Abstract

It has been shown that a repetitive motif with the sequence FKEL(F) within the Ki-67 antigen (pKi-67) serves as an epitope for the Ki-67 antibody and equivalent clones. However, no direct correlation between reactivity towards Ki-67 epitopes and reactivity in formalin-fixed paraffin-embedded (FFPE) tissue could be found. In this study our aim was the isolation and characterization of new monoclonal Ki-67 equivalent antibodies in an in vitro approach. To select pKi-67 reactive phage antibodies, we used a large naive Fab-phage library (Human Combinatorial Antibody Library; HuCAL). We implemented a panning strategy against two different overlapping peptides, both containing the 'FKELF' epitope. ELISA screening of randomly picked phage antibody clones after the third selection round yielded six highly reactive clones against the 'FKELF' epitope, of which five were found to be reactive in FFPE tissue, showing a Ki-67 equivalent staining pattern. Substitutional epitope analysis on peptide arrays of the new recombinant pKi-67 binders and of the established murine clones Ki-67, Mib-1 and Mib-5 were carried out to compare their fine specificities. The results suggest that the lysine residue in the epitope is critical for recognition of Ki-67 antigen in FFPE tissue.