In-line assays

Reactions can be analyzed almost instantaneously through in-line analyses, i.e., by analyzing the reaction using a probe inserted into the reactor or a reactor stream. In-line assays may be used for analyzing reactions run in dedicated equipment, which is often the case in manufacturing, or to assay reactions that are difficult to assay by conventional external methods (see below). Relatively few analytical techniques have been adapted for in-line assay. 

Fourier-transform infrared spectroscopy (FTIR) and pH measurements are the techniques most often adapted for in-line IPC. pH measurements are used for reactions that are run in water or have an aqueous component, e.g., an aqueous extraction. FTIR is especially good for monitoring continuous reactions [12] and reactions that would be dramatically changed by exposure to the atmosphere and temperature of the laboratory. Suitable reactions include low-temperature reactions, reactions run under pressure, reactions with gaseous or toxic materials (e.g., ethylene oxide), and reactions run under inert atmosphere. Further advantages of in-line assays are that no samples need to be prepared, and assay results can be generated within minutes. 

The result of a prior in-line assay is taken into account when deciding on the need for the determination of other parameters in the assayed sample. To this end, the result associated with the first analyte is compared with a pre-set threshold value specific for a given analytical problem and a binary answer (yes/no, present/absent, high/low) is obtained. Depending on the answer, other analytes may or may not be determined. A significant reduction in the total number of determinations is attained, thus expanding the analytical productivity of the laboratory. 

For additional evaluation of the effect of hydrophobization and the molecular weight of the polymers on the biological immuno-stimulating activity, we investigated the ex vivo cytokine (interIeukin-6 [IL-6], and tumor necrosis factor [TNFj-inducing activity from human peripheral whole blood cells of hydrophobized polymers by use of fractionated poly(M A-CDA) with narrow poly-dispersity. Since this assay uses the intact human cells, it shows more accurate results than in vitro assay using cultured cell line [25]. 

FIGURE 4.11 Relationship between the observed IC50 for allosteric antagonists and the amount of radioligand present in the assay according to Equation 4.13. Dotted line shows relationship for a competitive antagonist. 

Another major second messenger in cells is calcium ion. Virtually any mammalian cell line can be used to measure transient calcium currents in fluorescence assays when cells are preloaded with an indicator dye that allows monitoring of changes in cytosolic calcium concentration. These responses can be observed in real time, but a characteristic of these responses is that they are transient. This may lead to problems with hemi-equilibria in antagonist studies whereby the maximal responses to agonists may be depressed in the presence of antagonists. These effects are discussed more fully in Chapter 6. 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

The study of active transport mechanisms has grown substantially in recent years, with transport proteins such as P-gp, BCRP, and MRP-2 among the most studied [59]. Several types of in vitro assays to assess substrates of transporters have been established these include assays directed toward intestinal and biliary efflux [60]. Assays that measure passive and active transport are also used to assess penetration of the blood-brain barrier. In addition to the assays described above, transfected cell lines that overexpress transporters present in the blood-brain barrier are also employed [61]. 

Figure 7.13 Fractional velocity of as a function of tight binding inhibitor concentration in an assay for which the enzyme concentration is fixed so that [E j/Kf9 > 200 (i.e., zone C of Strauss and Goldstein, 1943). The point at which the linear concentration-response line intersects the x-axis indicates the concentration of active enzyme in the sample.

The palladium(II) phthalocyanines are satisfactory singlet oxygen generators. Thus, f>A values for tetra-t-butylphthalocyanine ((27), with stated replacement for Mg) are 2H, 0.22 Zn, 0.34 Pd, 0.54, i.e., increasing in line with the heavy atom effect.180 For palladium(II) tetra-t-butylphthalocyanine ((27), Pd instead of Mg) in benzene at 290 K, 4>f= 0.048 and X = 0.49.180 However, palladium(II) 2,3-dihydroxy-9,16,23-tri-t-butylphthalocyanine was synthesized by a mixed condensation (1 9 mix of appropriate dinitriles) as a likely amphiphilic sensitizer, but did not show bioactivity in an enzyme assay, possibly because of aggregation.180... 

While we recommend the application of control cell fines as reagent, assay, and EQA monitoring tools, it is important to emphasize that appropriate tissue controls are also continued to be used in parallel, as tissue is still considered the gold standard in laboratory assay control. For reliable results, it is also important that tissue or cell line controls are fixed and processed in the same manner as diagnostic material submitted for evaluation. 

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