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Heavy-Chain Complementarity-Determining Regions Determine Conformation Selectivity of Anti-A beta Antibodies
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
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2011 (English)In: Neurodegenerative Diseases, ISSN 1660-2854, Vol. 8, no 3, 117-123 p.Article in journal (Refereed) Published
Abstract [en]

Background/Aims: Amyloid-beta (Abeta) protofibrils are neurotoxic soluble intermediates in the Abeta aggregation process eventually forming senile plaques in Alzheimer's disease. This Abeta species is a potential biomarker for Alzheimer's disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent Abeta antibodies specific for Abeta protofibrils. Methods: Mice were immunized with Abeta protofibrils to generate hybridomas producing Abeta-specific monoclonal antibodies. Binding of antibodies to different Abeta conformations was investigated with inhibition ELISA. The antibodies' complementarity-determining region (CDR) sequences were determined and compared. Results: A majority of the antibodies were of the IgM class, all selectively binding to aggregated Abeta. Two IgG antibodies were generated: one with selective affinity for Abeta protofibrils and the other bound Abeta in all conformations. A high degree of similarity between the heavy-chain CDRs of the conformation-dependent antibodies was found, and all high-affinity Abeta antibodies displayed a high degree of sequence similarity in the light-chain CDRs. Conclusion: Sequence similarity in the heavy-chain CDRs is associated with conformation selectivity of the antibodies, while sequence similarity in the light-chain CDRs correlates with the affinity for Abeta.

Place, publisher, year, edition, pages
2011. Vol. 8, no 3, 117-123 p.
Keyword [en]
Alzheimer's disease, Amyloid-beta, Conformation dependent antibodies, Complementarity determining region
National Category
Medical and Health Sciences
Research subject
Geriatrics
Identifiers
URN: urn:nbn:se:uu:diva-132722DOI: 10.1159/000316530ISI: 000289100800003PubMedID: 20714111OAI: oai:DiVA.org:uu-132722DiVA: diva2:359006
Available from: 2010-10-26 Created: 2010-10-26 Last updated: 2011-04-19Bibliographically approved
In thesis
1. 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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