Affinity analysis of non-steady-state data obtained under mass transport limited conditions using BIAcore technology.

Abstract

Binding data obtained with Biacore instrumentation is often evaluated using a kinetic transport model where reaction rate constants and a mass transport coefficient are used to describe the interaction. Here the use of a simplified model, an affinity transport model, for determination of the affinity (K(D)) but not the kinetics (k(a), k(d)) has been investigated. When binding rates were highly governed by mass transport effects the two models returned the same affinity and gave similar residuals, but k(a) and k(d) values found with the kinetic transport model were unreliable. On the other hand the affinity transport model failed to describe the data when binding curves were less influenced by mass transport effects. Under such circumstances the kinetic transport model returned correct k(a) and k(d) values. Depending on the outcome of the analysis the affinity transport model can therefore be used to reduce uncertainties of the kinetic parameters or as an easy way to determine K(D) values from non-steady-state data. The use of the affinity transport model is illustrated with simulated data and with binding data obtained for the interaction between a 439 Da thrombin inhibitor and immobilized thrombin. Since it is more difficult to resolve high k(a) values for low molecular weight analytes, the affinity transport model may be particularly useful for affinity analysis involving fast reactions between such analytes and immobilized protein targets.