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| 2. |
- 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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| 3. |
- Paavilainen, Linda, et al.
(författare)
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Evaluation of monospecific antibodies : a comparison study with commercial analogs using immunohistochemistry on tissue microarrays
- 2008
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Ingår i: Applied immunohistochemistry & molecular morphology (Print). - 1541-2016 .- 1533-4058. ; 16:5, s. 493-502
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Tidskriftsartikel (refereegranskat)abstract
- Generation of monospecific antibodies (msAbs) (multiepitope) through affinity purification of polyclonal antisera is a plausible strategy for high-throughput production of affinity reagents toward large sets of proteins. These antibodies are generated using readily accessible gene sequence information from publicly available databases. The resulting antibodies have the potential to be used in a variety of assays, probing differentially presented and altered proteins with high sensitivity and specificity. In the present study, 48 msAbs were compared with corresponding commercial analogs. Immunohistochemical staining properties were evaluated on tissue microarrays, representing various normal human tissues from 144 different individuals. MsAbs showed similar immunostaining patterns as compared with corresponding commercial analogs in 44 out of totally 48 (92%) antibody pairs analyzed. Although only few antibody pairs showed major discrepancies, minor dissimilarities were frequently seen. Our results suggest that msAbs are reliable and valuable tools in antibody-based proteomics, enabling analysis of protein expression patterns in cells and tissues. High-throughput strategies employing such antibodies provide a consistent approach in the exploration of the human proteome.
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| 4. |
- Paavilainen, Linda, 1979-, et al.
(författare)
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The Impact of Tissue Fixatives on Morphology and Antibody-based Protein Profiling in Tissues and Cells
- 2010
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 58:3, s. 237-246
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Tidskriftsartikel (refereegranskat)abstract
- Pathology archives harbor large amounts of formalin-fixed, paraffin-embedded tissue samples, used mainly in clinical diagnostics but also for research purposes. Introduction of heat-induced antigen retrieval has enabled the use of tissue samples for extensive immunohistochemical analysis, despite the fact that antigen retrieval may not recover all epitopes, owing to alterations of the native protein structure induced by formalin. The aim of this study was to investigate how different fixatives influence protein recognition by immunodetection methods in tissues, cell preparations, and protein lysates, as compared with formalin. Seventy-two affinity-purified polyclonal antibodies were used to evaluate seven different fixatives. The aldehyde-based fixative Glyo-fixx proved to be excellent for preservation of proteins in tissue detected by immunohistochemistry (IHC), similar to formalin. A non-aldehyde-based fixative, NEO-FIX was superior for fixation of cultured cells, in regard to morphology, and thereby also advantageous for IHC. Large variability in the amount of protein extracted from the differently fixed tissues was observed, and the HOPE fixative provided the overall highest yield of protein. In conclusion, morphological resolution and immunoreactivity were superior in tissues fixed with aldehyde-based fixatives, whereas the use of non-aldehyde-based fixatives can be advantageous in obtaining high protein yield for Western blot analysis. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58:237-246, 2010)
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| 5. |
- Paavilainen, Linda, 1979-
(författare)
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Validation of antibodies for protein profiling : A study using immunohistochemistry on tissue microarrays
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The field of proteomics has rapidly expanded due to the completion of the human genome sequence. This thesis validates affinity-purified monospecific antibodies of polyclonal origin, for protein profiling in a broad spectrum of normal tissues and cells. Validation of antibodies is crucial for development of reliable binders for target proteins and this thesis evaluates the generation and application of large sets of msAbs in different settings. MsAbs were generated towards recombinant Protein Epitope Signature Tag (PrEST) antigens using a stringent affinity-purification strategy, presented in the first study. The specificity of msAbs was studied using reverse phase protein arrays and immunohistochemistry (IHC), and results presented over 90% success rate in the protein array analysis. In IHC, 81% of the msAbs displayed apparent specific staining in normal tissues. MsAbs were also compared with commercial analogs (cAbs) using IHC and Western blot. Results presented similar outcome between msAbs and cAbs in both applications, although interpretation suggested more extensive IHC staining patterns with msAbs than with monoclonal analogs. For antibody validation, an approach called paired antibodies was presented and involved the generation of two msAbs towards non-overlapping epitopes on the same protein. Similarities in protein detection between paired antibodies were studied using three different antibody-based methods. Similar results were observed in several applications, indicating that this strategy can be a useful tool for studying known and unknown proteins. Given the reliability of msAbs, they were also applied in a study investigating the impact of tissue fixatives on protein detection. The study showed that different fixation mechanisms appeared to affect protein recognition by indicating that aldehyde-based fixation, e.g. induced by neutral buffered formalin, was preferred for tissues used in IHC and non-aldehyde based fixation was applicable for tissues used in protein extraction analysis and Western blotting. Conclusively, validation results suggest that msAbs are reliable affinity binders that can be used as valuable tools for proteome-wide protein profiling in tissues and cells.
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| 6. |
- Uhlén, Mathias, et al.
(författare)
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A human protein atlas for normal and cancer tissues based on antibody proteomics
- 2005
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 4:12, s. 1920-1932
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, similar to 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.
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