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  • 1.
    Forsström, Bjorn
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Axnäs, Barbara Bislawska
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Danielsson, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Bohlin, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Dissecting antibodies withregards to linear and conformational epitopesManuskript (preprint) (Annet vitenskapelig)
  • 2.
    Forsström, Bjorn
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Axnäs, Barbara Bislawska
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Stengele, Klaus-Peter
    Buehler, Jochen
    Albert, Thomas J.
    Richmond, Todd A.
    Hu, Francis Jingxin
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton Paul
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays2014Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, nr 6, s. 1585-1597Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.

  • 3.
    Forsström, Björn
    et al.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Axnäs, Barbara Bislawska
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Danielsson, Hanna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Bohlin, Anna
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Dissecting Antibodies with Regards to Linear and Conformational Epitopes2015Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 3, artikkel-id e0121673Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.

  • 4.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Epitope mapping of antibodies towards human protein targets2011Doktoravhandling, med artikler (Annet vitenskapelig)
    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.

  • 5.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Brennan, Donal J
    Zendehrokh, Nooreldin
    Eberhard, Jakob
    Nodin, Björn
    Gaber, Alexander
    Pontén, Fredrik
    Johannesson, Henrik
    Smaragdi, Kristina
    Frantz, Christian
    Hober, Sophia
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Johnson, Louis B
    Påhlman, Sven
    Jirström, Karin
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    High nuclear RBM3 expression is associated with an improved prognosis in colorectal cancer2011Inngår i: Proteomics. Clinical applications, ISSN 1862-8354, Vol. 5, nr 11-12, s. 624-35Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 6.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Fernandez, Carmen Diez
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Johannesson, Henrik
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase2010Inngår i: NEW BIOTECHNOL, ISSN 1871-6784, Vol. 27, nr 2, s. 129-137Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.

  • 7.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Igel, Ulrika
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johannesson, Henrik
    Stadler, Charlotte
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    Sjoberg, Anna
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Schwenk, Jochen M.
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Johansson, Christine
    Uhlen, Mathias
    KTH, Skolan för bioteknologi (BIO), Centra, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Generation of monospecific antibodies based on affinity capture of polyclonal antibodies2011Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, nr 11, s. 1824-1835Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

  • 8.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopesManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties to obtain a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of in-bred rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen gene on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several in-bred rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 9.
    Hjelm, Barbara
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Forsström, Björn
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101). KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes2012Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 12, s. e45817-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  • 10.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Malm, Magdalena
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies2010Inngår i: Recent Patents on Biotechnology, ISSN 1872-2083, Vol. 4, nr 3, s. 171-182Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.

  • 11.
    Kronqvist, Nina
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Rockberg, Johan
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    New Ways for Discovery of Biopharmaceuticals: Emerging Techniques using Surface Display on Gram-positive Bacteria for Combinatorial Protein Engineering and Characterization2009Inngår i: Bioforum Europe, ISSN 1611-597X, Vol. 13, nr 6-7, s. 022-Artikkel i tidsskrift (Fagfellevurdert)
  • 12.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
    Epitope mapping of antibodies using bacterial surface display2008Inngår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, nr 12, s. 1039-1045Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We describe a method for mapping the epitopes recognized by antibodies, based on bacterial surface expression of antigen protein fragments followed by antibody-based flow-cytometric sorting. We analyzed the binding sites of both monoclonal and polyclonal antibodies directed to three human protein targets: (i) the human epidermal growth factor receptor 2 (HER2), (ii) ephrin-B3 and (iii) the transcription factor SATB2. All monoclonal antibodies bound a single epitope, whereas the polyclonal antibodies showed, in each case, a binding pattern with one to five separate epitopes. A comparison of polyclonal and monoclonal antibodies raised to the same antigen showed overlapping binding epitopes. We also demonstrated that bacterial cells with displayed protein fragments can be used as affinity ligands to generate epitope-specific antibodies. Our approach shows a path forward for systematic validation of antibodies for epitope specificity and cross-reactivity on a whole-proteome level.

  • 13.
    Rockberg, Johan
    et al.
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Löfblom, John
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Hjelm, Barbara
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Ståhl, Stefan
    KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi (stängd 20130101).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik (stängd 20130101).
    Epitope mapping using gram-positive surface display2010Inngår i: Current Protocols in Immunology, ISSN 1934-3671, nr SUPPL. 90, s. 9.9.1-9.9.17Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibodys binding characteristics will still be the main features determining the assays reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.

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