Human Phage Display Panning

E-ALPHA^® human phage display library was used to screen for clones that specifically bind to MUC16^ecto. Independent panning was carried out using 15 different phage sub-libraries. Individual scFv phage clones positive for MUC16^ecto were determined by FACS and the clones that possessed unique DNA coding sequences were subjected to further characterization. Positive phage clones were further validated for binding to MUC16^ecto overexpressing HEK293 cells. For phage display screening, unmodified HEK293, HEK293 expressing MUC16 (HEK-MUC16WT) or HEK293 cells expressing mutant MUC16 (HEK293-MUC16mut) were used. For FACS screening, phage clones were incubated with MUC16^ecto overexpressing HEK293 cells, then with anti-M13 mouse antibody. APC-labeled anti-mouse IgG secondary antibody was added to the reaction after washing. Binding was measured by FACS and expressed as mean fluorescence intensity (MFI). Cells incubated with secondary antibody alone, M13 K07 helper phage, and cells only were used as negative controls.