Assay time

Limitations encountered in routine use include (a) the use of radioactive materials, (b) limited sensitivity in the presence of high protein concentrations, (c) long assay time of up to 5 days,... 

Bran Recongnition Site Affinity K,(uM) Radioligand/Displacer Brain Region Assay Time, Temperature Buffer... 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Assay time Approximately 3 hours Approximately 50-60 tests/hour... 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

The combination of increased Pe and decreased %R allowed the permeation time to be lowered to 4 h, in comparison to the originally specified time of 15 h [547,550], a considerable improvement for high-throughput applications. The quality of the measurements of the low-permeability molecules did not substantially improve with sink conditions or the reduced assay times. 

The semi-empirical Yasuda-Shedlovsky technique of extrapolating a series of apparent pKa values obtained in several ratios of water/solvent to obtain an aqueous value is well established [32, 33], but three or more experiments are required and this adds significantly to assay times. A method of calculating aqueous pKas for various classes of organic acids and bases from single apparent pKa values obtained in water/solvent mixtures has been reported [34], and shows promise as a means of further speeding pKa measurement. 

One way to view the issue of sample throughput is to define the steps required for the process. If we add an in-life portion to the assay time for an ADME/PK study, the term drug metabolism discovery cycle time (DMDCT) applies to the amount of time from the beginning to the end of a study. Thus, DMDCT = in-life cycle time + assay cycle time. 

Sample pooling is also used in a process called cassette assay in which samples are pooled from multiple (typically 5 or 6) dosing experiments.24-83 87-88 Hsieh et al.89 showed that one could pool the plasma from six NCEs into one sample per time point to reduce sample assay time. Kuo et al.88 used a similar sample pooling approach for NCEs dosed into rats. The advantage is that cassette assay requires fewer samples. Two disadvantages are the need to dilute samples and the difficult set-up. 

FIGURE 7.9 (A) Upper traces show HPLC/MS/MS mass chromatograms for a set of six test compounds (total HPLC assay time ca. 3 min). (B) Lower traces show UPLC/MS/MS mass chromatograms for the same set of six test compounds (total UPLC assay time ca. 1 min). (Source Adapted from Yu, K. et al., Rapid Com-mun. Mass Spectrom., 2006, 20, 544. With permission of John Wiley Sons.)... 

An entire antibiotic screen can be carried out using 4 samples with the assay time being 15 min to 1 h depending upon whether the qualitative or quantitative mode is desired. 

Ultracentrifugation Minimal non-specific binding and osmotic volume shifts. Large plasma volumes required, long assay time, issues such as sedimentation, back diffusion and viscosity. Potential for lipoprotein contamination of plasma water layer. [28, 29]... 

With a format in which four compounds are tested per 96-well microplate, with robust host cells and rapid assay time, it is possible to assess up to 160 compounds/ week, even without automation. Beyond this throughput, compound supply and data collection become the rate-limiting factors. 

Some other sources have definitions that are different from the one given above [7,14]. The US Pharmacopeia [7] defines ruggedness as The ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same sample under a variety of normal test conditions, such as different laboratories, different analysts, different instruments, different lots of reagents, different elapsed assay times, different assay temperatures, different days, etc. Ruggedness is normally expressed as the lack of influence on test results of operational and environmental variables of the analytical method. Ruggedness is a measure of reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst . In fact this is nearly the definition of reproducibility. This definition is also followed by other authors [15]. 

This assay was developed using an incubation temperature of 30°C, although 37°C is used in other centres [32]. The assay at 30°C is linear with 50 pi plasma/assay tube for at least 60 min, and with a assay time of 60 min with at least 100 pi plasma/assay tube. When insufficient plasma is available, the assay can be performed with 25 pi plasma/assay tube (+25 pi demineralised water) for 120 min. If an assay temperature of 37°C is preferred, the assay time with 50 pi plasma/assay tube should be reduced to 30 min. Normal values measured at 37°C [31, 32] are higher (theoretically 1.7 times) than those measured at 30°C (Table 3.7.2). 

Activity with 1 x S subtract the corresponding blank value (or the individual background value if this is higher than the blank value) from the absorbance values of both reaction tubes with 1 x S (Table 3.7.1 tubes 3 and 4). Divide by the standard factor to obtain nmols/assay. Further divide by 60 min (assay time) to obtain nmol/min/assay. Multiply by 20 (1 ml/0.050 ml) to obtain nmol/min/ml plasma. Calculate the mean value of activity with 1 x S. 

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