where A 0 and A are the absorbances measured for the antibody in the absence and presence of antigen, respectively, and a 0 and i 0 respectively the total concentrations of antigen and antibody. Increasing concentrations (0.5-100 nM) of the VWF fragments or pVWF with or without being denatured were mixed with a constant amount of SZ34 (0.3 nM), in 1% (w/v) BSA-PBS, respectively, until equilibrium were reached (15 h incubation at 22uC). Then the proportion of SZ34 which remained unsaturated at each concentration of various VWF fragments and pVWF was measured by an ELISA method as following. The antigen/ antibody complexes were added to the microtiter plates coated with 100 ml of pVWF (7.5 mg/ml) and incubated at 37uC for 2 h. After being washed, the wells were incubated at 37uC for 2 h with HRP-labeled goat anti-mouse IgG (Sigma) in 1% (w/v) BSA-PBS. The plates were washed 5 times and tetramethyl benzidine (TMB) solution was added for color development. Absorbance was read at 450 nm and A 0 and A were then obtained, respectively.