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A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-7034-0850
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0002-5391-600X
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. 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.

Place, publisher, year, edition, pages
2007. Vol. 322, no 1-2, 40-49 p.
Keyword [en]
labeling; solid phase; protein A; antibody; Alexa fluor 555; Alexa fluor 647
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-4881DOI: 10.1016/j.jim.2007.01.023ISI: 000246422100005ScopusID: 2-s2.0-34147096033OAI: diva2:1744
QC 20100824Available from: 2008-09-16 Created: 2008-09-16 Last updated: 2010-08-24Bibliographically 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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Lundberg, EmmaSundberg, MårtenGräslund, TorbjörnUhlén, MathiasAndersson-Svahn, Helene
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