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| 2. |
- Janzi, M., et al.
(författare)
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Serum microarrays for large scale screening of protein levels
- 2005
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 4:12, s. 1942-1947
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Tidskriftsartikel (refereegranskat)abstract
- There is a great need for comprehensive proteomic analysis of large patient cohorts of plasma and serum samples to identify biomarkers of human diseases. Here we describe a new antibody-based proteomic approach involving a reverse array format where serum samples are spotted on a microarray. This enables all samples to be screened for their content of a certain serum protein in a single experiment using target-recognizing antibodies and fluorescently labeled secondary antibodies. The procedure is illustrated with the analysis of the IgA levels in 2009 spotted serum samples, and the data are compared with clinical routine measurements. The results suggest that it is possible to simultaneously screen thousands of complex clinical serum samples for their content of the relative amount of specific serum proteins of clinical relevance.
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| 3. |
- Lundberg, Emma, 1980-
(författare)
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Bioimaging for analysis of protein expression in cells and tissues using affinity reagents
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Nilsson, Peter, et al.
(författare)
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Towards a human proteome atlas : high-throughput generation of mono-specific antibodies for tissue profiling
- 2005
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 5:17, s. 4327-4337
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Tidskriftsartikel (refereegranskat)abstract
- A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.
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| 5. |
- Rimini, Rebecca, et al.
(författare)
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Validation of serum protein profiles by a dual antibody array approach
- 2009
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 73:2, s. 252-266
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, affinity-based technologies have become important tools for serum profiling to uncover protein expression patterns linked to disease state or therapeutic effects. In this study, we describe a path towards the production of an antibody microarray to allow protein profiling of biotinylated human serum samples with reproducible sensitivity in the picomolar range. With the availability of growing numbers of affinity reagents, protein profiles are to be validated in efficient manners and we describe a cross-platform strategy based on data concordance with a suspension bead array to interrogate the identical set of antibodies with the same cohort of serum samples. Comparative analysis enabled to screen for high-performing antibodies, which were displaying consistent results across the two platforms and targeting known serum components. Moreover, data processing methods such as sample referencing and normalization were evaluated for their effects on inter-platform agreement. Our work suggests that mutual validation of protein expression profiles using alternative microarray platforms holds great potential in becoming an important and valuable component in affinity-based high-throughput proteomic screenings as it allows to narrow down the number of discovered targets prior to orthogonal, uniplexed validation approaches.
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| 6. |
- Rockberg, Johan, et al.
(författare)
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Epitope mapping of antibodies using bacterial surface display
- 2008
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 5:12, s. 1039-1045
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Uhlén, Mathias
(författare)
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A human protein atlas
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276, s. 90-90
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 8. |
- Uhlén, Mathias, et al.
(författare)
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Generation and validation of affinity reagents on a proteome-wide level
- 2009
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 22:2, s. 57-64
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Tidskriftsartikel (refereegranskat)abstract
- There is a need for protein-specific affinity reagents to explore the gene products encoded by the genome. Recently, systematic efforts to generate validated affinity reagents on a whole human proteome level have been initiated. There are several issues for such efforts, including choice of antigen, type of affinity reagent, and the subsequent validation of the generated protein-specific binders. The advantages and disadvantages with the different approaches are discussed and the problems related to quality assessment of antibodies to be used in multi-platform applications are addressed. This review also describes the efforts to create a virtual resource of validated antibodies using a community-based portal and summarizes the status and visions for the publicly available human protein atlas (www.proteinatlas.org) showing the human protein profiles in a large number of normal and cancer tissues as well as a large set of human cell lines.
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| 9. |
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