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

Kamali-Moghaddam M, Pettersson FE, Wu D, Englund H, Darmanis S, Lord A, Tavoosidana G, Sehlin D, Gustafsdottir S, Nilsson LN, Lannfelt L, Landegren U - BMC Neurosci (2010)

Bottom Line: Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Pathology, Molecular Medicine, Uppsala University, Uppsala, Sweden. masood.kamali@genpat.uu.se

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.

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Related in: MedlinePlus

Schematic illustration of SP-PLA. (a) Capture antibodies are immobilized on a microparticulate solid support followed by (b) capture of target molecules from the biological sample. (c) Next, the beads are incubated with a pair of PLA probes - that is antibodies with attached oligonucleotides - where after excess probes and other reaction components are removed by washes. (d) Next a cocktail of reagents are added for probe ligation guided by a connector oligonucleotide, and for real-time PCR. (e) After a brief ligation step, (f) ligation products representing detected protofibrils are detected and quantified by real-time PCR.
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Figure 1: Schematic illustration of SP-PLA. (a) Capture antibodies are immobilized on a microparticulate solid support followed by (b) capture of target molecules from the biological sample. (c) Next, the beads are incubated with a pair of PLA probes - that is antibodies with attached oligonucleotides - where after excess probes and other reaction components are removed by washes. (d) Next a cocktail of reagents are added for probe ligation guided by a connector oligonucleotide, and for real-time PCR. (e) After a brief ligation step, (f) ligation products representing detected protofibrils are detected and quantified by real-time PCR.

Mentions: We have developed a solid-phase form of PLA (Figure 1) using the mAb158 antibody for sensitive and specific detection of Aβ protofibrils, and we have compared this assay to the previously established sandwich ELISA using the same antibody.


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

Kamali-Moghaddam M, Pettersson FE, Wu D, Englund H, Darmanis S, Lord A, Tavoosidana G, Sehlin D, Gustafsdottir S, Nilsson LN, Lannfelt L, Landegren U - BMC Neurosci (2010)

Schematic illustration of SP-PLA. (a) Capture antibodies are immobilized on a microparticulate solid support followed by (b) capture of target molecules from the biological sample. (c) Next, the beads are incubated with a pair of PLA probes - that is antibodies with attached oligonucleotides - where after excess probes and other reaction components are removed by washes. (d) Next a cocktail of reagents are added for probe ligation guided by a connector oligonucleotide, and for real-time PCR. (e) After a brief ligation step, (f) ligation products representing detected protofibrils are detected and quantified by real-time PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959092&req=5

Figure 1: Schematic illustration of SP-PLA. (a) Capture antibodies are immobilized on a microparticulate solid support followed by (b) capture of target molecules from the biological sample. (c) Next, the beads are incubated with a pair of PLA probes - that is antibodies with attached oligonucleotides - where after excess probes and other reaction components are removed by washes. (d) Next a cocktail of reagents are added for probe ligation guided by a connector oligonucleotide, and for real-time PCR. (e) After a brief ligation step, (f) ligation products representing detected protofibrils are detected and quantified by real-time PCR.
Mentions: We have developed a solid-phase form of PLA (Figure 1) using the mAb158 antibody for sensitive and specific detection of Aβ protofibrils, and we have compared this assay to the previously established sandwich ELISA using the same antibody.

Bottom Line: Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Pathology, Molecular Medicine, Uppsala University, Uppsala, Sweden. masood.kamali@genpat.uu.se

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.

Show MeSH
Related in: MedlinePlus