Ligand binding assay linearity

The primary performance measures of a ligand-binding assay are bias/trueness and precision. These measures along with the total error are then used to derive and evaluate several other performance characteristics such as sensitivity (LLOQ), dynamic range, and dilutional linearity. Estimation of the primary performance measures (bias, precision, and total error) requires relevant data to be generated from a number of independent runs (also termed as experiments or assay s). Within each run, a number of concentration levels of the analyte of interest are tested with two or more replicates at each level. The primary performance measures are estimated independently at each level of the analyte concentration. This is carried out within the framework of the analysis of variance (ANOVA) model with the experimental runs included as a random effect [23]. Additional terms such as analyst, instmment, etc., may be included in this model depending on the design of the experiment. This ANOVA model allows us to estimate the overall mean of the calculated concentrations and the relevant variance components such as the within-run variance and the between-run variance. 

This is a condition in which dilution of a spiked sample does not result in a biased measurement of the analyte concentration. Therefore, this gives an indication that samples can be diluted from above the assay range into the calibration range in a linear fashion. This is particularly important for ligand-binding assays (LBAs) where the range of quantification may be very narrow. The dilutional experiments should be based on the expected highest concentrations of study samples. 

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. 

In competitive binding assays involving monovalent labeled ligands and monovalent inhibitors the slope on a semilog plot is linear and covers a 2-log range between about 10% and 90% inhibition if binding at one site does not affect binding at a second site such behavior is termed noncooperative. 

Evaluation of parallelism for ligand-binding PK assays is very similar to that for biomarker PD assays since most PK assays that employ commercial kit methods are likely to be for endogenous compounds. This describes the dilution of incurred samples rather than dilution of spiked samples. The dilution of spiked samples is termed dilution linearity [3]. 

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