Binding studies by biolayer interferometry (BLI)

Binding kinetics measurements were performed using biolayer interferometry on a ForteBio Red Octet 96 instrument (Sartorius, Inc., Göttingen). Briefly, individual human antibodies were diluted to 10  µg/mL, loaded onto Protein A biosensors (Octet, 185010) for 30 s, and washed to remove any unbound material before conducting measurements in PBST. The immobilized antibodies were incubated with varying concentrations of F1 to capture the kinetic data. All binding data were collected at 30°C. A total of 4–6 concentrations of antigens were used. The baseline and dissociation steps were carried out in a buffer only. The kinetic data were fit to a simple 1:1 binding model to determine the dissociation constant (K_D) using the association (Kon) and dissociation (Koff) rates. Binding assays were performed in triplicate, and average K_D values were calculated.