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

e range of standards (Figure 4).

The high-sensitivity standards yield a much lower fluorescence intensity signal since the concentration range is much lower. When using the low range standards, reading the assay with the high PMT voltage may improve the sensitivity. Table 5 shows a comparison of the recovery of a low range GM-CSF assay run using the low and high PMT voltage settings. The low PMT setting yielded acceptable recovery down to only 3.13 pg/ml. When the same assay was run at the high PMT setting, accurate recovery down to 0.2 pg/ml was achieved.


Guidelines for Running Cytokine Assays
The overall assay performance must be taken into consideration when determining how to set up both the cytokine assay standards and the Bio-Plex array reader. In Table 6, the guidelines for optimizing the Bio-Plex system (including assays and the array reader) are shown. The standards as well as the PMT voltage chosen for reading the assay may significantly affect results.


Selecting a Custom Set of Standards
The two sets of standards and RP1 target values discussed in this tech note have been validated using the Bio-Plex system and have been shown to yield optimal results. Efficient assay performance may also be achieved using a custom set of standards as long as a few simple rules are followed.

1. Select a minimum of 8 standard curve points.
The logistic regression model requires a minimum of 5 standards for the 4PL and 6 standards for the 5PL to fulfill the analysis requirements (Motulsky 1996). A total of 8 points are recommended to ensure an accurate fit, with a maximum of 12 points (Baud 1993)
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