2. Choose standards evenly distributed across the entire range of concentrations.
As noted earlier, the response of a Bio-Plex assay is nonlinear; there is a flattening of the response near zero and at the high end of the curve with a linear portion in the middle as shown in Figure 2. Due to the nature of this response, the chosen standards concentrations should be evenly distributed across the entire range (Nix and Wild 2001). The regression routine will then yield the most accurate representation of the assay response.
3. Choose a 5PL regression routine.
When conditions are optimal, the upper and lower portions of the curve are symmetrical, and the 4PL logistic routine is appropriate. In reality, many conditions, such as a high-dose hook effect and inadequate washing, can cause asymmetry in the curve (Nix and Wild 2001). The 5PL regression contains an additional parameter to accommodate asymmetry in the curve, and thus is the more robust type of logistic regression for Bio-Plex assays.
4. Prepare standards using serial dilutions.
When selecting the range of a custom set of standards, determine the lowest and highest desired concentrations, then determine what dilution series should be used to yield a total of 8 points within the range. For example, if the low and high desired values are 5 and 10,000 pg/ml respectively, a threefold dilution series would yield 8 points (10,000, 3,333, 1,111, 370, 123.4, 41.1, 13.7, and 4.6). A dilution series is simple to prepare and less prone to errors compared to preparation of individual concentrations.