Quantitative dose response assay

Extracts that exhibited significant inhibitory activity, defined as >50% inhibition of CPE at 100 (xg/mL, were advanced into secondary screening, which includes confirmation of activity observed during the primary screen using an expanded range of concentrations (dose response), plaque inhibition assay, and a one-step growth inhibition and testing of additional influenza viruses. As shown in Fig. 1.2a, the quantitative dose response assay was used to assess the potency of the most two... 

A quantitative approach was taken to evaluate the effect of various concentrations of water extracts of the two crosslinked solids and individual liquid components on cell growth of L-929 cells in vitro. Both cells and adhesive materials were prepared as described above. For this assay, dilutions of an aqueous extract of the solids were prepared for a dose-response evaluation. The following weight per volume ratios were used 4,000, 500, 100, 50, 4, 3, 2, 1 fig per 20-mL water. Extraction was performed for 4 hr at 60 °C followed by 20 hr at room temperature. Supernatants were transferred to clean containers. The negative control consisted of sterile, triple distilled water, and the positive control was 40 mg/mL dextran sulfate. 

The overall process and critical activities for robotic implementation of automated HTS from intake of the researcher s benchtop assay through to the identification of a validated, optimized lead series of compounds around a bona fide chemical scaffold are shown in Fig. 2. The downstream steps of hit confirmation/ validation and structure-activity relationship (SAR) elucidation and hit to lead (HTL) have been included to emphasize that the same robotic assay used for production HTS can and should be used by the HTS team to perform high-volume hit confirmation and first-line automated dose-response analysis of confirmed hits to obtain quantitative potency (IC50) rankings to elucidate the nascent SAR of emergent hit series... 

The single most important entity in the study of the dose-response relationship is the bioassay system in which the chemical will be studied. Since the most essential feature of the results will be the quantitative data idiich are derived, the rules governing the accuracy and precision of the assay should approach as nearly as possible those achieved in measurements in chemical systems. Since biological systems are not machines, accuracy and precision can be difficult problems in bioassays. However, biological systems frequently are the match of chemical systems when it comes to sensitivity since the dose or concentration of chemical to which the bioassay systems may respond is often exceedingly low. 

Quantitative risk assessment requires extrapolation from results of experimental assays conducted at high dose levels to predicted effects at lower dose levels which correspond to human exposures. The meaning of this high to low dose extrapolation within an animal species will be discussed, along with its inherent limitations. A number of commonly used mathematical models of dose-response necessary for this extrapolation, will be discussed. Other limitations in their ability to provide precise quantitative low dose risk estimates will also be discussed. These include the existence of thresholds incorporation of background, or spontaneous responses modification of the dose-response by pharmacokinetic processes. 

To measure CYP inhibition, metabolite formation is quantitated in the absence or presence of a potential inhibitor as outlined in Figure 10.4. Metabohte concentration in the presence of inhibitor can be measured at a single concentration of inhibitor or at many to generate a dose-response curve, depending on the tradeoff between accuracy and throughput that s considered acceptable for the assay in question. Data are normally expressed either as percent inhibition at one concentration (e.g. 40% 3 i.M) or as an IC50 value. An estimate of the IC50 can be obtained from one-point data as well. ... 

The results of the biosynthesis experiments suggest that NAD depletion is due to an increased rate of NAD consuming reactions. NAD can be consumed by ADP-ribosyl transfer reactions which can be inhibited by 3-aminobenzamide (10). When 5 mM 3-aminobenzamide was added to the culture medium, total inhibition of NAD depletion was observed (data not shown). Since ADP-ribosyl transfer reactions can be catalyzed by nuclear poly(ADP-ribose) polymerase and by cytoplasmic mono(ADP-ribosyl) transferases (11), the inhibition of these enzymes by 3-aminobenzamide was quantitatively compared. Fig. 2 shows dose response curves for the inhibition of purified poly(ADP-ribose) polymerase (12) and mono(ADP-ribosyl) transferase (13) by this compound. The activity of poly(ADP-ribose) polymerase was much more sensitive to inhibition with a 50% inhibitory concentration (IC50) of approximately 5.5 xM under the assay conditions used compared to an IC50 of 2000 iM for the mono(ADP-ribosyl) transferase. The effect of different concentrations of 3-aminobenzamide on NAD depletion in CF-3 cells incubated with Ca + depleted medium was also determined (Fig. 2). These cell experiments indicated an IC50 similar to that of mono(ADP-ribosyl) transferases and argue that NAD depletion in CF-3 cells was due to a stimulation of cellular mono(ADP-ribosyl)ation. 

In all assays, aliquots are withdrawn from the receiving phase in defined time intervals and the concentration of the analyte is determined via a calibration curve (Figure 3b). Alternatively, the concentration in the receiving phase can be continuously monitored when a pump and a flow cuvette are available. For quantitative analysis, kinetic traces are measured for increasing carrier concentrations, and the obtained dose-response curves are analyzed with the Hill equation to reveal the EC50, which is the effective concentration needed to observe 50% of the maximal activity, and the Hill coefficient n (Figure 3c, Section 4.2). 

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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