Concentration-dependent binding and inhibition of HK binding to cultured endothelial cells by mAb C11C1. HUVECs cultured to confluence on a fibronectin-coated 96-well Immulon 2HB plate. Mixtures were incubated with HUVECs in binding buffer for 30 minutes at 4°C. Binding was measured as described in “Materials and methods” in the presence of 50 μM ZnCl2. Nonspecific binding of HK or factor XII was determined using Eu-labeled HK or factor XII (Eu-HK, Eu-XII) in the absence of ZnCl2 and was subtracted from total binding to determine specific binding. The inset linear graph shows the concentration-dependent binding of Eu-HK to HUVECs as assessed using 200 μL of each Eu-HK concentration/well. The bar graph shows 100 μL of mixtures of Eu-HK (31 nM, ▪, □) or EU-XII (43 nM, ▧) and various indicated concentrations of mAb C11C1 were added to each well. Control antibody used with Eu-HK is normal purified mouse IgG (Sigma I-52381). The data are plotted as percent bound and comparisons were made to the TRF value obtained in the absence of antibody (0 nM). Mean ± SEM, n = 3. For the concentrations of mAb C11C1, 214 nM (P = .013), and for 1429 nM (P = .004).
