Detection Limit; Linearity And The Linear Dynamic Range; Luminescence Read Mode - Molecular Devices SpectraMax iD3 User Manual

SpectraMax iD3 Multi-Mode Microplate Reader User Guide
For optimal 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.

Detection Limit

The detection limit is the smallest sample concentration that can be measured reliably above
the blank. Determining the detection limit requires taking a number of blank measurements
and calculating an average value and standard deviation for the blanks. The detection
threshold is defined as the average blank plus three standard deviations. If the average
sample value measures above the threshold, the sample can be detected at a statistically
significant level.
The detection limit can be described by the following equation:
Det Limit =
where conc
blank replicates, and blank and Std are average values of the replicates for the blank and
standard wells.
Determining the detection limit for an assay requires multiple blanks to calculate their
standard deviation.

Linearity and the Linear Dynamic Range

Within a wide range at moderately high concentrations, blanked raw data is proportional to
the quantity of label in a sample.
The linear dynamic range (LDR) is defined by:
LDR = log
where LDR is expressed as a log, and max conc lin is the highest concentration in the linear
range that can be quantified.
When the standard curve after blank reduction is not linear in concentration at the lower
end, there might be an incorrect or contaminated blank.
When the standard curve levels are off at the highest concentrations, this can be addressed
to the inner filter effect: excitation does not reach as deep into the sample for lower
concentrations, without being more significantly attenuated (absorbance) layer by layer.

Luminescence Read Mode

In luminescence read mode, no excitation is necessary as the species being measured emit
light naturally. For this reason, the lamp does not flash, so no background excitation
interference occurs.
98
3 Stdev
blank
std – blank
conc
std
is the concentration of the standard, StDev
std
( )
max conc lin
10 detec on limit
is the standard deviation of the
Blank
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