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Evaluation of monospecific antibodies: a comparison study with commercial analogs using immunohistochemistry on tissue microarrays
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2008 (English)In: Applied immunohistochemistry & molecular morphology (Print), ISSN 1541-2016, Vol. 16, no 5, 493-502 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2008. Vol. 16, no 5, 493-502 p.
Keyword [en]
antibody proteomics, monospecific antibody, immunohistochemistry, tissue microarray, protein atlas
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-103803DOI: 10.1097/PAI.0b013e31817c645eISI: 000259648200014PubMedID: 18685494OAI: oai:DiVA.org:uu-103803DiVA: diva2:218728
Available from: 2009-05-20 Created: 2009-05-20 Last updated: 2009-08-27Bibliographically approved
In thesis
1. Validation of antibodies for protein profiling: A study using immunohistochemistry on tissue microarrays
Open this publication in new window or tab >>Validation of antibodies for protein profiling: A study using immunohistochemistry on tissue microarrays
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 54 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 474
antibody validation, monospecific antibodies, protein profiling, immunohistochemistry, tissue microarrays, western blot, fixatives
National Category
Cell and Molecular Biology
Research subject
urn:nbn:se:uu:diva-107471 (URN)978-91-554-7588-8 (ISBN)
Public defence
2009-09-25, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsv 20, Uppsala, 13:15 (English)
Available from: 2009-09-04 Created: 2009-08-12 Last updated: 2009-09-04Bibliographically approved

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