GE HEALTHCARE Biacore T100, Handbook, Page: 203 / 238

Biacore T100 Software Handbook BR-1006-48 Edition AE
203
Affinity in solution 11
11 Affinity in solution
Determination of affinity in solution provides an alternative to steady state
affinity measurements (see Chapter 9) for interactions that take a long time to
reach equilibrium or for any other reason are difficult to determine with a direct
binding assay. In principle, the affinity in solution approach uses Biacore to
determine the free concentration of one interactant in equilibrium mixtures
containing known total interactant concentrations.
11.1 Conventions and background
11.1.1 Experimental setup
The interactants in affinity in solution determination are denoted A and B:
A + B = AB
Experiments are set up so that a fixed concentration of B is mixed with variable
concentrations of A and allowed to reach equilibrium. The free concentration of
B is then determined by injecting the sample over a ligand that binds B but not
A or the complex AB (the interactant A or a derivative thereof is usually suitable
as ligand). It is assumed that the measurement itself does not significantly
disturb the equilibrium in the sample.
The experimental setup requires a calibration curve with known concentrations
of B determined over the same sensor surface, in order to calculate the free B
concentrations in the samples.
11.1.2 Evaluation principles
The equilibrium constant for a 1:1 interaction is given by
or
K
D
A
free
B
free
⋅
AB
-----------------------------
=
K
D
A
tot
AB
–
( )
B
tot
AB
–
( )
AB
--------------------------------------------------------
=