uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Interference from Heterophilic Antibodies in Amyloid-beta Oligomer ELISAs
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)
Show others and affiliations
2010 (English)In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 21, no 4, 1295-1301 p.Article in journal (Refereed) Published
Abstract [en]

Amyloid-beta (Abeta) oligomers of different sizes and forms have recently been the focus for many Alzheimer's disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Abeta species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked. HA, which recognize immunoglobulins from other species, are present in human plasma and cerebrospinal fluid (CSF) and may cause interference in sandwich immunoassays like enzyme-linked immunosorbent assays (ELISAs) by cross-binding the capture and detection antibodies of the assay. They thus may generate a false positive signal. Here we show that when assessing the Abeta oligomer content in plasma samples from 44 individuals with a sandwich ELISA, none of the 21 positive signals remained when the assay was repeated in the presence of factors blocking HA. Similarly, in CSF samples from 104 individuals, the signals from the 22 positive samples were strongly reduced when analyzed after anti-HA treatment. Taken together, HA interference is a problem that needs to be addressed when measuring low levels of an antigen in human plasma and CSF samples.

Place, publisher, year, edition, pages
IOS Press , 2010. Vol. 21, no 4, 1295-1301 p.
Keyword [en]
Alzheimer's disease, Heterophilic antibodies, ELISA
National Category
Neurology
Research subject
Geriatrics
Identifiers
URN: urn:nbn:se:uu:diva-132732DOI: 10.3233/JAD-2010-100609ISI: 000283049700021PubMedID: 20693634OAI: oai:DiVA.org:uu-132732DiVA: diva2:359011
Note

Title also written as: Interference from Heterophilic Antibodies in Amyloid-β Oligomer ELISAs

Available from: 2010-10-26 Created: 2010-10-26 Last updated: 2017-12-12Bibliographically 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
2. Amyloid-β Protofibrils in Alzheimer´s Disease: Focus on Antibodies, Inflammation and Astrocytes
Open this publication in new window or tab >>Amyloid-β Protofibrils in Alzheimer´s Disease: Focus on Antibodies, Inflammation and Astrocytes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Soluble amyloid-beta (Aβ) aggregates, including Aβ protofibrils, play a central role in Alzheimer’s disease (AD) and constitute a potential diagnostic biomarker and a therapeutic target. Aβ protofibrils promote synapse dysfunction and neurodegeneration, but the mechanisms behind these effects remain unclear. The aim of this thesis was to increase the knowledge of Aβ protofibrils in AD pathology.

When measuring low abundant antigens, such as soluble Aβ aggregates, in plasma and CSF by immunoassays, there is a possibility of interference by heterophilic antibodies (HA). In paper I, we show that HA generate false positive signals, by cross-binding the assay antibodies, when plasma and CSF from AD patients and healthy controls were analyzed for soluble Aβ aggregates, using sandwich ELISAs.

Natural anti-Aβ antibodies exist in AD patients and healthy individuals. Circulating Aβ and anti-Aβ antibodies may form immune complexes, masking epitopes on the anti-Aβ antibody, which makes the anti-Aβ antibody concentration difficult to measure. In paper II, the ELISpot technique enabled us to successfully measure B cell production of anti-Aβ antibodies. Our results show that anti-Aβ protofibril antibody production is present in both AD patients and healthy individuals, but is significantly higher in AD patients, indicating that the immune system attempt to eliminate the toxic Aβ species.

Insufficient lysosomal degradation is proposed to cause sporadic AD. In paper III, we used a co-culture system of astrocytes, neurons and oligodendrocytes, to clarify the role of astrocytes in Aβ protofibril clearance. Astrocytes are the most prominent glial cell type in the brain, but their role in AD remains elusive. We found that astrocytes effectively engulf, but inefficiently degrade Aβprotofibrils. This result in a high intracellular load of toxic, partly N-terminally truncated Aβ and lysosomal dysfunction. Moreover, we found that secretion of microvesicles, containing N-terminally truncated Aβ, induce neuronal apoptosis. In paper IV, we show that treatment with the protofibril selective antibody mAb158 lead to enhanced Aβ clearance and thereby prevent Aβ neurotoxicity.

Taken together, this thesis contributes with important knowledge on the role of Aβ protofibrils in AD pathogenesis and technical aspects that should be considered when measuring Aβ in human tissues.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1147
Keyword
Alzheimer's Disease, Amyloid-beta, Protofibrils, ELISA, ELISpot, Astrocyte, Monoclonal Antibody, Immunofluorescence
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-264647 (URN)978-91-554-9373-8 (ISBN)
Public defence
2015-12-04, Rudbecksalen, Rudbeckslaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2015-11-13 Created: 2015-10-15 Last updated: 2016-01-14Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMedInterference from Heterophilic Antibodies in Amyloid-beta Oligomer ELISAs.

Authority records BETA

Sehlin, DagSöllvander, Sofia

Search in DiVA

By author/editor
Sehlin, DagSöllvander, Sofia
By organisation
Geriatrics
In the same journal
Journal of Alzheimer's Disease
Neurology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 548 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf