Figure 3.

Kinetics of scFv double and triple mutant proteins’ binding to the Fu-bc subunit protein. The Fu-bc subunit protein was coupled to the SPR biosensor chip, and scFv double (Mutant11–14) and triple (Mutant14–18) mutants were applied at graded concentrations (25–500 nM) over the coupled surface (from t = 0 to t = 300 s), followed by buffer washout (dissociation) for another 180 s and measurement of net binding (in RU). The association (k_a) and dissociation rate constants (k_d) were derived by fitting the recorded sensograms to (1:1) Langumir-binding rate equations, and the affinity constant K_D was derived by dividing the dissociation constant (k_d) with the association constant (k_a). The overlay of curves for different concentrations of scFv protein was fitted into a numerical model by global analysis using Autolab kinetic evaluation software 5.1. The color of each fitted curve shown is a representative response of different concentrations of analytes (i.e., green, 25 nM; golden, 50 nM; violet, 100 nM; blue, 200 nM; and red, 400 nM). Fu-bc, fusion and bc loop; scFv, single chain variable fragment antibody; SPR, surface plasmon resonance.