End point assays

In these assays aU of the metabolite/anatyte is converted into some chromo-phoric compound. These have an advantage in that any variation in factors that may affect the time to completion (e.g. temperature, amount of coupling 

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The underlying assumption in any end-point assay is that the time point measured is well within the initial velocity phase of the reaction, so that product formation or substrate disappearance is a linear function of time. If this is true, then the... 

End-point assays can also use coupled enzyme systems in which the product of the test reaction provides the substrate for the subsequent auxiliary and indicator reactions. The choice of pH is less critical than with the measurement of enzyme activity frequently a compromise pH is used and... 

BCA molecules complex with the cuprous ion to yield a water-soluble purple product that has an absorbance at 562 nm. This method only requires 30 min of incubation at 37°C but has the disadvantage of not being a true end-point assay because the color will keep developing with time. In reality, the rate of color development is slowed sufficiently following incubation to permit large numbers of samples to be assayed in a single run. 

Clinical chemistry Enzyme rate assays, colorimetric assays, end-point assays, immunoassays Upstone, 2000 ... 

The precise configuration can be chosen according to the laboratory workload. Table 2.2 clearly illustrates the capacities of the various models. These instruments will support a wide range of analytical methods including end-point assays at one or two wavelengths and with one or two steps as well as rate analyses. 

Mulitplex Makes multiple measurements of different end points for different processes a single end point assay will not identify most cytotoxidties... 

The measurements can be made at various levels from that of subcellular organelles, to single cells, cell populations and even tissues. They can be made on either fixed cells or on live cells that are incubated under physiological conditions. They can be made in end point assays or kinetically, in real time. Compared to previous manual methods, automation provides a marked improvement in the capacity for sample and experiment throughput, the precision of measurement and in the sheer number and diversity of parameters measureable for an experiment. Consolidation of the technical capabilities allo vs unparalleled vhthin-experiment, cross-comparisons of biochemical, morphological and functional parameters. Compared to flo v cytometry it offers substantially greater analytic capability for morphometric and kinetic parameters, although for substantially lo ver numbers of cells. 

Additional assays have surfaced on both ends of the scale of complexity. Single end point assays such as those for a host of receptor binding and activation assays and enzyme activity modulation assays have been developed and applied to alternative developmental toxicity testing. These assays can be carried out in a... 

Therefore, a validation exercise with a variety of compounds with unknown mechanisms of developmental toxicity has only limited value if only used to derive an overall predictability rate of a single assay. It is more useful to elucidate the applicability domain of the assay in terms of the mechanisms of development covered, and to validate that aspect by testing compounds that do or do not affect the applicability domain. For single end point assays such as specific receptor activation assays, this exercise is relatively straightforward. For more complex assays such as those involving embryonic cell differentiation, the understanding of the applicability domain is more complex, as extensive research with the embryonic stem cell test has learned (27, 47). Whole embryo cultures are probably more straightforward in terms of applicability domain as they involve the entire embryo in a limited window of development, but such assays are complex and not animal free. 

Since the BCA protein assay is not a true end-point assay, the amount of color produced varies with the incubation time and the incubation temperature. While this allows considerable flexibility in optimizing the BCA assay for each application, it also requires that the optimized procedure be followed exactly every time the assay is done. 

The dimerization behavior caused by changes in the assay temperature has two effects on the behavior of the protease. First, increased temperature increases the catalytic rate of the enzyme, resulting in increased initial rates in the assay, particularly when the enzyme is used directly from a concentrated stock. Second, increased temperature pushes the dimenmonomer equilibrium in the direction of monomer, an apparently less active species, decreasing the steady-state rate of the reaction. In the 60-min end point assay, these effects combine to give optimal enzyme activity at approx 25°C, but further temperature optimization may be useful if initial rates or longer incubation times are used. 

AK end-point assays were initiated by the addition of 100 pL magnesium acetate or 100 pL ADP, as appropriate, to well E of each 5-well strip in turn. With 30 s between additions and allowing IVt min per sample for conversion of ADP to ATP by released AK, a rack of 15 samples could be processed in 15 min. ATP production was determined by the addition of 100 pL of bioluminescence reagent (Celsis, Newmarket, UK) with light output measured over 10 s after a 1 s delay. 

Screening in 96- or 384-well plate formats allows precise fitness measurements. The accuracy of the detection system, kinetic assays (in contrast to end point assays), the possibility to normalize activity values (e. g., using measured cell concentrations), and better control over cell growth and protein expression contribute to the improved... 

With turbidimetric end-point assays, turbidity is measured after a fixed incubation period. By inclusion of standards alongside dilutions of the material under test, a standard curve can be created and the endotoxin concentration can be read off this curve for the material under test. Equipment using microtiter plates is commercially available to minimize the amounts of LAL reagent necessary and to maximize the number of test sets per set of standards. The period of incubation over which turbidimetric end-point assays is conducted is critical. All samples will be equally turbid with overlong incubation if incubation times are too short none of the samples will be measurably turbid. It is not possible to stop the reaction to take the readings. 

C-guanidine flux assay Scintillation counter 96-weII (20000), channel or delay Toxin to activate flux through inactivation hfeasurement of ion rather than relatively high-throughput, amenable to automation End-point assay, continuous functional recording... 

Various authors have considered the amount of enzyme required for an end point assay (15-17). While it is advisable not to be too wasteful, most common coupling enzymes are not too expensive and a few preliminary experiments will soon determine the minimum amount needed to give reUable data in a reasonable time. Putting the problem quantitatively, our ideal amount of enzyme should lead to the reaction being virtually complete (from 100% to 1% of substrate remaining) in a reasonable time (say, lO min). Since the enzyme will slow down as the substrate is used up, we caimot just calculate the amount of enzyme needed from its given activity (V) divided into the substrate available instead we need to consider the integrated rate equation for the process. Remember that V is a measure of the amount of enzyme added (V = kcat o) stid so when we choose the amount of enzyme to add, we choose a V value. 

In some methods, substrates are used which yield coloured products, e.g. p-nitrophenylphosphate (in the Bessey-Lowry-Brock method), thymolphthalein phosphate or phenolphthalein phosphate. These substrates can be used for the kinetic measurement of enzymic activity, unlike the previous methods which are end-point assays. 

Despite their extensive use, assays employing labeled analytes suffer from several disadvantages. First, analytes need to be conjugated to an enzyme, fluorophore or a radioactive moiety, which increases the cost of the assay. Second, assays that employ reporters typically require multiple washing and incubation steps and in most cases are end-point assays. Finally, in many instances, conjugation of reporters near the active site of the receptor can interfere with receptor-analyte binding. 

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