Effect of CD16A-Ig on complement-mediated hemolytic activity. (A) Rabbit anti-DNP-IgG opsonized rRBCs were incubated with or without various concentrations of CD16A-Ig and then 1:5 diluted mouse serum was added. Hemolytic activity was then carried out as described in the text. Cells treated with buffer alone served as a control. Cells lysed with water represented 100% hemolysis. The supernatant was read at 450nm. Data are representative of 3 independent experiments. (B) Clearance of FcγR-Ig dimers from circulation. A group of mice (n = 3) were injected with dimers and blood samples were collected at various time points and diluted in PBS/EDTA. Plasma was collected to detect the FcγR-Ig dimers by sandwich ELISA. Plates were coated with 50 μL of 10 μg/mL anti-hCD16A (CLBFcgran-1) and anti-hCD32A (IV.3) mAbs overnight at 4°C. The wells were then blocked with PBS/5mM EDTA/1% BSA. After washing, 50 μL of the plasma samples were added into the wells and incubated for another 1 hour. The wells were washed and 50 μL of HRP-conjugated anti–human Fc specific antibody were added and incubated for another 1 hour. HRP substrate was added to the washed wells and read at 450 nm. The plasma from a group of normal mice injected with PBS served as a specificity control. As positive controls, purified CD16A-Ig and CD32A-Ig were used while BSA coated wells served as negative controls. Purified CD32A-Ig was used as a standard to quantify the level of dimers in the blood. Data are means plus or minus SD of triplicates.