Kinetic Exclusion Assay (KinExA) Theory

This page describes the Kinetic Exclusion Assay (KinExA) theory. We show how kinetically excluding the dissociation of ligand-receptor complex in measurements underlies our ability to perform exact solutions to the classic binding equations shown in the Kd mathematics.

The KinExA method measures the concentration of uncomplexed receptor (R) molecule in a mixture of receptor, ligand (L), and LR complex. The concentration of uncomplexed R is measured by exposing the solution phase mixture to solid phase immobilized L for a very brief period of time. The "contact time" between the solution phase mixture and the solid phase immobilized L is kept short enough that dissociation of LR complex is insignificant. When the possibility of significant dissociation of LR complex is kinetically excluded, only uncomplexed ("free") R can bind to the solid phase. The amount of free R that binds to the solid phase (measured by fluorescence emission from a secondary label) is directly proportional to the concentration of free R in the solution phase sample.

KinExA technology is based on the following conditions:

1. The concentration of L bound to the solid phase is in excess. When the concentration of solid phase immobilized L is in excess, the concentration of "free" R captured on the solid phase is directly proportional to the concentration of uncomplexed R in the solution phase sample.

2. The "contact time" between the solution phase mixture and the solid phase immobilized L is kept short enough that dissociation of LR complex is insignificant.  When the possibility of significant dissociation of LR complex is kinetically excluded, only uncomplexed ("free") B-component can bind to the solid phase.

3. The fluorescent labeling entity is in excess, and the unbound label is washed from the cell. The resulting fluorescent signal is directly proportional to the free R in the original solution phase mixture.

Results of using the KinExA method are the following:

1. The solid phase is used only as a probe to measure the concentration of uncomplexed R in the solution phase sample.

2. The fluorescence signal (in Volts) is directly proportional to the concentration of uncomplexed R in the solution phase sample.

3. The fluorescence signal is linearly related to the concentration of uncomplexed R in the solution phase sample.