Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against...

Phage-display of antibody libraries is a powerful tool to select antibodies for specific epitopes. We describe a strategy for selecting highly specific scFv-clones that discriminate between various conformational states of cell surface receptors. This approach adapts the M13 pIII phage-display technology toward a cell suspension-based strategy, which allows panning against complex, multimeric, fully functional cell membrane epitopes without alteration of structure due to purification or immobilization. As the functional properties are preserved, phage can be specifically depleted or selected for neo-epitopes exposed after physiological alterations of the targeted molecules. This subtractive strategy allows highly specific selection for single-chain antibodies directed against functionally regulated epitopes on the cell surface molecules that can be tailored for diagnostic and therapeutic applications. Using this protocol, activation-specific single-chain antibodies can be obtained within 4-6 weeks.