Molecular Devices SpectraMax Paradigm User Manual page 52

SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Data Qualification and Validation
When validating the data of a fluorescence polarization measurement and the assay, the two
factors to look at are the precision value and the Z´ parameter.
The FP precision value is a measure of replicate uniformity determined by the standard
deviation of replicates at a label concentration of 1 nM. Since the precision of a measured
signal also depends on the read time, the read time must also be specified. A longer read
time leads to a lower (better) precision value.
Z´ is the standard statistical parameter in the high-throughput screening community for
measuring the quality of a screening assay independent of test compounds. It is used as a
measure of the signal separation between the positive controls and the negative controls in
an assay.
The value of Z´ can be determined using the following formula:
Z´ = 1 –
where SD is the standard deviation, c+ is the positive control, and c– is the negative control.
A Z´ value greater than or equal to 0.4 is the generally acceptable minimum for an assay.
Higher values might be desired when results are more critical.
Z´ is not linear and can be made unrealistically small by outliers that skew the standard
deviations in either population. To improve the Z´ value, you can increase the quantity of
label in the sample, if acceptable for the assay, or increase the read time per well.
The assay window is dependent on the fluorophore lifetime and relative size of the receptor
to the ligand. Precision values are better (lower) at higher signals, which normally come from
higher label concentrations.
For a given assay window, Z´ is a downward sloping linear function. That is, as precision
values get higher (worse), the Z´ value gets lower (worse).
Precision is dependent upon assay characteristics (sample volume, label concentration) and
read time. In many assays, the characteristics are defined and cannot be changed. In this
case, the only way to improve precision is to increase the read time per well.
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3(SD ) + 3(SD )
c+ c–
| Mean – Mean |
c+ c–
5014038 E