Characterization of PrEST-based antibodies towards human Cytokeratin-17

• Published in: Journal of immunological methods Vol. 342; no. 1-2; pp. 20 - 32
• Main Authors: Larsson, K., Eriksson, C., Schwenk, J.M., Berglund, L., Wester, K., Uhlén, M., Hober, S., Wernérus, H.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.03.2009; Elsevier
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antibody; Antibody Specificity; Bioengineering; Biological and medical sciences; Bioteknik; Cytokeratin-17; Epitope Mapping; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Keratin-17 - immunology; MEDICIN; MEDICINE; Molecular immunology; Peptide Mapping; PrEST; Protein Array Analysis; Proteomics - methods; Rabbits; Techniques; TECHNOLOGY; TEKNIKVETENSKAP; Tissue Array Analysis; PrEST; Epitope mapping; Antibody; Cytokeratin-17; ABP; Human; Cytokeratin; Keratin; Antigenic determinant; Cytoskeleton; Intermediate filament; Immunological method
• ISSN: 0022-1759; 1872-7905; 1872-7905
• DOI: 10.1016/j.jim.2008.11.013

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.