Estimated KDfor IgG of each patient’s ancestor and mutated antibodies from 3 independent experiments (circles), including the representative experiment shown in (B), which used RF antibody TJ4 with known KDas the reference antibody,Equation 1, and threshold IgM fluorescence intensities determined by visual inspection (seeSTAR Methods). RF antibodies TJ5 and TJ0 with known KDwere included as validation antibodies. Only one data point for TJ0 and two data points are shown for P4 ancestor, as sufficient membrane IgM- for IgG-dextran binding was only achieved in 1 and 2 out of 3 experiments, respectively (seeFigure S6B;Table S6). Bar columns represent the mean estimated KDfor each antibody.
