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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

Detection of Aβ protofibrils using SP-PLA. (A) Assays were performed in buffer (blue diamonds), in 10% CSF (red squares), and in 50% CSF (green triangles). (B) Comparison of SP-PLA (red squares) versus ELISA (blue diamonds) for detection of Aβ protofibrils in 10% CSF. The X-axes show concentrations of Aβ protofibrils in pg/ml and the Y-axes show cycle threshold values for real-time PCR. In B the right Y-axis indicates the OD A450nm for ELISA. The vertical red and blue lines indicate the limit of detection for SP-PLA and ELISA, respectively, calculated as 2-fold standard deviations above the background signals. Error bars indicate standard deviations from the mean for triplicates for each reaction.
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Figure 2: Detection of Aβ protofibrils using SP-PLA. (A) Assays were performed in buffer (blue diamonds), in 10% CSF (red squares), and in 50% CSF (green triangles). (B) Comparison of SP-PLA (red squares) versus ELISA (blue diamonds) for detection of Aβ protofibrils in 10% CSF. The X-axes show concentrations of Aβ protofibrils in pg/ml and the Y-axes show cycle threshold values for real-time PCR. In B the right Y-axis indicates the OD A450nm for ELISA. The vertical red and blue lines indicate the limit of detection for SP-PLA and ELISA, respectively, calculated as 2-fold standard deviations above the background signals. Error bars indicate standard deviations from the mean for triplicates for each reaction.

Mentions: The SP-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 ELISA protein assays [14]. Using the microparticle-based SP-PLA, Aβ was first captured by monoclonal antibodies immobilized on magnetic particles. Thereafter, the Aβ protofibrils were detected using a pair of PLA probes both utilizing the same monoclonal antibody as the capture reagent. This form of the assay thus ensures that only aggregates of Aβ are detected. Figure 2A illustrates the detection of Aβ protofibrils spiked in buffer, and in 10% and 50% CSF. The dynamic range of the assay on synthetic protofibrils extended over more than six orders of magnitude, and the limit of the detection was approximately 0.1 pg/ml for Aβ protofibrils spiked in buffer, and 0.27 pg/ml for Aβ protofibrils spiked in 10% and 50% CSF. These results represent a 4 to 10-fold greater sensitivity than the mAb158 ELISA. Figure 2B illustrates a comparison between SP-PLA and mAb158 ELISA for detection of synthetic Aβ protofibrils spiked in 10% CSF, where SP-PLA demonstrated a 4-fold lower limit of detection and a dynamic range four orders of magnitude greater than that of mAb158 ELISA.


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)

Detection of Aβ protofibrils using SP-PLA. (A) Assays were performed in buffer (blue diamonds), in 10% CSF (red squares), and in 50% CSF (green triangles). (B) Comparison of SP-PLA (red squares) versus ELISA (blue diamonds) for detection of Aβ protofibrils in 10% CSF. The X-axes show concentrations of Aβ protofibrils in pg/ml and the Y-axes show cycle threshold values for real-time PCR. In B the right Y-axis indicates the OD A450nm for ELISA. The vertical red and blue lines indicate the limit of detection for SP-PLA and ELISA, respectively, calculated as 2-fold standard deviations above the background signals. Error bars indicate standard deviations from the mean for triplicates for each reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Detection of Aβ protofibrils using SP-PLA. (A) Assays were performed in buffer (blue diamonds), in 10% CSF (red squares), and in 50% CSF (green triangles). (B) Comparison of SP-PLA (red squares) versus ELISA (blue diamonds) for detection of Aβ protofibrils in 10% CSF. The X-axes show concentrations of Aβ protofibrils in pg/ml and the Y-axes show cycle threshold values for real-time PCR. In B the right Y-axis indicates the OD A450nm for ELISA. The vertical red and blue lines indicate the limit of detection for SP-PLA and ELISA, respectively, calculated as 2-fold standard deviations above the background signals. Error bars indicate standard deviations from the mean for triplicates for each reaction.
Mentions: The SP-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 ELISA protein assays [14]. Using the microparticle-based SP-PLA, Aβ was first captured by monoclonal antibodies immobilized on magnetic particles. Thereafter, the Aβ protofibrils were detected using a pair of PLA probes both utilizing the same monoclonal antibody as the capture reagent. This form of the assay thus ensures that only aggregates of Aβ are detected. Figure 2A illustrates the detection of Aβ protofibrils spiked in buffer, and in 10% and 50% CSF. The dynamic range of the assay on synthetic protofibrils extended over more than six orders of magnitude, and the limit of the detection was approximately 0.1 pg/ml for Aβ protofibrils spiked in buffer, and 0.27 pg/ml for Aβ protofibrils spiked in 10% and 50% CSF. These results represent a 4 to 10-fold greater sensitivity than the mAb158 ELISA. Figure 2B illustrates a comparison between SP-PLA and mAb158 ELISA for detection of synthetic Aβ protofibrils spiked in 10% CSF, where SP-PLA demonstrated a 4-fold lower limit of detection and a dynamic range four orders of magnitude greater than that of mAb158 ELISA.

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