GE HEALTHCARE Biacore T100, Handbook

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9 Kinetics and affinity analysis
9.5 Quality assessment for affinity evaluation
172
Biacore T100 Software Handbook BR-1006-48 Edition AE
or to residuals averaged over a moving time window. The latter option smooths
the experimental residual display, making it easier to observe the general shape
of the residual curves. Movable horizontal
Limit guides can be displayed to
mark the extent of the residual variation and aid visual interpretation. (Note that
the limit guides are not related to the guidelines shown on the residual tab for
QC purposes (Section 9.4.1), and do not in any way imply acceptance limits.)
Figure 9-4. Kinetic data check comparison to residuals (left) and averaged residuals (right).
9.5 Quality assessment for affinity evaluation
Steady state affinity evaluations are performed by fitting a plot of R
eq
against
concentration C to a model representing equilibrium 1:1 binding. The closeness
of fit is reported as a chi-squared value, calculated in the same way as for
kinetics. Note however that the number of points in the steady state affinity plot
is very much lower than for kinetic evaluation, so that chi-squared is a more
sensitive indicator of fitting quality.
The plot of R
eq
against C approaches a limiting value (equivalent to R
max
) at very
high concentrations. Robust evaluation of the data requires that the plot shows
sufficient curvature for reliable estimation of R
max
. As a rule of thumb, the
evaluation is acceptable only if the calculated K
D
value is less than half the
highest analyte concentration used. (For a 1:1 interaction, the K
D
value is equal
to the analyte concentration that gives 50% saturation of the binding sites, so
that R
eq
=0.5R
max
. In other words, reliable evaluation is only obtained if the
surface is more than 50% saturated at the highest analyte concentration.)
To help in this assessment, the calculated K
D
value is indicated as a vertical line
at the corresponding analyte concentration. The line is red and broken if the
value is greater than half the highest concentration.