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Selection of Standards for Bio-Plex Cytokine Assays, Rev A

ate some typical recovery values for broad range and high-sensitivity standards.


Effect of PMT Voltage on Assay Performance
Bio-Plex assays are analyzed on a Bio-Plex system that contains a PMT. The PMT detects assay signals in the reporter or RP1 channel. The PMT voltage on the Bio-Plex system is automatically adjusted during CAL2 calibration using the RP1 target value from a bead with stable fluorescence. This method yields a reproducible signal output from day to day and between instruments. Two RP1 target values are recommended for use with Bio-Plex assays, a low target value for the broad range standards and a high target value for the high-sensitivity standards. Following calibration, the low RP1 target value yields a low PMT voltage and the high RP1 target value yields a higher PMT voltage. Table 4 compares the resulting PMT voltage on a Bio-Plex system following calibration with the low and high target values.

The PMT voltage is increased in assays in which higher sensitivity is desired. However, when using the broad range standards, the signals of the high concentration standards on certain cytokines may become saturated and result in a reduced dynamic range. Figures 3 and 4 show a broad range standard IL-8 assay analyzed with the high and low PMT settings. The standard points are labeled with the corresponding recovery. In Figure 3, the recovery at the two highest points is beyond the range of the regression, due to a saturated signal at the high end of the standard curve, and thus OOR (out of range) is displayed. When the same assay is analyzed at the low PMT setting, there is no saturation and the standards recovery is accurate across the entir
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