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Site-specifically conjugated anti-HER2 Affibody® molecules as one-step reagents for target expression analyses on cells and xenograft samples
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-7034-0850
Affibody AB.
Affibody AB.
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-8993-048X
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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.

Place, publisher, year, edition, pages
2007. Vol. 319, no 1-2, 53-63 p.
Keyword [en]
ErbB-2; HER2/neu; Affibody molecule; flow cytometry; immunofluorescence; immunoprecipitation
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-4880DOI: 10.1016/j.jim.2006.10.013ISI: 000244215200007ScopusID: 2-s2.0-33846244218OAI: diva2:1742
QC 20100824Available from: 2008-09-16 Created: 2008-09-16 Last updated: 2013-06-12Bibliographically approved
In thesis
1. Bioimaging for analysis of protein expression in cells and tissues using affinity reagents
Open this publication in new window or tab >>Bioimaging for analysis of protein expression in cells and tissues using affinity reagents
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.
Trita-BIO-Report, ISSN 1654-2312 ; 2008:14
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
urn:nbn:se:kth:diva-4862 (URN)978-91-7415-096-4 (ISBN)
Public defence
2008-09-26, FD5, AlbaNova Universitetscentrum, Kungl Tekniska Högskolan, Roslagstullsbacken 21, Stockholm, 10:00 (English)
QC 20100824Available from: 2008-09-05 Created: 2008-09-05 Last updated: 2010-08-24Bibliographically approved

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