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Here N is the number of determinations and K is the number of sample sets.

The LOD determined by this approach would avoid the inclusion of analytical false negative and false positive values. Due to assay conditions being variable, each bioanalytical laboratory should determine the LOD using the CLSI approach instead of using the LOD stated in a kit brochure. If the LOD is used as the lower limit for data inclusion instead of the LLOQ, the user should justify such a choice and be aware of the risk being taken with the higher variability at the LOD to LLOQ range, interpreting the data with caution.

Selectivity and Matrix Effect Selectivity is the ability of the assay to discriminate the analyte unequivocally in the presence of components that may be expected to be present in the sample and alter assay results. Lack of selectivity can result in signal inhibition or enhancement. The results can appear as false or negative positives (an over or underestimation of the analyte concentration). In general, signal suppression from binding proteins occurs more often for LBAs than that of enhancement, resulting in a negative bias.

Interference in an assay is the product of concentration of the interfering substance times the cross-reactivity (or binding inhibition). However, the concentration-response relationship of LBA is nonlinear, and often the magnitude of cross-reactivity (or inhibition) is not evenly dispersed over the entire assay range. Thus, a standard curve prepared in a protein buffer solution has higher optical density (OD) responses than that in biological matrices, while the differences in responses are variable over the standard curve. Moreover, a standard curve prepared in biological matrices from one person would have different responses than that of other people. Therefore, the estimate of interference should not simply extrapolate across the entire assay range or be represented by an IC50 value.

Matrix interference should be surveyed in samples from normal and diseased donors, especially in samples from the anticipated patient population. Spike recovery using samples from persons in the target population should be performed. A later selectivity evaluation of LBA involves a parallelism test of high-concentration endogenous samples from many people diluted at >4 dilution factors with the standard diluent.

Specificity Specificity reflects the ability of an assay to distinguish between the analyte of interest and other structurally similar components of the sample. For LBAs a specificity test should include test of interference from components that may specifically affect the binding reaction. For example, inter ference from the drug compound may alter ligand interactions with the capture reagent. If the ligand reagent of an LBA is the target drug or its analog, the presence of the drug compound at certain concentrations can interfere with the assay. In addition, biomarker homologs or endogenous molecules of the same family may contribute to specificity problems. If reference material of the potential interferent is available, specificity can be tested using various amounts of the test material spiking into various levels of VSs (Lee and Ma, 2007).

Specificity for the biomarker in the presence of the drug under study should also be tested, using samples from endogenous pools (such as the SCs), with and without the addition of the drug compound at various concentrations spanning the expected therapeutic range. Figure 5 illustrates a specificity experiment of a target biomarker (A) and its proximal biomarker (B), each tested with three sample pools at pM levels against various nanomolar drug concentrations. As expected, the test drug inhibited the quantification of the target biomarker at concentrations greater than 2nM. For the proximal to E o in

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