Standard Assay

Luminescence activity. The specific luminescence activities (quanta/s emitted from 1ml of a solution of A280nm,icm 1.0) of luciferases A, B and C are in a range of 1.2 4.1 x 1016 photons/s when measured with the standard assay buffer (20 mM Tris-HCl, pFl 7.8, containing 1M NaCl, 0.05% BSA, and 0.14 xg/ml of coelenterazine, at 24°C). These are the highest specific activities of coelenterazine luciferases. 

Assays of earthworm luciferin and luciferase (Bellisario et al., 1972). The standard assay mixture contains luciferin, luciferase... 

The measurement of ER has become a standard assay in the clinical management of breast cancer. The presence of ERa identifies those breast cancer patients with a lower risk of relapse and better clinical outcome. Receptor status also provides a guideline for those tumors that may be responsive to hormonal intervention. But only about half of ER-positive patients respond to hormonal therapies. Of those who respond initially, most will eventually develop an estrogen unresponsive disease following a period of treatment even though ERa is often still present. Mutant receptors and constitutively active r eceptors as well as hormone-independent activation of the ERa are discussed. The involvement of ER 3 isoforms is under investigation. 

The design of an assay is, in large measure, prospective quality assurance. The factors that are likely to affect the results of the assay must be defined and controlled to the greatest extent possible. Once the general outlines of an assay have been established, key features should be examined, including optimization of sample preparation, sample stability, choice of standards, assay range, assay repeatability, optimization of separation, and optimization of detection. 

Work with those who submit samples to limit submissions to those likely to yield useful information. In a typical sample queue, some are outside of the parameters required for a standardized assay, having a concentration too low for the precision required or containing matrix components incompatible with the assay. Others may be low on the list of project priorities. Analytical morale gains if every sample run leads to results that are valued. 

The gold-standard assay used for all chemokine receptor inhibitors that reach clinical-phase trials is the chemotaxis functional assay. This assay relies on the ability of chemokines to recruit cells expressing their respective receptor to areas of inflammation. In vitro, this assay was first described in detail by Taub et al. (16) for 24/48-well plates currently, this can be achieved by using 96-well plates. Cells are incubated in the upper chamber with an antagonist for a particular receptor (at different concentrations or with buffer) and challenged to migrate to the lower chamber, which has the relevant chemokine. After 2 to 4 hours of incubation at 37°C, the upper chamber inlet is removed and the cells in the lower chamber quantified by fluorescence with, for example, Calcein AM (Invitrogen, Carlsbad, CA). 

The enzyme activity was assayed by measuring the production of optically active mandelonitrile synthesized from benzaldehyde and cyanide. The standard assay solution contained 300 gmo citrate buffer (pH 3.5-6.0), 50 /rmol of benzaldehyde, 100 /rmol potassium cyanide and 100 jA of the enzyme in a final volume of 1.0 mL. The reaction was started by an addition of 100 fx of the enzyme solution and incubated at 25 °C for 1-120 min. Aliquots (100 jiY) were withdrawn at various reaction times and the reaction was stopped by the addition of 0.9 mL of organic solvent (9 1 hexane iso-propanol by volume). The mandelonitrile formed was extracted and the supernatant, obtained by centrifugation (15,000 x g, 1.0 min at 4 °C), was assayed by HPLC. A blank reaction was also performed without enzyme and the amount of mandelonitrile obtained was deducted from the biocatalyzed reaction product. One unit of the enzyme is defined as the amount of the enzyme that produces 1 /imol of (R)-mandelonitrile under reaction conditions in 1 min. 

For pH and thermoinactivated samples, the conditions were equal to those in the standard assay, except that the concentration of tartrate buffer, pH 3.0, was 0.3 M. 

Effect of Temperature and pH on Activity and Stability. When Xylanase II was subjected to the standard assay at pH 5 but at several different temperatures, the highest activity was found after incubation for 30 min at 60°C. From 21 C to 45 C the energy of activation from a linear plot of In (activity) vs. T was 41.6 2.1 kJ/mol, where the range is the standard deviation. When the standard assay was conducted at various pHs and 50°C, the highest activity was at pH 6.05. Activities half the maximum were found at pHs 4.4 and 8.0. 

As proof of principle, Lehn and coworkers individually synthesized all acyl hydrazone combinations from the 13 DCL building blocks and measured their inhibition of acetylthiocholine hydrolysis by ACE in a standard assay. They then established a dynamic deconvolution approach whereby the pre-equilibrated DCL containing all members is prepared, frozen, and assayed. Thirteen sublibraries were then prepared containing all components minus one hydrazide or aldehyde component, and assayed. Active components in the DCL were quickly identified by an increase in ACE activity, observed in sublibraries missing either hydrazide 7 or dialdehyde i, pointing to the bis-acyl hydrazone 7-i-7 as the most likely active constituent. This was in line with the individual assay data recorded earlier resynthesis of this compound characterized it as a low nanomolar inhibitor of the enzyme. 

In most kinetic investigations, one assumes the enzyme remains stable over the course of the measurement. When this is the case, corrective measures must be taken to obtain valid kinetic data. A useful test for any enzyme system is to plot enzyme activity versus time. This is readily accomplished by using a standardized assay (usually at optimal or saturating substrate concentrations) to measure the enzyme s specific activity periodically during the course of some experiment. This approach may fail to detect a reduction in activity characterized by lower affinity for substrate however, use of a subsaturating substrate concentration in a time-course study will reveal this behavior. 

Figure 2. Illustration of the importance of the choice of reaction conditions on the determination of initial velocity. Shown are four conditions applied to examine the rate behavior of Escherichia coli NAD+-dependent coenzyme A-linked aldehyde dehydrogenase (Reaction NAD+ + CoA-SH + Acetaldehyde = NADH + Acetyl-S-CoA + H+). All assay mixtures contained enzyme, 0.5 mM NAD+, 8 /jlW CoA-SFI, 16 mM acetaldehyde, and 22.5 mM Tris buffer at pFI 8.1. (a) Time-course observed when enzyme was added to the standard assay (b) time-course observed when enzyme was added to standard assay augmented with 10 mM 2-mercaptoethanol (c) time-course observed when enzyme was first preincubated for 15 min with 8 /jlW CoA-SH, 16 mM acetaldehyde, 10 mM 2-mercaptoethanol, and 22.5 mM Tris buffer at pH 8.1, and the reaction was initiated by addition of NAD+ (d) time-course observed when enzyme was preincubated with lOmM 2-mercaptoethanol for 15 min andthen addedtostandard assay augmented with 10 mM 2-mercaptoethanol. The data are most compatible with the idea that the enzyme has an active-site thiol group that must be reduced to express full catalytic activity during assay.

Drug/Lab test interactions Therapeutic levels of rifampin inhibit standard assays for serum folate and vitamin 612-... 

While there is no standard assay design for teratogenicity or developmental toxicity screening, there are many similarities in the types of assays that have been described in the literature (4-9, 22-25). One version of these (4) is described here, and an overview of the assay design is shown in Fig. 3. Variations on this assay (alone or in combination with other techniques) can also be used for more descriptive characterization of the effects of a test substance or evaluation of the mechanisms of developmental toxicity. 

Fig. 2.2.4.2 Determination of the optimum temperature of recombinant (R)-specific ADH. The enzymatic activity was measured with acetophenone using the standard assay.

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