Molecular Devices SpectraMax Paradigm User Manual page 35

Analyzing Luminescence Data
The conversion rate of photons to counts is individual for each reader. Therefore, raw data
from the same plate can seem significantly different from one instrument to the next. Also,
the data format used by other manufacturers might not be counts per second and can be
different by several orders of magnitude. It is important to know that the number of counts
and the size of figures is in no way an indication of sensitivity. See
Concentrations or qualitative results are derived from raw data with a standard curve or by
comparison with reference controls. A standard curve consists of, at a minimum, a blank
sample and a reference standard sample of known concentration. The raw data can then be
expressed in equivalent concentration of a reference label. The raw data is normalized to
counts per second by dividing the number of counts by the read time per well.
Analyzing and validating luminescence data generally consists of the following:
Background Correction on page 35
Detection Limit on page 36
Sample Volumes and Concentration of Reactants on page 36
Data Optimization on page 37
Background Correction
The light detected in a luminescent measurement generally has two components: specific
light from the luminescent reaction and an approximately constant level of background light
caused by various factors, including the plate material and impurities in the reagents. The
background can be effectively measured using blank replicates. Blanks should include the
luminescent substrate (chemical energy source) but not the luminescence agent (generally
an enzymatic group which makes the substrate glow).
A blank well contains everything used with the sample wells except the label and sample-
specific compounds. Do not use an empty well for a blank.
The blank sample reveals the offset underlying each data sample. This offset does not carry
information on the label, and is generally subtracted before data reduction is done.
For optimum results, Molecular Devices recommends that you run replicates for all blanks,
controls, and samples. In this case, the blank value that can be subtracted is the average
value of all blanks.
To help eliminate background luminescence from a microplate that has been exposed to
light, Molecular Devices recommends dark adaptation of the microplate by placing the
sample-loaded microplate in the instrument for several minutes before starting the read.
5014038 E
Chapter 2: Read Modes and Read Types
Detection Limit on page 36.
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