Bivalent interaction is essential for high avidity, quinine-dependent binding of mAb 314.1 to platelets. mAb 314.1 Fab and IgG, labeled with Alexa Fluor 633 and Alexa Fluor 488, respectively, were incubated with platelets at the indicated concentrations in the presence and absence of 0.1 mM quinine. After incubation, platelet-bound Fab and IgG were measured by flow cytometry without an intermediate washing step. Specific MFI values were obtained by subtracting the signal obtained without the drug from that obtained with the drug. Binding isotherms obtained with (A) mAb 314.1 Fab and (B) 314.1 IgG in a representative study. (C,D) Corresponding Scatchard plots. Lines shown are the linear fit for data points obtained in replicate studies shown in panels A and B. In the experiments shown, KD values for quinine-dependent binding of 314.1 Fab and IgG were 1.9 × 10−5 and 1.4 × 10−10 mol/L, respectively.
