GE HEALTHCARE Biacore T100, Руководство пользователя

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Biacore T100 Software Handbook BR-1006-48 Edition AE
147
Concentration analysis 8
Remove the check-mark from the Include column to exclude a sample from the
evaluation, or from the Cycle# column in the expanded list to exclude individual
cycles. Remember that each sample must be represented by at least two cycles
with different flow rates for the evaluation to be possible.
It is important that the sensorgrams at the lowest and highest flow rates
(recommended 5 and 100 µl/min respectively) are sufficiently separated. If the
curves are close together or coincide, this indicates that there is not sufficient
mass transport limitation in the binding for reliable concentration
measurement. The table column
QC ratio provides a guidance value for this
assessment: sufficient mass transport limitation is indicated by a value for the
QC ratio of about 0.13 or higher. Exclude samples where the ratio is less than this
value.
The QC ratio is calculated as follows:
Under conditions of complete mass transport limitation, the binding rate is
proportional to the cube root of the flow rate, so the quotient Q has a value of 1.
When there is no mass transport limitation, the binding rate is independent of
the flow rate so Q has a value equal to the cube root of the flow rate ratio. The
range of possible theoretical values for Q will thus depend on the flow rates used
(for flow rates of 5 and 100 µl/min, the value is 0.37). The QC ratio is calculated
from the measured value for Q normalized to a scale of 0–1:
The recommended acceptance value of 0.13 or higher for the QC ratio is
empirically determined, and corresponds to a ratio of initial binding rates at 100
and 5 µl/min of about 1.3 or higher.
Occasionally, the measured binding rate may be lower at the higher flow rate,
leading to a negative value for the QC ratio. This occurs as a result of
experimental variation when the binding rate is not affected by flow rate (so the
binding rates should in theory be equal and the QC ratio should be 0). Samples
where the QC ratio is negative do not show sufficient mass transport limitation
for calibration-free concentration measurements.
quotient Q
initial rate at high flow rate
initial rate at low flow rate
-------------------------------------------------------------------
low flow rate
high flow rate
---------------------------------- 3
×
=
Q
max
1
=
Q
min
low flow rate
high flow rate
---------------------------------- 3 =
QC ratio
Q
measured
Q
min
–
Q
max
Q
min
–
-----------------------------------------
=