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Principles of Curve Fitting for Multiplex Sandwich Immunoassays, Rev B

calculating the concentration of each standard and then comparing it to the actual concentration using the formula: Observed concentration/ expected concentration x 100. This method yields information about the relative error in the calculation of samples. Backcalculation of standards is automatically performed with Bio-Plex Manager software, and the results are displayed in the report table in the (Obs/Exp) * 100 column (Figure 4). It is most desirable to have each standard fall between 70 and 130% of the actual value, although more stringent ranges may be applied if greater accuracy is desired. The limitation of using backcalculation as the sole method of evaluating goodness of fit is the existence of a bias toward the concentrations of the standards. More specifically, only the standard concentrations are used to assess the quality of the fit; the portions of the curve between each of the standard points are ignored (Nix and Wild 2001).

Spiked Recovery
Spiked recovery is used to assess the overall accuracy of an assay (Davies 2001). This method incorporates variables in assay preparation as well as the regression analysis. Samples are spiked with known concentrations of cytokine and analyzed to determine the closeness of the calculated value to the actual value. The chosen concentrations are usually between the concentrations of the standards, thus removing the bias inherent to the backcalculation of standards method. The results are assessed in the same manner as the standards recovery, using the Obs/Exp x 100 formula. A spiked recovery value between 80 and 120% is considered acceptable. Spiked recovery results may be analyzed using Bio-Plex Manager software by format
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