Ligand binding assay calibration curve

Findlay, J.W.A., and Dillard, R.F. (2007) Appropriate calibration curve fitting in ligand binding assays. The AAPS Journal, 9, E260 E267. 

Due to the method principle, ligand-binding assays are inherently non-linear. Thus, four- and five-parameter mathematic models are used to create calibration curves, and consequently a higher number of calibration points is needed to define the curve most accurately. Especially in the asymptotic parts of the calibration curve, a sufficient number of calibrators must be placed to define upper and lower limits of quantification with pre-defined accuracy and precision. Unless it is shown that matrix constituents have no impact on detection signals, calibration curves must be prepared in an authentic matrix. 

The relative binding response is interpolated from a calibration curve in order to compute concentration. As an inhibition assay, the response is inversely related to biotin concentration and exhibits a sigmoidal dose-response relationship that is typical of most ligand-binding assays. With respect to specificity, the routine compliance assay is targeted to the quantitation of free biotin only in nutritional dairy products, and therefore does not include biocytin (Indyk et al. 2000). However, in milk and supplemented infant formulas, the overwhelming majority of biotin is present in the free form. 

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