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High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
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2011 (English)In: Proteomics. Clinical applications, ISSN 1862-8354, Vol. 5, no 11-12, 624-35 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis. Experimental design: One polyclonal antibody and four monoclonal anti-RBM3 antibodies were generated and epitope mapped using two different methods. Bacterial display revealed five distinct epitopes for the polyclonal antibody, while the four mouse monoclonal antibodies were found to bind to three of the five epitopes. A peptide suspension bead array assay confirmed the five epitopes of the polyclonal antibody, while only one of the monoclonal antibodies could be mapped using this approach. Antibody specificity was confirmed by Western blotting and immunohistochemistry, including siRNA-mediated knock-down. Two of the antibodies (polyclonal and monoclonal) were subsequently used to analyze RBM3 expression in tumor samples from two independent colorectal cancer cohorts, one consecutive cohort (n=270) and one prospectively collected cohort of patients with cancer of the sigmoid colon (n=305). RBM3-expression was detected, with high correlation between both antibodies (R=0.81, p<0.001). Results: In both cohorts, tumors with high nuclear RBM3 staining had significantly prolonged the overall survival. This was also confirmed in multivariate analysis, adjusted for established prognostic factors. Conclusion and clinical relevance: These data demonstrate that high tumor-specific nuclear expression of RBM3 is an independent predictor of good prognosis in colorectal cancer.

Place, publisher, year, edition, pages
2011. Vol. 5, no 11-12, 624-35 p.
National Category
Biomedical Laboratory Science/Technology
URN: urn:nbn:se:kth:diva-59105DOI: 10.1002/prca.201100020ISI: 000298334000006PubMedID: 21956899ScopusID: 2-s2.0-84855183064OAI: diva2:475102
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
QC 20120110Available from: 2012-01-10 Created: 2012-01-10 Last updated: 2012-06-15Bibliographically approved
In thesis
1. Epitope mapping of antibodies towards human protein targets
Open this publication in new window or tab >>Epitope mapping of antibodies towards human protein targets
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis, based on five research papers, presents results from development and evaluation ofmethods for identifying the interaction site of antibodies on their antigens and the functional investigation of these in different assays. As antibodies have proven to be invaluable tools in diagnostics, therapy and basic research, the demand of characterizing these binding molecules has increased. Techniques for epitope mapping in a streamlined manner are therefore needed, particularly in high throughput projects as the Human Protein Atlas that aims to systematically generate two antibodies with separate epitopes towards all human proteins. 

In paper I we describe an approach to map the epitopes of polyclonal and monoclonal antibodies for the first time using staphylococcal display. This method was combined with peptide scanning and alanine scanning using suspension bead arrays, to create a streamlined approach of highresolution characterization of epitopes recognized by antibodies as demonstrated in paper II. Single epitopes were identified for the monoclonal antibodies and several (one to five) separate epitopes scattered throughout the antigen sequence were determined for each polyclonal antibody. Further, antibodies of different species origin showed overlapping binding epitopes.

In paper III we studied the epitope patterns of polyclonal antibodies generated with the same antigen in different animals. Although common epitope regions could be identified the exact epitope pattern was not repeated, as some epitopes did not reoccur in the repeated immunizations. In paper IV, a potential biomarker for colon cancer, RBM3, was investigated using validated antibodies by epitope mapping and siRNA analysis.

Finally, in paper V, a method for generating epitope-specific antibodies based on affinity purification of a polyclonal antibody is described. The generated antibodies were used in several immunoassays and showed a great difference in functionality. Paired antibodies with separate epitopes were successfully generated and could be used in a sandwich assay or to validate each other in immunohistochemistry.

Taken together, in these studies we have demonstrated valuable concepts for the characterization of antibody epitopes.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. xi, 46 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2012:1
antibody, antibody validation, biomarker, epitope mapping, peptide array, proteomics, RBM3, staphylococcal surface display
National Category
Medical Biotechnology Biochemistry and Molecular Biology
urn:nbn:se:kth:diva-59529 (URN)978-91-7501-222-3 (ISBN)
Public defence
2012-01-27, FD5, Albanova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
QC 20120111Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2012-01-11Bibliographically approved

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Hjelm, BarbaraHober, SophiaUhlén, Mathias
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