Figure 4.

(a) FITC-labeled OBI binding to Daudi cells with two increasing concentrations of 5 and 15 nM during the association phase, dissociation was measured in plain medium. (n = 4) (b) InteractionMap analysis for OBI binding to Daudi cells, input data from all four binding curves in (a) was used for the calculation. The analysis results in two defined interaction components that mainly differ in their binding stability (x-axis). The more stable component is contributing more to the overall interaction pattern, as indicated by warmer colors in the plot. However, the difference in contribution is less between the two components than for RTX (compare to Figure 1b). (c) Daudi cells were incubated with FITC-OBI in the concentration range from 5 to 60 nM until binding levels are close to equilibrium followed by replacement with the same concentration of unlabeled OBI to observe the release of bound FITC-OBI. (d) Binding of 60 nM FITC-labeled OBI-Fab to Daudi cells in the presence (red) and absence (blue) of 60 nM unlabeled OBI-Fab. (e) Interaction Map analysis for the binding traces displayed in (c). With increasing concentration the main interaction component shifts to the right on the x-axis, indicating that OBI binding becomes overall less stable with increasing concentrations. From 7 nM on, a second interaction component becomes visible with separates completely from the main interaction at 10 nM. This secondary interaction component is more stable and does not change with further concentration increase, but has a minor contribution to the overall interactions, as indicated by the color scale.