( Diana Davis PhD Aiguo...)Principles of Curve Fitting for Multiplex Sandwich Immunoassays, Rev B

Diana Davis, PhD, Aiguo Zhang, PhD, Chloe Etienne, Ivan Huang, and Michele Malit, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA

Introduction
Bio-Plex cytokine assays are bead-based multiplex sandwich immunoassays. The concentrations of analytes in a Bio-Plex cytokine immunoassay are quantitated using a calibration or standard curve. More specifically, a series of known concentrations of an analyte is used to construct a plot of signal intensity vs. concentration. The plot is mathematically modeled to derive an equation that may be used to predict the concentrations of unknown samples. The type of mathematical or curve-fitting model as well as the fit of the model have a direct effect on the accuracy of the results. Therefore, curve fitting is a critical component of immunoassay performance. In this technical note, curvefitting methods as well as methods used to determine the quality of the curve fitting are discussed. Advantages and disadvantages of each method and the effects on the predicted cytokine concentrations are illustrated using representative Bio-Plex cytokine assay data.

Methods of Curve Fitting
Linear Regression
The simplest method for determining concentrations from a standard curve is to construct a plot of the concentration vs. the response using the linear portion of the response curve. This method has been used traditionally to quantitate results of ELISA and other immunoassays (Nix and Wild 2001). An example of a GM-CSF Bio-Plex cytokine assay plotted using a linear regression is shown in Figure 1. The R² value is used to determine the overall goodness of the linea
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