To find the 'true' kinetic parameters, measurements should take place at a very low ligand density, in fact a ligand density that approaches zero.
Of course, there is no signal. Most SPR instruments in the market are very reproducible, but data do contain systematic errors, because of:
The Result is: An "apparent" kd and KD is measured. This is also illustrated in an article in Nature, see graphic.
The AutoVysion generates always the real kinetic and equilibrium constants by extrapolation to zero ligand density.
For determining the equilibrium constant the functional concentration and molecular weight of the analyte should be known. The concentration is being measured on the high ligand density side of the way, the binding rate is limited by the diffusion of the analyte towards the surface. The binding rate is then proportional to the analyte concentration. The instrument enables to measure the analyte concentrations also when the affinity of the analyte to the ligand is weak. (More details will be published soon).
The AutoVysion applies a calibrated flow cell for determination of the functional antibody concentration in combination with the kinetic association and equilibrium constants of an analyte. Under a strict and constant back-and-forth flow rate, mass transport limitation (MTL) occurs on a high density ligand surface. Within 10% of Rmax the signal is linear at the high ligand density area of the gradient and the slope is directly correlated to the concentration of the analyte within certain margins. Default value is the molecular weight of antibodies (150 kD). For other analytes the initial slope will be directly proportional to the molecular weight which can be set in the analysis software of the AutoVysion.