Kinetic Analysis of Single-Domain Antibodies

A Bio-Rad ProteOn XPR36 system was used to assess binding kinetics of the sdAb to target (L1 or SEB) immobilized on channels of a standard GLC sensor chip as detailed previously [18 (link), 19 (link)]. Briefly, the antigen was immobilized to the sensor chip surface on four rows at saturation concentrations of 10 µg/mL using EDC/NHS chemistry. The chip was turned 90 degrees, then dilutions of each sdAb (ranging from 100 to 0 nM) were flowed across the chip for 120 s at 100 µL/min, and the association was recorded. Next, dissociation was monitored as buffer was flowed over the chip for 600 s. The L1 surface was regenerated flowing through 50 mM glycine (pH 2.5) between individual samples. The one shot kinetics were determined from each of the antigen coated rows using five concentrations of single domain antibody, and kinetic parameters were calculated using the standard Langmuir binding model available on the ProteOn Manager RM 2.1 software (Bio-Rad). Typically the four values for K_D are within 20 %, and the range of values from the four measurements was always within a factor of 2.