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Bioimaging for analysis of protein expression in cells and tissues using affinity reagents
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-7034-0850
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The detection and analysis of biomolecules, such as proteins, are of great interest since these molecules are fundamental for life and our health. Due to the complexity of biological processes, there is a great advantage of studying proteins in their natural context, for example by using bioimaging. The objective of this doctoral thesis has been to develop, implement and evaluate techniques for the use of proteinspecific affinity reagents in diverse bioimaging platforms for analysis of protein expression in situ in cells and tissues.

To be able to visualize a desired protein in situ using affinity reagents, reporter labels are needed. A novel technique for labeling of antibodies on solid phase was developed. This method offers simultaneous purification, concentration and labeling of an antibody sample, giving highly predictable and reproducible results, in a miniaturized format.

Another study demonstrates the use of an alternative affinity reagent, the Affibody molecule, in bioimaging as well as other immunoassays. As a relevant proof-of-principle, an Affibody molecule binding the HER2 receptor was site-specificly labeled and employed for analysis of HER2 protein expression in cells and tissue using immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation and flow cytometry.

Furthermore, it is shown how antibody-based bioimaging approaches can be applied for systematic analysis of protein expression in terms of subcellular localization and expression levels in cell lines. The systematic subcellular localization of nearly 500 proteins was performed using IF and confocal microscopy. Global analysis of expression levels of nearly 2000 proteins in a panel of cell lines using IHC and automated image analysis, revealed that most proteins are expressed in a cell size dependent manner. Two normalization approaches were evaluated and found to allow for protein profiling across the panel of morphologically diverse cells, revealing patterns of protein over- and underexpression, and proteins with stable as well as with lineage specific expression were identified.

Finally, the value of antibody-based, bioimaging proteomics as a platform for biomarker discovery is demonstrated. The identification and in depth study of a candidate biomarker for colorectal cancer, SATB2, is described using both IHC and IF bioimaging. Results from extended analyses of tumor biopsies showed that detection of SATB2 protein using IHC provides a clinically relevant diagnostic tool with high specificity and sensitivity to aid in diagnosis of colorectal cancer. Furthermore, the study demonstrated a potential prognostic role of SATB2, as decreased expression was associated with a significantly shorter overall survival in patients with advanced colorectal cancer.

Place, publisher, year, edition, pages
Stockholm: KTH , 2008. , vii, 86 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:14
Keyword [en]
Affibody, antibody, biomarker, cell line, cell microarray, colorectal cancer, confocal microscopy, HER2, immunohistochemistry, immunofluorescence, light microscopy, protein expression, protein localization, SATB2, tissue microarray
National Category
Industrial Biotechnology Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-4862ISBN: 978-91-7415-096-4 (print)OAI: oai:DiVA.org:kth-4862DiVA: diva2:138
Public defence
2008-09-26, FD5, AlbaNova Universitetscentrum, Kungl Tekniska Högskolan, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100824Available from: 2008-09-05 Created: 2008-09-05 Last updated: 2010-08-24Bibliographically approved
List of papers
1. A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase
Open this publication in new window or tab >>A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase
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2007 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 322, no 1-2, 40-49 p.Article in journal (Refereed) Published
Abstract [en]

Fluorescently labeled antibodies are very important tools in cell biology, providing for specific and quantitative detection of antigens. To date, fluorophore labeling of antibodies has been performed in solution and has been limited by low-throughput methods requiring a substantial amount of pure antibody sample at a high concentration. We have developed a novel solid-phase labeling protocol for small amounts (i.e. micrograms) of antibodies with fluorescent dyes. Protein A affinity medium was used as solid support in a micropipette tip format. This solid-phase approach, including the advantage of the strong and specific interaction between Protein A and antibodies, allows for simultaneous purification, labeling and concentration of the antibody sample, making it possible to start with unpure antibody samples at low concentrations. We have optimized the protocol with regard to reaction pH, time, temperature and amount of amine reactive dye. In addition, we have evaluated the stability and activity of the labeled antibodies. To evaluate the reproducibility and robustness of this method we labeled eight antibodies with amine reactive fluorescent dyes followed by evaluation of antibody specificity on protein arrays. Interestingly, this gave an extremely high conformity in the degree of labeling, showing the robustness of the method. The solid-phase method also gave predictable and reproducible results and by varying the amount of reactive dye, the desired degree of labeling can easily be achieved. Antibodies labeled using this solid-phase method were similar in stability and activity to antibodies labeled in solution. This novel solid-phase antibody labeling method may also be applicable for other conjugation chemistries and labels, and has potential for throughput applications.

Keyword
labeling; solid phase; protein A; antibody; Alexa fluor 555; Alexa fluor 647
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4881 (URN)10.1016/j.jim.2007.01.023 (DOI)000246422100005 ()2-s2.0-34147096033 (Scopus ID)
Note
QC 20100824Available from: 2008-09-16 Created: 2008-09-16 Last updated: 2010-08-24Bibliographically approved
2. Site-specifically conjugated anti-HER2 Affibody® molecules as one-step reagents for target expression analyses on cells and xenograft samples
Open this publication in new window or tab >>Site-specifically conjugated anti-HER2 Affibody® molecules as one-step reagents for target expression analyses on cells and xenograft samples
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2007 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 319, no 1-2, 53-63 p.Article in journal (Refereed) Published
Abstract [en]

Affibody (R) molecules are a class of small and robust affinity proteins that can be generated to interact with a variety of antigens, thus having the potential to provide useful tools for biotechnological research and diagnostic applications. In this study, we have investigated Affibody-based reagents interacting specifically with the tyrosine kinase receptor HER2. A head-to-tail dimeric construct was site-specifically conjugated with different fluorescent and enzymatic groups resulting in reagents that were used for detection and quantification. The amount of cell surface expressed HER2 oil eleven (11) well characterized cell lines was quantified relative to each other by flow cytometry and shown to correlate well with results from parallel analyses of HER2 mRNA levels measured by real-time PCR. Further, immunofluorescence I microscopy studies of the cell lines and immunohistochemical analyses of cryosections of HER2 expressing SKOV-3 xenografts showed strong staining of the plasma membrane of tumor cells with little background staining. Full-length HER2 protein Could also be efficiently recovered from a cell extract by an immunoprecipitation procedure, using an Affibody ligand-based resin.

These novel non-IgG derived reagents could be used to detect and quantify HER2 expression. By adapting the methods for use with Affibody molecules binding to other cell surface receptors, it is anticipated that also these receptors can be detected and quantified in a similar manner.

Keyword
ErbB-2; HER2/neu; Affibody molecule; flow cytometry; immunofluorescence; immunoprecipitation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4880 (URN)10.1016/j.jim.2006.10.013 (DOI)000244215200007 ()2-s2.0-33846244218 (Scopus ID)
Note
QC 20100824Available from: 2008-09-16 Created: 2008-09-16 Last updated: 2013-06-12Bibliographically approved
3. Toward a confocal subcellular atlas of the human proteome
Open this publication in new window or tab >>Toward a confocal subcellular atlas of the human proteome
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2008 (English)In: Molecular and cellular proteomics, ISSN 1535-9476, Vol. 7, no 3, 499-508 p.Article in journal (Refereed) Published
Abstract [en]

Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

Keyword
HIGH-THROUGHPUT MICROSCOPY; FLUORESCENCE MICROSCOPY; AUTOMATED MICROSCOPY; MEMBRANE-PROTEINS; LOCALIZATION; CELLS; TISSUES
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4882 (URN)10.1074/mcp.M700325-MCP200 (DOI)000254076400004 ()2-s2.0-41549148628 (Scopus ID)
Note
QC 20100824Available from: 2008-09-16 Created: 2008-09-16 Last updated: 2010-09-14Bibliographically approved
4. The correlation between cellular size and protein expression levels: Normalization for global protein profiling
Open this publication in new window or tab >>The correlation between cellular size and protein expression levels: Normalization for global protein profiling
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2008 (English)In: Journal of Proteomics, ISSN 1874-3919, Vol. 71, no 4, 448-460 p.Article in journal (Refereed) Published
Abstract [en]

An automated image analysis system was used for protein quantification of 1862 human proteins in 47 cancer cell lines and 12 clinical cell samples using cell microarrays and immunohistochemistry. The analysis suggests that most proteins are expressed in a cell size dependent manner, and that normalization is required for comparative protein quantification in order to correct for the inherent bias of cell size and systematic ambiguities associated with immunohistochemistry. Two reference standards were evaluated, and normalized protein expression values were found to allow for protein profiling across a panel of morphologically diverse cells, revealing putative patterns of over- and underexpression. Using this approach, proteins with stable expression as well as cell-line specific expression were identified. The results demonstrate the value of large-scale, automated proteome analysis using immunohistochemistry in revealing functional correlations and establishing methods to interpret and mine proteomic data.

Keyword
Immunohistochemistry; Protein quantification; Normalization; Expression profiling
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4883 (URN)10.1016/j.jprot.2008.06.014 (DOI)000260816700005 ()2-s2.0-51749118509 (Scopus ID)
Note
QC 20100728. Uppdaterad från in press till published (20100728).Available from: 2008-09-16 Created: 2008-09-16 Last updated: 2010-07-28Bibliographically approved
5. Tissue specific expression of the transcription factor SATB2 in colorectal carcinoma
Open this publication in new window or tab >>Tissue specific expression of the transcription factor SATB2 in colorectal carcinoma
2008 (English)Article in journal (Other academic) Submitted
Keyword
SATB2; colorectal cancer; antibody-based proteomics; diagnostics; prognostic factors
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4884 (URN)
Note
QS 20120314Available from: 2008-09-16 Created: 2008-09-16 Last updated: 2012-03-14Bibliographically approved

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