Activation-specific binding of single-chain antibody MAN-1 in flow cytometry. (A) MAN-1 binding to Mac-1–expressing CHO cells. The expression level of the CHO cell line expressing the native and the GFFKR-deleted Mac-1 are evaluated with anti-CD11b and anti-CD18 mAbs (open curves) in comparison to matched isotype controls (shaded curves). (B) MAN-1 binding to PMA-activated but not to nonactivated human monocytes. (C) Binding of various concentrations of MAN-1 to PMA-activated monocytes in comparison to resting monocytes. The binding of MAN-1 in panels A, B, and C is detected with an Alexa Fluor 488–labeled anti–His-tag antibody and compared with the binding of an nonspecific single-chain antibody. Typical examples are shown, as routinely assessed after each scFv purification (n > 20).