Characterization of PrEST-based antibodies towards human Cytokeratin-17
2009 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 342, no 1-2, 20-32 p.Article in journal (Refereed) Published
Antibody-based proteomics efforts depend on validated antibodies to ensure correct annotation of analyzed proteins. We have previously argued that a low sequence identity to other proteins is a key feature for antigens used in antibody generation. Thus, a major challenge for whole-proteome studies is how to address families of highly sequence related proteins within the context of generating specific antibodies. In this study, two non-overlapping parts of human Cytokeratin-17, a protein belonging to the intermediate filament family of highly sequence-related proteins, were selected as a model system to study the specificity and cross reactivity of antibodies generated towards such a target. These recombinantly produced Protein Epitope Signature Tags (PrESTs) were immunized in five rabbits each and the batch-to-batch variations in the obtained immune responses were studied by mapping of linear epitopes using synthetic overlapping peptides. The obtained results showed a similar but not identical immune response in the respective antibody groups with a limited number of epitopes being identified. Immunohistochemical analysis of the affinity purified monospecific antibodies on tissue micro arrays resulted in a general recognition of human cytokeratins for all analyzed binders whereas antibodies identified as binding to the most unique parts of the PrESTs showed the most Cytokeratin-17 like staining. The data presented here support the strategy to use sequence identity scores as the main criteria for antigen selection but also indicate the possibility to instead produce a single antibody recognizing a defined group of proteins when the intended targets overall sequence identity score is too high. This type of group-specific antibodies would be an important tool for antibody-based projects aiming for a complete coverage of the human proteome.
Place, publisher, year, edition, pages
2009. Vol. 342, no 1-2, 20-32 p.
Cytokeratin-17, PrEST, Epitope mapping, Antibody
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-103930DOI: 10.1016/j.jim.2008.11.013ISI: 000264685300003PubMedID: 19108777OAI: oai:DiVA.org:uu-103930DiVA: diva2:219041