Figure 6. Comparison of ligand-binding activities between GPIb-IX/ND and glycocalicin.
4 μg/ml WM23 IgG was immobilized onto microtiter wells to capture GPIb-IX/ND (●) and glycocalicin (■). Empty Nanodiscs (▲) was also added to the WM23-coated wells as a control. (A) 0–6.4 nM full-length VWF or (B) A1A2A3 tri-domains was incubated with GPIb-IX/ND or glycocalicin or control wells in the presence of 0.2 U/ml botrocetin. The binding was detected by anti-VWF antibody and HRP-conjugated secondary antibody. (C) Difference in capturing efficiency. 4 μg/ml WM23 IgG F(ab′)2 fragment was coated to microtiter wells to capture GPIb-IX/ND or glycocalicin. Empty Nanodiscs was also added to the wells coated with WM23 F(ab′)2 fragment as a control. SZ2 (1 μg/ml) was incubated and detected by HRP-conjugated goat-anti-mouse antibody. “Non-specific binding” is the binding of SZ2 to the control wells. Absorbance at 450 nm was measured from 3 independent experiments and presented as the mean ± SD. For some of the points, the error bars are smaller than the symbols. One hundred percent in (C) is defined as the binding of SZ2 to GPIb-IX/ND captured by WM23 F(ab′)2 fragment.