Subtractive panning (depletion/selection) strategy for the generation of conformation-specific single-chain antibodies. The first round was performed on human monocytes. A depletion step was performed in which phages bound to nonactivated Mac-1 or to the monocytes cell surface were separated by centrifugation and discarded. The supernatant was used for the next selection step in which the nonbinding phages in the supernatant were discarded and the binding phages were rescued and eluted. The obtained phages were then amplified in Escherichia coli and used for the next round. In the following 3 rounds (only 2 are pictured), the cell background was changed to Mac-1–expressing CHO cells to avoid enrichment of phages binding to activation-specific monocyte epitopes other than those on Mac-1.