Interspot vs. Channel Referencing
In order for interspot referencing to be utilized with multiplex SPR, it must provide kinetic data equivalent to that obtained using a separate channel to determine nonspecific binding.
To demonstrate the equivalence of these two referencing methods, a model system was constructed using interleukin-2 (IL-2) as the analyte and IL-2 antibody as the ligand. Five vertical channels were created on a sensor chip using the MCM, with each channel containing the ligand at a different density. An injection of buffer only into the last vertical channel created a channel reference devoid of ligand.
The IL-2 analyte was then injected into the horizontal channels at six different concentrations to obtain complete binding kinetic data in one experiment (One-Shot Kinetics). The data was referenced using either the vertical reference channel or the horizontal interspots (where no ligand was immobilized) and is presented in the Table.
The global association and dissociation kinetic rate constants (ka and kd), as well as the equilibrium dissociation constants (KD) are virtually identical, whether interspot or channel referencing is used. In addition, when comparing these three values across the five different ligand surfaces, all the CVs are between 2–7%.
This data also illustrates the lack of dependence of kinetic data constant values on the amount of ligand bound to the sensor chip (ligand density), since the kinetic constants were virtually identical at all five densities, using either interspot or channel referencing. Thus, multiplexed SPR can produce reliable binding analysis data across a wide range of ligand densities.