Performance characteristics, ligand binding

Performance characteristics of ligand-binding assays are estimated using calculated concentrations from the spiked validation samples during prestudy validation and/or from the quality control samples during in-study validation. 

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. 

Figure 5.11 illustrates the basic performance of the on-line MS assay. For comparison, a homogenous fluorescence assay has been set up in parallel. For this purpose, the carrier flow was split after the second microcoU reactor, with 90% of the total flow being directed to a fluorescence detector (Fig. 5.11a) and 10% to the MS (Fig. 5.11b). The affinity interaction between streptavidin and biotin was chosen to study the characteristics of an on-line MS biochemical assay. Fluorescein-biotin was used as reporter ligand for both fluorescence and MS in the SIM mode (m/z 390) detection. In the fluorescence mode, the homogeneous biochemical assay is based on the quenching of the fluorescein-biotin fluorescence upon binding to streptavidin.

Binding Characteristics. For ligand-receptor binding assays, we used an EDTA/Tris-citrate (1 mM, pH 7.4)-dialyzed mitochondrial fraction of housefly heads, the so-called 2 fraction, which was prepared by a modification of Squires s method (4). The final pellet was frozen at -25°C and then suspended in 50 mM Tris-citrate buffer (pH 7.4 at 3°C) for assays. 4-w-Propyl[2,3-3h]-2,6,7-trioxa-l-phosphabicyclo[2.2.2]— octane 1-oxide ([ HjPr-BP, 41 Ci/mmol) was used as a ligand for the reason described above. Radioreceptor assays were performed by filtration techniques with glass fiber filters. Specific [ HjPr-BP binding was taken as that displaced by excess unlabeled Pr-BP(10 yM). 

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