among subjects with mild cognitive impairment, but they are not suitable to follow disease progression or to monitor drug intervention. We demonstrate that the proximity ligation assay (PLA) can provide even more sensitive detection of synthetic Ab protofibrils
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