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Validation of antibodies for tissue based immunoassays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
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 [en]
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: urn:nbn:se:uu:diva-251344ISBN: 978-91-554-9258-8 (print)OAI: oai:DiVA.org:uu-251344DiVA: diva2:806808
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
List of papers
1. Scalable In Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers
Open this publication in new window or tab >>Scalable In Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, e32927- p.Article in journal (Refereed) Published
Abstract [en]

Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-174583 (URN)10.1371/journal.pone.0032927 (DOI)000303062000022 ()
Available from: 2012-05-29 Created: 2012-05-22 Last updated: 2017-12-07Bibliographically approved
2. Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection
Open this publication in new window or tab >>Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection
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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.

Keyword
antibody, biotin, conjugation, protein detection, tissue microarray
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-211012 (URN)10.1369/0022155413502360 (DOI)000326066300001 ()
Available from: 2013-11-20 Created: 2013-11-19 Last updated: 2017-12-06Bibliographically approved
3. The Transcriptomic and Proteomic Landscapes of Bone Marrow and Secondary Lymphoid Tissues
Open this publication in new window or tab >>The Transcriptomic and Proteomic Landscapes of Bone Marrow and Secondary Lymphoid Tissues
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, e115911- p.Article in journal (Refereed) Published
Abstract [en]

Background: The sequencing of the human genome has opened doors for global gene expression profiling, and the immense amount of data will lay an important ground for future studies of normal and diseased tissues. The Human Protein Atlas project aims to systematically map the human gene and protein expression landscape in a multitude of normal healthy tissues as well as cancers, enabling the characterization of both housekeeping genes and genes that display a tissue-specific expression pattern. This article focuses on identifying and describing genes with an elevated expression in four lymphohematopoietic tissue types (bone marrow, lymph node, spleen and appendix), based on the Human Protein Atlas-strategy that combines high throughput transcriptomics with affinity-based proteomics. Results: An enriched or enhanced expression in one or more of the lymphohematopoietic tissues, compared to other tissue-types, was seen for 693 out of 20,050 genes, and the highest levels of expression were found in bone marrow for neutrophilic and erythrocytic genes. A majority of these genes were found to constitute well-characterized genes with known functions in lymphatic or hematopoietic cells, while others are not previously studied, as exemplified by C19ORF59. Conclusions: In this paper we present a strategy of combining next generation RNA-sequencing with in situ affinity-based proteomics in order to identify and describe new gene targets for further research on lymphatic or hematopoietic cells and tissues. The results constitute lists of genes with enriched or enhanced expression in the four lymphohematopoietic tissues, exemplified also on protein level with immunohistochemical images.

National Category
Other Medical Sciences not elsewhere specified
Identifiers
urn:nbn:se:uu:diva-243679 (URN)10.1371/journal.pone.0115911 (DOI)000347239900109 ()25541736 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, KAW2008.0143
Available from: 2015-02-19 Created: 2015-02-11 Last updated: 2017-12-04Bibliographically approved
4. Estrogen receptor beta profiling in human tissues following extensive antibody validation
Open this publication in new window or tab >>Estrogen receptor beta profiling in human tissues following extensive antibody validation
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(English)Manuscript (preprint) (Other academic)
Keyword
Estrogen receptor beta, ER-beta, IHC, antibody validation, TMA
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-251340 (URN)
Available from: 2015-04-21 Created: 2015-04-15 Last updated: 2015-10-01

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