Characterization of PrEST-based antibodies towards human Cytokeratin-17.
J Immunol Methods
; 342(1-2): 20-32, 2009 Mar 15.
Article
em En
| MEDLINE
| ID: mdl-19108777
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteômica
/
Queratina-17
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Anticorpos Monoclonais
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Especificidade de Anticorpos
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2009
Tipo de documento:
Article