Diana Davis, Aiguo Zhang, Jim Torrence, and Emily Dale, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA
Bio-Plex cytokine assays are bead-based quantitative multiplex immunoassays. The concentrations of analytes in these assays are quantitated using a calibration or standard curve. A regression analysis is performed to derive an equation that is used to predict the concentrations of unknown samples. The specific concentrations of the standards used to construct the standard curve have a direct effect on the quality of the results. Therefore, selection of appropriate standards is a critical component of immunoassay performance. In this tech note, the effect of the selection of standards on the accuracy, sensitivity, and dynamic range of the results is discussed. The effect on assay performance of reading assays on a Bio-Plex system set at two different photomultiplier tube (PMT) voltages as well as criteria for selecting a custom set of standards are also addressed.
Regression Analysis for Bio-Plex Assays
Because the typical response of an immunoassay is nonlinear, the nonlinear or logistic regression routine is the preferred method for sandwich immunoassay data analysis (Baud 1993). The four-parameter (4PL) and five-parameter (5PL) logistic equations contain parameters (the midpoint of the curve, the slope where the curve is linear, and the upper and lower points) that are used to predict the response of the assay (relative median fluorescence intensity, or MFI; Figure 1). Use of logistic regression yields the widest range of predictable concentrations for Bio-Plex immunoassays (Nix and Wild 2001).