Phage displayed antibodies specific for a cytoskeletal antigen. Selection by competitive elution with a monoclonal antibody

Abstract

The display of repertoires of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents with defined specificities. Here we report the selection of antibody fragments against the cytoskeletal fraction of T24 bladder cancer cells. To focus selection to a specific antigen, we eluted bound phage with a mouse monoclonal antibody directed against cytokeratins. Our initial studies proved that such a selection procedure with a library, carrying the mouse antibody fragment repertoire, resulted in phage specificity for the antigen against cytokeratin 8, recognized by the mouse monoclonal antibody. To facilitate detection of reactive clones, monoclonal antibodies against phage epitopes were developed. A human synthetic library (> 10(8) clones) was used for selection by competitive elution after binding to T24 cytoskeleton. About 50% of the phage reacted in ELISA with cytoskeletons of T24 cells, while with noncytokeratin containing cells no reaction was observed. Immunofluorescence studies and Western blotting with a number of these clones showed reactivity against cytokeratin. We conclude that the competitive elution method can be used as a rapid technique to obtain immunoreactive phages, and eventually human single chain antibodies directed against defined epitopes which were formerly characterized and validated by mouse monoclonal antibodies.