( reporter validation test is designed t...)Correlation of Bio-Plex Suspension Array Reader Validation With Multiplex Cytokine Assay Performance, Rev A

reporter validation test is designed to confirm that the reporter channel is correctly measuring assay signals by simulating the results of a typical assay. This test utilizes a set of beads containing various intensities of a dye similar to R-phycoerythrin, the fluorochrome used in Bio-Plex assays.*

The fluorescence of each bead within the set is expressed as molecules of equivalent soluble fluorescence, or MESF, a unit that corresponds to the fluorescence intensity of a given number of pure fluorochrome molecules in solution (Henderson et al. 1998). The beads are analyzed on the Bio-Plex system and a plot of fluorescence intensity versus concentration is created. The plot is used to calculate a series of parameters that are typically used to evaluate assay performance, including dynamic range, accuracy, linearity, sensitivity, and slope. Table 2 lists each of the parameters and their respective definitions.

Methods and Results
Effect of Misaligned Optics on Reporter Channel Performance
If the Bio-Plex system is moved or jarred, its optics may become misaligned, adversely affecting assay results. To demonstrate this phenomenon, the optics path of the reporter channel of a Bio-Plex system was deliberately misaligned to different degrees. The degree of misalignment of the system was verified using the reporter channel %CV of the optics validation test (7.6%, 10.5%, 16.0%, and 18.0%). A reporter channel %CV of <10% is considered acceptable. At each degree of misalignment, a reporter validation test was performed and each of the reporter validation parameters (dynamic range, linearity, accuracy, slope, and sensitivity) was evaluated to assess the ef
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