Single-chain antibody MAN-1 inhibits Mac-1 binding to the ligands heparin, fibrinogen, and ICAM-1, but not C3bi. (A) Inhibition of static adhesion of Mac-1–expressing CHO cells to immobilized fibrinogen and heparin by MAN-1. Adhesion of CHO cells transfected with the GFFKR-deleted Mac-1 to immobilized fibrinogen and heparin was evaluated. Nontransfected CHO cells served as negative control. Mean and standard deviation is given for triplicate experiments. Adherent cells were quantified with a phosphatase-substrate assay, and the absorbance was read at 405 nm. (B) Static adhesion of Mac-1–expressing CHO cells to ICAM-1–expressing CHO cells is inhibited by MAN-1, whereas adhesion to immobilized C3bi is not inhibited. Cells expressing the GFFKR-deleted, activated Mac-1 adhere more strongly to immobilized C3bi than nonactivated Mac-1 cells or a CHO cell control. Binding can be inhibited by an activation-nonspecific anti–Mac-1 antibody, but not by scFv MAN-1. Adherent cells were quantified with a phosphatase substrate assay, and absorbance was read at 405 nm. Mean and standard deviation is given for triplicate experiments. Adhesion of Mac-1–expressing CHO cells to immobilized ICAM-1–expressing CHO cells were counted based on their clearly distinguishable round shape on a flat monolayer of ICAM-1–expressing cells. Six visual fields were counted. Experiments were performed in triplicates. All static adhesion assays were performed at least 5 times. Representative results are shown.