GE HEALTHCARE Biacore T100, Handbook

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9 Kinetics and affinity analysis
9.7 Curve fitting principles
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Biacore T100 Software Handbook BR-1006-48 Edition AE
In some situations, the fitting algorithm may be unable to find a fit for the
experimental data with the initial parameter values as specified in the model.
This may happen typically if the concentration unit is incorrect: for example if
the unit is set to mM instead of nM in the keyword table. On occasion, however,
it can be necessary to adjust the starting values for fitting parameters, accessed
through the
Parameters button in the fitting dialog (step 4 in Section 9.2).
9.7.2 Local and global parameters
Parameters in the fitting equations are treated as either local or global
variables or constants :
• Local parameters are assigned an independent value for each curve in the
data set. Typical local parameters are concentration (which is different for
different curves) and bulk refractive index contribution (which may be
expected to vary between curves).
• Global parameters have one single value that applies to the whole data
set. Typical global parameters are the rate constants for the interaction,
which should in principle have the same value for all curves in the data set.
• Constants have a fixed value that is not changed in the fitting procedure.
An example is the analyte concentration. Constants may also be local
(separate values for each curve) or global (one value for the whole data
set).
The local/global status of parameters can be changed through the Parameters
button in the fitting dialog (step 4 in Section 9.2), without making any changes
to the model.
Evaluating kinetics or affinity with global rate constants gives a more robust
value for the rate constants, although the curves may fit the experimental data
more closely if all parameters are fitted locally. This is because local fitting
allows variation between the constants obtained from different curves: when
the constants are fitted globally, this variation appears in the closeness of fit
rather than the reported values. Rate constants are always global in predefined
kinetic models.
In general, kinetic constants should be fitted as global parameters and bulk
refractive index contribution as a local parameter. The analyte binding capacity
of the surface R
max
is a global parameter by default in the predefined models
(this assumes that the ligand activity is unchanged between cycles in the assay).
It is however justified to use a local R
max
if there is reason to believe that the
ligand activity may vary between cycles (e.g. in a capture assay, if the capture
level varies between cycles).