Development of Surface-FIDA towards a diagnostic tool for Alzheimer's disease
Development of Surface-FIDA towards a diagnostic tool for Alzheimer's disease
Alzheimer’s disease (AD) is the most common neurodegenerative disorder. Definitive methods for an early and accurate pre-symptomatic diagnosis are not available. One important hallmark of AD is the accumulation of extracellular amyloid plaques in the brain, which predominantly consist of aggregated...
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Personal Name(s): | Wang-Dietrich, Lei (Corresponding author) |
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Contributing Institute: |
Strukturbiochemie; ICS-6 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2012
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Physical Description: |
VI, 103 S : zahlr. Ill. u. graph. Darst |
Dissertation Note: |
Universität Düsseldorf, Diss., 2012 |
ISBN: |
978-3-89336-801-3 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
Addenda |
Series Title: |
Schriften des Forschungszentrums Jülich. Reihe Gesundheit Health
55 |
Subject (ZB): | |
Publikationsportal JuSER |
Alzheimer’s disease (AD) is the most common neurodegenerative disorder. Definitive methods for an early and accurate pre-symptomatic diagnosis are not available. One important hallmark of AD is the accumulation of extracellular amyloid plaques in the brain, which predominantly consist of aggregated amyloid-$\beta$ (A$\beta$) peptides. A$\beta$ soluble oligomers, but not monomers are presumed as the main neurotoxic species in AD pathology, and are thus considered to be important biomarkers, which mirror fundamental pathogenic events in AD. A method for sensitive and specific detection and quantification of A$\beta$ oligomers in body fluids, such as cerebrospinal fluid (CSF), is highly desired. In the present work, a method called surface-fluorescence intensity distribution analysis (Surface-FIDA) with single aggregate sensitivity was adopted and optimized for the detection of A$\beta$ aggregates. To enhance the signal-to-noise ratio and to avoid the loss of A$\beta$ aggregates during the washing steps, a successfully optimized Surface-FIDA was developed in this work. First, the assay chip (microtiter plate with glass bottom) was coated with carboxymethyl dextran (CMD). The capture antibody was then covalently linked to the glass surface via CMD as a spacer. This subsequently allowed the A$\beta$ aggregates to be caught specifically by the capture antibody. In the end, at least two different detection antibodies labeled with different fluorescent dyes were applied. The measurements were performed on the confocal laser scanning microscope. By using the same monoclonal antibody as the capture and detection antibody, selective detection of A$\beta$ aggregates was achieved. Monomers were not detected and do not disturb this assay. Moreover, using different isoform specific detection antibodies simultaneously, the assay was able to visualize the different A$\beta$ isoforms (A$\beta$1-40, A$\beta$1-42 and A$\beta$pGlu3-42) in heterogeneous aggregates. The lowest detection limit of soluble oligomers can reproducibly be as low as 80 fg. Thus, using the optimized protocol, both the sensitivity and specificity of this assay have been improved in comparison to a protocol using non-covalent binding of capture antibodies. Finally, the optimized protocol was applied for detecting A$\beta$ oligomers in human CSF. The levels of oligomeric A$\beta$ were not age-dependent for the age-range studied (56-94 years), whereas the oligomeric A$\beta$ levels in CSF samples were significantly increased for AD samples compared to controls. A negative correlation between the levels of oligomers in CSF and mini-mental state examination scores was observed. The results support that A$\beta$ oligomers in CSF could be used as biomarker for AD. This makes the developed Surface-FIDA for detection of A$\beta$ oligomers a promising assay with potential benefits for the diagnosis and therapy monitoring of AD. |