Selection of phage-displayed antibodies specific for a cytoskeletal antigen by competitive elution with a monoclonal antibody

Abstract

A phage-display combinatorial library of VL and VH sequences of mouse antibodies was constructed, which contained 4.5 x 10(7) independent clones. From this library pools of phage were selected by up to four biopanning rounds on cytoskeletal preparations of ovarian carcinoma cells (OVCAR-3). Phage of these pools were then allowed to bind to a cytoskeleton preparation of bladder carcinoma cells (T24). The binding phage were challenged by a monoclonal antibody (mAb) directed against an epitope on cytokeratin 8. Displaced phage were rescued and screened for anti-cytokeratin immunoreactivity by ELISA, indirect immunofluorescence and Western blotting. About 50% of the phage selected by competition with the cytokeratin mAb reacted with the cytoskeletal preparations of T24 cells in ELISA. In contrast, in non-cytokeratin-containing cells, no reaction was observed. Immunofluorescence and Western blotting studies with a number of these clones showed reactivity against cytokeratin. We conclude that the phage-display competitive elution method can be used as a rapid technique to obtain immunoreactive phages, and eventually single-chain Fv (scFv) antibodies directed against defined epitopes, which were formerly characterized and validated by mAbs.