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
Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Rudbeck Laboratory)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Rudbeck Laboratory)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Rudbeck Laboratory)
Show others and affiliations
2013 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 61, no 11, 773-784 p.Article in journal (Refereed) Published
Abstract [en]

Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.

Place, publisher, year, edition, pages
2013. Vol. 61, no 11, 773-784 p.
Keyword [en]
antibody, biotin, conjugation, protein detection, tissue microarray
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-211012DOI: 10.1369/0022155413502360ISI: 000326066300001OAI: oai:DiVA.org:uu-211012DiVA: diva2:665486
Available from: 2013-11-20 Created: 2013-11-19 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Validation of antibodies for tissue based immunoassays
Open this publication in new window or tab >>Validation of antibodies for tissue based immunoassays
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In situ protein detection in human tissues using antibodies reveals the cellular protein localization, and affinity-based proteomic studies can help to discover proteins involved in the development of diseases. However, antibodies often suffer from cross-reactivity, and the lack of positive and negative tissue controls for uncharacterized proteins complicates the mapping of the proteome. The aim of this thesis is thus to improve the methodology for validating antibodies used for immunostaining on formalin-fixed paraffin-embedded tissues.

Two of the papers include comparisons between mRNA-expression and immunostaining of corresponding protein. In paper I, ISH and IHC staining patterns were compared on consecutive TMA-slides. The study of well-characterized genes showed that ISH could be used for validation of antibodies. ISH was further used for antibody evaluation, and could validate four out of nine antibodies showing potentially interesting staining patterns. In paper III, transcriptomic data generated by RNA-sequencing were used to identify tissue specific expression in lymphohematopoietic tissues. An increased expression in one or more of these tissues compared to other tissue types was seen for 693 genes, and these were further compared to the staining patterns of corresponding proteins in tissues.

Antibody labeling is necessary for many immunoassays. In paper II, two techniques for antibody-biotinylation were compared, aiming to find a stringent labeling method for antibodies used for immunostaining on TMAs. The ZBPA-method, binding specifically to Fc-part of antibodies, was found to be superior to the Lightning Link-biotinylation kit targeting amine groups, since labeling of amine groups on stabilizing proteins in the antibody buffer causes unspecific staining.

The localization of the estrogen receptor beta (ERβ) in human normal and cancer tissues was studied in paper IV. Thorough evaluation of 13 antibodies using positive and negative control cell lines showed that only one antibody, PPZ0506, is specific for ERβ in all three immunoassays used. Contradictory to previously published data, tissue profiling using PPZ0506 showed that ERβ is expressed in a limited number of normal and cancer tissues.

In conclusion, the present investigations present tools for validation of antibodies used for large-scale studies of protein expression in tissues.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1109
Keyword
Antibody validation, Conjugation, Estrogen receptor-beta, IHC, ISH, Lymphohematopoietic tissues, Proteomic, RNAseq, TMA, Transcriptomic
National Category
Medical and Health Sciences Biological Sciences
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-251344 (URN)978-91-554-9258-8 (ISBN)
Public defence
2015-06-13, Fåhreussalen, Rudbecklaboratoriet, hus C5, Dag Hammarskjölds väg 20, Uppsala, 12:30 (English)
Opponent
Supervisors
Available from: 2015-05-21 Created: 2015-04-15 Last updated: 2015-07-07

Open Access in DiVA

No full text

Other links

Publisher's full text

Authority records BETA

Andersson, SandraPontén, FredrikAsplund, Anna

Search in DiVA

By author/editor
Andersson, SandraPontén, FredrikAsplund, Anna
By organisation
Molecular and Morphological PathologyScience for Life Laboratory, SciLifeLabDepartment of Immunology, Genetics and Pathology
In the same journal
Journal of Histochemistry and Cytochemistry
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 570 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