Quantitative inhibition assays

One of the most popular bioassay for interferons is termed the cytopathic effect inhibition assay . This assay is based upon the ability of many interferons to render animal cells resistant to viral attack. It entails incubation of the interferon preparation with cells sensitive to destruction by a specific virus. That virus is then subsequently added, and the percentage of cells that survive thereafter is proportional to the levels of interferon present in the assay sample. Viable cells can assimilate certain dyes, such as neutral red. Addition of the dye followed by spectrophotometric quantitation of the amount of dye assimilated can thus be used to quantitate percentage cell survival. This type of assay can be scaled down to run in a single well of a microtitre plate. This facilitates automated assay of large numbers of samples with relative ease. 

Direct kinetic assays are the only valid methods for the measurement of activators and inhibitors and calibration plots of the percentage activation or inhibition by known amounts of the substance can be made. Examples of inhibition assays include the quantitation of organophosphorus pesticides using the inhibition of cholinesterase (EC 3.1.1.7) while manganese can be measured in amounts as low as 1 X 10-12 mol using its activating effect on isocitrate dehydrogenase (EC 1.1.1.41). 

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... 

Figure 6. Quantitative inhibition assays of anti-l serum Ma with oligosaccharides (27).

Smalley, J., Marino, A. M., Xin, B., Olah, T., and Balimane, P. V. (2007). Development of a quantitative LC-MS /MS analytical method coupled with turbulent flow chromatography for digoxin for the in vitro P-gp inhibition assay. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 854 260-267. 

Fig. 4. Various ways of quantitation of TAC in inhibition assays measurement of induction time, absorbance (fluorescence) after fixed time, and area the kinetic curve of time course of changes in absorbance or fluorescence. Dashed line, reference solid line, sample measured. Differences between the areas under curves for sample and reference (protection area) indicated only.

Reversal of inhibition caused by 14 was assayed quantitively using compound 3, 6-benzylaminopurine and diphenylurea, the latter two of which are about 1/10 and 1/1000 as active as 3, respectively, in promoting the growth of tobacco callus (13,81). Not unexpectedly, 6-benzylaminopurine was only about 1/3 as potent as 3 in reversing inhibition by )L4 and diphenylurea was only 1/500 as effective. Thus, the ability of the cytokinins to reverse inhibition paralleled their activity as cytokinins, consistent with the... 

After Zemplen treatment under usual conditions, the sparingly water-soluble hexamers 49 and 51 were obtained in quantitative yields. Their modest solubility in water has however not jeopardized their biological evaluations in solid-phase competitive inhibition assays as well as in their cross-linking abilities with a model plant lectin, Concanavalin A, known to form cross-linked lattices in the presence of multiantennary glycans (39). 

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the... 

Structural Insights from Quantitative Precipitin Inhibition Assays... 

Although the toxin s effects on the microfilament system are easily visualized by fluorescence microscopy, it may be important to determine the changes in the ratio of cellular F- and G-actin quantitatively. Because G-actin dissolves in Triton X-100, whereas F-actin (or at least a major fraction of F-actin) does not, fractionation by detergent solubility can be used to study changes in the G- and F-actin content of cells. A different approach is the determination of G-actin content by the DNAse inhibition assay according to Blikstad (Blikstad et al., 1978). The action of DNAse in cleaving DNA is inhibited by monomeric G-actin but not by F-actin. Thus, the extent of inhibition of DNAse is proportional to the concentration of G-actin in cell lysates. 

Inhibitors may be quantitated using enzyme activity assays if the unknown inhibitor sample does not contain the enzyme employed in the inhibition assay. Standards containing constant enzyme and saturating substrate concentrations are prepared with varying concentrations of inhibitor. The resulting activity versus [inhibitor] calibration curves, for reversible and irreversible inhibitors, are shown below in Figure 2.12. 

Figure 7 Quantitative inhibition assays in oligosaccharide microarray. (A) Determination of the concentration of soluble a-methyl mannose to inhibit 50% of Con A binding to spotted mannose. (B) Determination of the concentration of a-methyl mannose to inhibit 50% of Con A binding to spotted glucose. Each data point represents the meaniSD for 10 spots from 2 independent experiments.

Thompson WL, Wannemacher RW Jr. Detection and quantitation of T-2 mycotoxin with a simplified protein synthesis inhibition assay. Appl Environ Microbiol. 1984 48(6) 1176 1180. 

Prior to P450 reaction phenotyping or inhibition experiments, it is important to determine enzyme kinetic parameters such as Km and Umax for the formation of selected metabolites that are subjected to quantitative analysis by LC-MS. For example, -warfarin is catalyzed by CYP2C9 to a specific metabolite, 7-hydroxy-5 -warfarin (Fig. 15.13). Thus, a CYP2C9 inhibition assay is developed based on the reaction. In the assay, Y-warfarin is incubated with HLM in the presence of a test compound, followed by quantification of 7-hydroxy-5 -warfarin by LC—MS (Zhang et ah, 2001). To set up this assay in our lab, enzyme kinetics for the formation of 7-hydroxy-iS-warfarin in HLM was determined. In this experiment, warfarin was incubated at concentrations from 0 to 250 >M with HLM at optimized conditions. Rates of 7-hydroxy-S -warfarin formation at various substrate concentrations were determined as shown in Figure 15.13a, from which Km and Umax values were calculated. The warfarin assay represented an analytical challenge since the turnover of warfarin in the HLM system was extremely low. To be able to quantitatively determine low concentrations of 7-hydroxy-5 -warfarin in the incubations, a very sensitive LC—MS method that used MRM with a 4000 QTRAP has been developed (Fig. 15.13a). 

Figure 4.2 Quantitation of antigen by competitive (inhibition) assay. An antigen is fixed (attached) and binds a specific antibody from solution. However, additional antigen is provided in solution and antibody binding to the bound antigen is reduced in direct proportion to the concentration of free antigen. The bound antibody is then detected by binding a second, labeled (enzyme, isotope, etc.) antibody.

The first quantitative immunoassays were based on the use of radioactive reporter molecules, mainly and " C. These radioimmunoassays (RIAs) have gradually been replaced by nomadioactive reporter molecules or labels, e.g., enzymes, fluorescent, and luminescent molecules and combinations of these. The nonradio-active labels facilitated development of automated immunoassays, and they also contributed to the design of assays with substantially reduced detection limits. However, nomadioactive assay have an advantage only in immunometric sandwich assays in inhibition assays, the use on nonradioactive labels has not provided any improvement in assay sensitivity as compared to RIA [1]. The development in immunoassay technology has been reviewed by Ekins [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. 

Kontrawelert, R, Francis, D.L., Brooks, RM., Ghosh, R. (1989) Application of an enzyme-linked immunosorbent-inhibition assay to quantitate the release of KS peptides into fluids of the rat subcutaneous air pouch model and the effects of chondroprotective drugs on the release process. Rheumatology International, 9 (2), 77-83. 

EP Frenkel, R Plough, RL Kitchens. Measurement of tissue vitamin B12 by radioisotopic competitive inhibition assay and quantitation of tissue cobalamin fractions. Methods Enzymol 67 31-40, 1980. 

Van Lenten, L., and Ashwell, G., 1972, The binding of desialylated glycoproteins by plasma membranes of rat liver—development of a quantitative inhibition assay, J. Biol. Chem. 247 4633-4640. 

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