Characterization of PrEST-based antibodies towards human Cytokeratin-17.
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.
Larsson
K
K
School of Biotechnology, Division of Proteomics, Albanova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
Eriksson
C
C
Schwenk
J M
JM
Berglund
L
L
Wester
K
K
Uhlén
M
M
Hober
S
S
Wernérus
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D018604:Epitope Mapping; D006801:Humans; D053537:Keratin-17 / Q000276:immunology*; D010449:Peptide Mapping; D040081:Protein Array Analysis; D040901:Proteomics / Q000379:methods*; D011817:Rabbits; D046888:Tissue Array Analysis
D000911:Antibodies, Monoclonal; D053537:Keratin-17