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Sensitive ELISA detection of amyloid-β protofibrils in biological samples
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
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2007 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 103, no 1, 334-345 p.Article in journal (Refereed) Published
Abstract [en]

Amyloid-β (Aβ) protofibrils are known intermediates of the in vitro Aβ aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Aβ protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Aβ protofibril immunoassay, Aβ conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Aβ protofibrils without interference from Aβ monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Aβ protofibrils in both cell and animal models, proving that Aβ protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Aβ protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Aβ protofibrils in AD and has the potential of becoming an important diagnostic assay.

Place, publisher, year, edition, pages
2007. Vol. 103, no 1, 334-345 p.
Keyword [en]
Alzheimer's disease, Amyloid-β protofibril, Conformation-dependent antibody, Protofibril-specific ELISA
National Category
Medical and Health Sciences
Research subject
Geriatrics
Identifiers
URN: urn:nbn:se:uu:diva-98511DOI: 10.1111/j.1471-4159.2007.04759.xISI: 000249949700030PubMedID: 17623042OAI: oai:DiVA.org:uu-98511DiVA: diva2:174744
Available from: 2009-02-24 Created: 2009-02-24 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Soluble amyloid-β aggregates in Alzheimer’s disease
Open this publication in new window or tab >>Soluble amyloid-β aggregates in Alzheimer’s disease
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Soluble oligomeric aggregates of the amyloid-β (Aβ) peptide are suggested to initiate Alzheimer's disease (AD), leading to impaired synapse signalling, widespread neuronal death and loss of cognitive functions. These aggregates seem tightly linked to disease progression, and have therefore gained much attention as potential novel disease markers. In this thesis soluble oligomeric Aβ aggregates in general, and the Aβ protofibril species in particular, have been investigated with the aim to quantify and determine their role in AD pathogenesis.

Sandwich-ELISAs specifically measuring Aβ42 peptides are widely used both in AD research and as complements for clinical diagnosis. Here it was demonstrated that presence of soluble Aβ aggregates disturbs such analyses, making it difficult to interpret the results. This discovery was made through analyses of samples from cell- and mouse models carrying the AD causing 'Arctic' APP mutation. When analyzed by ELISA, Aβ42 levels were reduced in Arctic samples, in contrast to levels measured by denaturing SDS-PAGE Western blot. The same divergence in Aβ42-levels between analyses was observed in CSF samples from Down syndrome infants. The discrepancy between methods was hypothesized to be due to presence of soluble Aβ aggregates leading to impaired ELISA detection caused by epitope masking. This was confirmed by developing a protofibril specific ELISA, by which samples from Arctic cell- and mouse models were demonstrated to have enhanced Aβ protofibril levels.

AD patients have reduced ELISA-measured Aβ42-levels in CSF compared to healthy controls. To test if this reduction was due to oligomeric Aβ species present in AD CSF, Aβ42-levels were analyzed under both denaturing and non-denaturing conditions. These two measures were combined and an Aβ42 oligomer ratio established. Higher ratios were found in AD patients than healthy controls, implying that Aβ oligomers are present in CSF during Alzheimer pathogenesis. The observations from AD patients and young Down syndrome individuals suggest that Aβ42 oligomer formation is an early mechanism of AD pathogenesis, which potentially could be used as a biomarker to monitor disease development.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 69 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 430
National Category
Geriatrics
Research subject
Geriatrics
Identifiers
urn:nbn:se:uu:diva-98512 (URN)978-91-554-7441-6 (ISBN)
Public defence
2009-04-17, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-03-25 Created: 2009-02-24 Last updated: 2009-03-27Bibliographically approved
2. Aβ Conformation Dependent Antibodies and Alzheimer's Disease
Open this publication in new window or tab >>Aβ Conformation Dependent Antibodies and Alzheimer's Disease
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Soluble intermediates of the amyloid-β (Aβ) aggregation process are suggested to play a central role in the pathogenesis of Alzheimer’s disease (AD) by causing synaptic dysfunction and neuronal loss. In this thesis, soluble Aβ aggregates have been studied with a particular focus on the Aβ protofibril, which has served as the antigen for developing conformation dependent monoclonal antibodies.

Antibodies generated from mice immunized with Aβ protofibrils were characterized regarding Aβ binding properties and the amino acid sequences of their antigen binding sites. A conformation dependent IgG antibody, mAb158, was further characterized and found to bind to Aβ protofibrils with a 200-fold higher affinity than to monomeric Aβ without affinity for soluble amyloid-β precursor protein (AβPP) or other amyloidogenic proteins. A sandwich enzyme-linked immunosorbent assay (ELISA) based on mAb158 was used to measure soluble Aβ protofibrils in brain extracts from AβPP-transgenic mice. Low levels of protofibrils could also be detected in human AD brain. However, positive signals generated from measurements in AD and control CSF samples were attributed to interference from heterophilic antibodies (HA), generating false positive signals by cross-binding the assay antibodies; consequently, a study on HA interference in Aβ oligomer ELISAs was initiated. A large set of plasma and CSF samples from AD and non-AD subjects were analyzed with and without measures taken to block HA interference, revealing that virtually all signals above the assay limit of detection were false and generated by HA interference.

Many types of soluble Aβ aggregates have been described and suggested to impair neuron and synapse function. To investigate the soluble Aβ pool, synthetic Aβ and brain extracts from AβPP-transgenic mice and AD patients were ultracentrifuged on a density gradient to separate Aβ by size under native conditions. Four distinct gradient fractions were defined based on the appearance of synthetic Aβ in atomic force microscopy (AFM) and immunoreactivity in our protofibril specific sandwich ELISA. Interestingly, most Aβ from AD patients and AβPP-transgenic mice separated in the same fraction as toxic synthetic protofibrils.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 69 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 623
Keyword
Alzheimer's disease, amyloid-beta, protofibrils, conformation, monoclonal antibody, ELISA
National Category
Medical and Health Sciences
Research subject
Geriatrics
Identifiers
urn:nbn:se:uu:diva-132736 (URN)978-91-554-7949-7 (ISBN)
Public defence
2010-12-17, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 2004-2167
Available from: 2010-11-25 Created: 2010-10-26 Last updated: 2011-01-13Bibliographically approved

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Englund, HilleviSehlin, Dag

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