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Estrogen receptor beta profiling in human tissues following extensive antibody validation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
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(English)Manuscript (preprint) (Other academic)
Keyword [en]
Estrogen receptor beta, ER-beta, IHC, antibody validation, TMA
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-251340OAI: oai:DiVA.org:uu-251340DiVA: diva2:806684
Available from: 2015-04-21 Created: 2015-04-15 Last updated: 2015-10-01
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
2. Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
Open this publication in new window or tab >>Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody.

Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity.

Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 82 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1272
Keyword
Mass spectrometry, proteomics, microarray, protein, antibody, antigen, affinity, validation
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-259623 (URN)978-91-554-9300-4 (ISBN)
Public defence
2015-09-25, C4:305, BMC, Husargatan 3, Uppsala, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2015-09-03 Created: 2015-08-10 Last updated: 2015-10-01

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