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Wash the cuvette with PBS/T buffer and re-establish the baseline.
Repeat the binding cycle with further concentrations of the ligate or different ligate.
Data analysis
Kinetic parameters of an interaction were determined by IAsys FASTfit software which can be used to analyze data obtained using the IAsys system. The on-rate constant was determined by analyzing the initial 90% of the binding of varying concentrations of ligate to immobilized ligand. If the association curve had two phases, the measured on-rate constant obtained from first phase of biphasic association analysis was used. A plot of on-rate constants versus the concentrations of the antibody was obtained. The slope of the plot is the association rate constant (Ka) and the intercept value on the y-axis is the dissociation rate (Kd). The equilibrium constant can then be calculated from association and dissociation rate constants (KD = Kd/Ka).
Kd can also be obtained by analysis of the dissociation curve. The off-rate constant was determined by analyzing the initial 90% of the binding of varying concentrations of ligate to immobilized ligand. The average of the off-rate is Kd. The equilibrium constant can also be calculated from association rate constant obtained by on-rate analysis and dissociation rate constant obtained by off-rate analysis (KD = Kd/Ka).
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