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Open Access Highly Accessed Methodology article

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

Masood Kamali-Moghaddam1*, Frida Ekholm Pettersson2, Di Wu1, Hillevi Englund2, Spyros Darmanis1, Anna Lord2, Gholamreza Tavoosidana1, Dag Sehlin2, Sigrun Gustafsdottir1, Lars NG Nilsson2, Lars Lannfelt2 and Ulf Landegren1

Author affiliations

1 Department of Genetics and Pathology, Molecular Medicine, Uppsala University, Uppsala, Sweden

2 Department of Public Health and Caring Sciences, Molecular Geriatrics, Uppsala University, Uppsala, Sweden

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Citation and License

BMC Neuroscience 2010, 11:124  doi:10.1186/1471-2202-11-124

Published: 5 October 2010

Abstract

Background

Protein 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.

Results

For 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.

Conclusions

The 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.