Figure 3.

Demonstration of affinity improvement of B30 over parental R3bH01.

Human BDNF was directly immobilized on a CM5 Biacore sensor chip surface. A dilution series of parental antibody R3bH01 (A) and the affinity optimized antibody B30 (B) were injected, allowed to associate, followed by dissociation in HBS-EP+ running buffer. All data were fit to a 1:1 Langmuir binding model using T200 evaluation software v1.0. Data shown are representative curve fits for at least 2 independent dilution series experiments. Biacore measurements shown were performed at 37ºC. C: Binding of Fab B30 to Human BDNF by Kinetic Exclusion Assay. The B30 Fab, ranging in concentration from 2 nM to 343 fM was mixed with a fixed concentration of human BDNF, either at 60 pM for a K_D controlled curve or 400 pM for a receptor-controlled curve. All solutions of Fab and antigen were incubated overnight at room temperature to attain equilibrium. Data points are a measurement of percentage free human BDNF at each concentration of B30 Fab. D: N-curve analysis was used to globally fit the curve data to obtain an equilibrium dissociation constant, or K_D value.