LUMAT LB 9507 - Calculation of a Variable Reference Value; For Standardization of the Logit Transformation

Appendix LB 9507
6
When using a manually smoothed spline with a smoothing factor
which is larger than the one that was calculated automatically it is
recommended that you print out the curve plot, since there is
always the danger that the deviation from the original points may
become too large.
A.1.4 Calculation of a Variable Reference Value
for Standardization of the Logit Transformation
This is possible with LIA and ILMA curves according to the
formula
X: Conc. value
B: RLU value
with b ~ 1, since the transformation has to yield a linear curve.
The reference value X
50
is (also) used only as an internal variable
for calculation.
B
max
can then be calculated by using three different standard
points (X
i
B) in an iterative approximation.
The value pairs should be distributed rather evenly over the curve,
the highest value being the one with the highest standard
concentration and the lowest one a value which is not equal or
adjacent to the value with the lowest standard concentration, so
that errors caused by an inaccurate measurement of the non-
specific binding do not affect the result too much.
If the approximation does not lead to a satisfactory result, use
plausible substitution values for the maximum binding (either 20
% or nearly 100 %). If in special cases only two value pairs are
available for calculation, the slope factor is equated with 1.
B
B
b
- NSB
- B
=
X
X
50max
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