Kinetic exclusion analysis (KinExA) of the solution binding properties of the 4LCA antibody and BoNT/A LC.

<p>(A) The equilibrium binding affinity was determined by titrating recombinant BoNT/A LC into solutions of fixed 4LCA concentration. At equilibrium, free 4LCA was captured by passage through a bead matrix conjugated to LC and measured by binding of a fluorescent secondary antibody. The data were fit using the manufacturer's software to a bimolecular binding model, giving an optimized KD value of 31±5×10⁻¹² M. (B) The association rate constant (k_on) was measured by mixing 4LCA (200 pM) with LC (10 pM) and measuring the concentration of free antibody concentration over time. The average k_on value for 4LCA was estimated to be 2.3±0.1×10⁶ M⁻¹ s⁻¹, which gives a calculated k_off value of 7.1×10⁻⁵ s⁻¹.