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


Standard Curve Ranges for Bio-Plex Assays
Two sets of standards are recommended for Bio-Plex cytokine assays, a high-sensitivity range (0.23,200 pg/ml) and a broad range (1.9532,000 pg/ml). The specific concentrations for the two sets of standards are shown in Table 1. The highsensitivity standards are recommended for use when the concentrations of the unknowns fall within the physiological range of cytokines in serum. The highsensitivity standards are suitable for quantitation of low levels of cytokines and provide the greatest sensitivity. The broad range standards yield a wide dynamic range and are used when quantitation of a wide range of cytokine concentrations is desired or when the range of expected concentrations of cytokines is unknown. The broad range standards enable the detection of the widest range of concentrations and yield a wide dynamic range. A comparison of the high-sensitivity and broad range standard curves plotted as concentration versus fluorescence intensity is shown in Figure 2.


Effect of Range on Standards Recovery
The overall dynamic range, accuracy, and sensitivity of results obtained using high-sensitivity vs. broad range standards are quite different. These differences may be illustrated using a method known as backcalculation of standards, or standards recovery (Davies 2001). In this method, a regression model is applied to a set of data and the resulting equation is used to calculate the concentrations of the standards using the response values (Nix and Wild 2001). The analysis is performed by calculating the concentration of each standard and then comparing it to the actual concentration usi
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