Binding specificity of hFcγRs for monoclonal IgG. (A) Histograms show the binding of anti-FLAG mAb (thin line) or its isotype control (solid gray), and the binding of monoclonal human IgG subclasses in complex with PE-F(ab′)2 anti–human F(ab′)2 to FLAG-tagged hFcγRs on CHO transfectants. Concentrations of human IgG and PE-F(ab′)2 anti–human F(ab′)2 are indicated. Solid gray histograms represent the binding of PE-F(ab′)2 anti–human F(ab′)2 alone; 2 independent experiments gave similar results. N.T. indicates not tested. (B) FcγRIIIAV158 is a low-affinity receptor for IgG2. Histograms show the binding of heat-aggregated IgG2 or IgG2-F′2 IC to FLAG-tagged hFcγRIIIA on CHO transfectants. Concentrations of human IgG and PE-F(ab′)2 anti–human F(ab′)2 are indicated. Solid gray histograms represent the binding of PE-F(ab′)2 anti–human F(ab′)2 alone. Affinity constants (×105 M−1) were determined from SPR analysis on immobilized FcγRIIIA ectodomains using the same concentrations of IgG2 as in Figure 3A. The standard deviation of the affinity constant (KA) determination is indicated.
