Limits...
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.

Show MeSH

Related in: MedlinePlus

Detection of endogenous Aβ protofibrils using SP-PLA. Brain homogenates from five transgenic ArcSwe mice expressing elevated levels of protofibrils and five control mice were examined using SP-PLA (A) and the mAb158 ELISA (B). The blue and red rectangles indicate the median for control and transgenic-mice, respectively, revealing a significant difference between the two groups of mice. (C) Results of SP-PLA of brain homogenates from transgenic and non-transgenic mice, spiked in 10% human CSF. Error bars indicate standard deviations from the mean for triplicates for each reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2959092&req=5

Figure 5: Detection of endogenous Aβ protofibrils using SP-PLA. Brain homogenates from five transgenic ArcSwe mice expressing elevated levels of protofibrils and five control mice were examined using SP-PLA (A) and the mAb158 ELISA (B). The blue and red rectangles indicate the median for control and transgenic-mice, respectively, revealing a significant difference between the two groups of mice. (C) Results of SP-PLA of brain homogenates from transgenic and non-transgenic mice, spiked in 10% human CSF. Error bars indicate standard deviations from the mean for triplicates for each reaction.

Mentions: The Aβ protofibrils in the experiments reported so far were all prepared by in vitro aggregation of synthetic Aβ peptides. In order to investigate the ability of the SP-PLA to detect biologically derived soluble Aβ aggregates we used brain homogenates from five APPArc-Swe mice [16], previously shown to have elevated levels of protofibrils [10,11]. In a blind-test, SP-PLA revealed concentrations of soluble Aβ aggregates in brain homogenates from the five transgenic mice, at levels ranging from 11 to 84 pg/ml. The samples from non-transgenic mice displayed background signals at 2 to 4 pg/ml (Figure 5A) and a similar difference was observed using the mAb158 ELISA. We note, however, that in both transgenic mice and controls the concentrations of protofibrils estimated by ELISA by reference to a standard dilution series of the in vitro aggregated form of the peptide were roughly 10-fold higher than those recorded by SP-PLA (Figure 5B). The differences in estimated concentrations in the two assays could be due to the requirement for antibody recognition of three identical determinants in SP-PLA while two determinants are bound in the sandwich ELISA, potentially rendering SP-PLA specific for larger aggregates. When we used a homogenous form of PLA where two recognition events are required to generate detection signals [12,13] the estimated concentrations of Aβ protofibrils - and possibly other lower molecular weight Aβ species, oligomers - in the tested samples were in the same range as those determined by ELISA (data not shown), supporting the notion that the SP-PLA form of the assays is limited to larger soluble Aβ aggregates. To establish the feasibility of demonstrating the presence of Aβ oligomers/protofibrils in human CSF we spiked brain homogenates either from a transgenic or a non-transgenic mouse in 10% human CSF. Figure 5C illustrates successful detection of endogenous Aβ aggregates from the transgenic mouse but not from the non-transgenic mouse after spiking the preparations in human CSF. The results illustrate the potential of the assay to detect low levels of Aβ oligomers in human bodily fluids.


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 endogenous Aβ protofibrils using SP-PLA. Brain homogenates from five transgenic ArcSwe mice expressing elevated levels of protofibrils and five control mice were examined using SP-PLA (A) and the mAb158 ELISA (B). The blue and red rectangles indicate the median for control and transgenic-mice, respectively, revealing a significant difference between the two groups of mice. (C) Results of SP-PLA of brain homogenates from transgenic and non-transgenic mice, spiked in 10% human CSF. 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 5: Detection of endogenous Aβ protofibrils using SP-PLA. Brain homogenates from five transgenic ArcSwe mice expressing elevated levels of protofibrils and five control mice were examined using SP-PLA (A) and the mAb158 ELISA (B). The blue and red rectangles indicate the median for control and transgenic-mice, respectively, revealing a significant difference between the two groups of mice. (C) Results of SP-PLA of brain homogenates from transgenic and non-transgenic mice, spiked in 10% human CSF. Error bars indicate standard deviations from the mean for triplicates for each reaction.
Mentions: The Aβ protofibrils in the experiments reported so far were all prepared by in vitro aggregation of synthetic Aβ peptides. In order to investigate the ability of the SP-PLA to detect biologically derived soluble Aβ aggregates we used brain homogenates from five APPArc-Swe mice [16], previously shown to have elevated levels of protofibrils [10,11]. In a blind-test, SP-PLA revealed concentrations of soluble Aβ aggregates in brain homogenates from the five transgenic mice, at levels ranging from 11 to 84 pg/ml. The samples from non-transgenic mice displayed background signals at 2 to 4 pg/ml (Figure 5A) and a similar difference was observed using the mAb158 ELISA. We note, however, that in both transgenic mice and controls the concentrations of protofibrils estimated by ELISA by reference to a standard dilution series of the in vitro aggregated form of the peptide were roughly 10-fold higher than those recorded by SP-PLA (Figure 5B). The differences in estimated concentrations in the two assays could be due to the requirement for antibody recognition of three identical determinants in SP-PLA while two determinants are bound in the sandwich ELISA, potentially rendering SP-PLA specific for larger aggregates. When we used a homogenous form of PLA where two recognition events are required to generate detection signals [12,13] the estimated concentrations of Aβ protofibrils - and possibly other lower molecular weight Aβ species, oligomers - in the tested samples were in the same range as those determined by ELISA (data not shown), supporting the notion that the SP-PLA form of the assays is limited to larger soluble Aβ aggregates. To establish the feasibility of demonstrating the presence of Aβ oligomers/protofibrils in human CSF we spiked brain homogenates either from a transgenic or a non-transgenic mouse in 10% human CSF. Figure 5C illustrates successful detection of endogenous Aβ aggregates from the transgenic mouse but not from the non-transgenic mouse after spiking the preparations in human CSF. The results illustrate the potential of the assay to detect low levels of Aβ oligomers in human bodily fluids.

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