Bio-Rad Bio-Plex System Validation and Calibration Tools - Manual (Page 9)

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Slope of the Reporter Channel Response
Definition
The slope of the regression line resulting from the plotting of reporter channel
mean fluorescent values against assigned reporter channel validation bead MESF
values is related to the dynamic range of the instrument. The slope of the
regression line is a function of the response of the reporter channel
photomultiplier tube. The acceptable range for the slope is 0.0593–0.0799.
Impact on Assay Performance
The slope of the regression line is directly related to the dynamic range of the
instrument. The slope yields direct information about the response of the
photomultiplier tube. If the photomultiplier tube signal saturates at low
fluorescence values, the dynamic range of the instrument is affected. The slope
of the line impacts the dynamic range and the range in turn impacts the
quantitatable range of an assay. If the validation kit yields a value for the slope
that is not within specifications, assay results could be adversely affected.
Sensitivity of Reporter Channel
Definition
Every instrument has an inherent level of noise due primarily to the electronics.
The sensitivity of the Bio-Plex array reader is defined as the lowest detectable
signal above instrument noise. Noise can be attributed to the laser, the
photomultiplier tube, the amplification electronics or the fluidics. The acceptable
sensitivity using the Bio-Plex array reader is <200 MESF.
Impact on Assay Performance
The sensitivity using the Bio-Plex validation kit is expressed in terms of MESF.
The fluorescence is traceable to R-PE, the primary molecule used in Bio-Plex
assays. The typical background or zero standard of a Bio-Plex cytokine assay
falls at a median fluorescence intensity of 100. The background of a "blank" bead
from the validation kit exhibits a median fluorescence intensity of 8. A plot of the
median RP1 fluorescence intensity versus the MESF units illustrates that the
instrument is approximately 10 times more sensitive than a Bio-Plex cytokine
assay (See Figure 1). This is a desired result, as the sensitivity of the instrument
should not directly limit assay sensitivity.
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MESF
(R
PE)
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