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Santos, A. N. et al. Detection of amyloid-β oligomers in human cerebrospinal fluid by flow cytometry and fluorescence resonance energy transfer. J. Alzheimers Dis. 11, 117-125

ACGT ProGenomics AG, Halle, Germany.
Journal of Alzheimer's disease: JAD (Impact Factor: 3.61). 04/2007; 11(1):117-25.
Source: PubMed

ABSTRACT The neuropathology of Alzheimer's disease (AD) has been linked recently to non-fibrillar forms of neurotoxic amyloid-beta (Abeta) oligomers of which high levels are observed in the brain of AD patients. This suggests that Abeta oligomers play a key role in the early events of AD, underlining their potential for the early diagnosis of the disease. We have developed an extremely sensitive assay for the detection of oligomeric and fibrillar structures of Abeta that is based on multiparametric analysis of data obtained by flow cytometry and fluorescence resonance energy transfer (Fret). The assay readily detects Abeta oligomers in human cerebrospinal fluid (CSF) as verified by dot blot of the isolated particles. By measuring 174 CSF samples of non-demented control patients with various neurological disorders a high reliability and reproducibility of the method could be demonstrated.

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Available from: Alexander Navarrete Santos, Aug 13, 2015
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    • "Some of these groups have chosen to adapt the traditional ELISA by using aggregate-specific antibodies [29] [30] [31] [32], or by using a single monoclonal antibody for both antigen capture and detection [29,32–37]. Others groups apply techniques like a bio-barcode assay [38], surfacefluorescence intensity distribution analysis [39] or fluorescence resonance energy transfer in combination with flow cytometry [40]. With the exception of the assays using aggregate-specific antibodies, all these methods still rely on the binding of multiple antibodies and are vulnerable to the effects of epitope masking and steric hindrance. "
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    ABSTRACT: According to the predominant theories, soluble amyloid-beta (Aβ) aggregates are the principal neurotoxic agents in Alzheimer’s disease pathology, making them a popular target for the development of therapeutics and diagnostic markers. One of the most commonly used methods for determining the concentration of Aβ is ELISA. However, ELISA was developed for monomeric proteins and may be ill-suited for detecting aggregates. Therefore, we investigated the effect of aggregation on the ELISA measurement and developed a novel chemical pre-treatment method, designed to disaggregate Aβ peptides, to improve the ELISA measurement of the total Aβ concentration.
    02/2015; 4. DOI:10.1016/j.mex.2015.02.011
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    • "CSF total tau, phosphorylated tau at threonine 181, and A␤ 42 concentrations were determined using ELISA (Innotest hTAU-Ag, INNOTEST ® PHOSPHO-TAU [181P], INNOTEST ® ß-AMYLOID(1-42), Innogenetics, Gent, Belgium) respectively as previously described [24] [25] [26]. Flow cytometric analysis of the blinded samples was done as previously described [17]. "
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    • "CSF total tau, phosphorylated tau at threonine 181, and A␤ 42 concentrations were determined using ELISA (Innotest hTAU-Ag, INNOTEST ® PHOSPHO-TAU [181P], INNOTEST ® ß-AMYLOID(1-42), Innogenetics, Gent, Belgium) respectively as previously described [24] [25] [26]. Flow cytometric analysis of the blinded samples was done as previously described [17]. "
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    ABSTRACT: Oligomers of the amyloid-β peptide (Aβ) are thought to be the most toxic form of Aβ and are linked to the development of Alzheimer's disease (AD). Here, we used a flow cytometric approach for the detection and assessment of oligomers in cerebrospinal fluid (CSF) from AD patients and other neurological disorders. 30 CSF samples from patients suffering from AD (n = 14), non-demented controls (n = 12), and other neurological disorders (dementia with Lewy bodies, n = 2; vascular dementia, n = 1; primary progressive aphasia, n = 1) were analyzed for the presence of Aβ-oligomers by flow cytometry. The CSF levels of total tau (t-tau), phosphorylated tau (p-tau), and amyloid-β (Aβ)42 were determined using ELISA. CSF Aβ-oligomer levels in AD patients were elevated in comparison to the non-AD group (p = 0.073). The ratio Aβ-oligomers/Aβ42 was significantly elevated in AD subjects compared to non-AD subjects (p = 0.001). Most important, there was a negative correlation between the amount of Aβ-oligomers and the Mini-Mental Status Exam score (r = -0.65; p = 0.013) in AD patients. The detection of Aβ-oligomers using flow cytometry analysis seems to be useful in assessing the stage of AD. This is a novel and important finding as none of the currently used CSF biomarkers are clearly associated with dementia severity.
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