Assays design

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. 

Standards are used for a number of purposes. An external standard contains a mixture of substances typically observed in the sample. Knowledge of the concentration of the standard substances allows calibration of the detector to compensate for run-to-run or day-to-day variability. External standards should not be subjected to hydrolysis or other sample processing steps, except as necessary for detection, since this would add other sources 

a chromatographic profile or fingerprint of trace unknowns can be established and monitored, so that if product performance unexpectedly changes, there will be a starting point for troubleshooting. The effects of experimental variables on sample recoveries should be measured directly by controlled variation of an experimental factor, using the reference standard, or suitable external standards, or spiked addition of an external standard to the reference standard. A detailed example of the use of internal and external standards is presented in Chapter 4. 

Considerable attention should be paid to obtaining samples truly representative of the production process early in method development. Production processes generate mixtures that are far more complex and variable than may be generally realized therefore, a separation developed using an early production sample may prove to be inadequate for a later sample. Minor peaks observed in the chromatogram, whether known or unknown, serve as a record of the consistency of the process and can be used to monitor process changes. Sometimes a particular peak can be associated with a desirable or undesirable property of the product and used for controlled process optimization. 

Before discussing the various assays which have developed using synthetic peptide substrates, one should review briefly the basic principles for these procedures. In order to introduce substrate assays into a laboratory, a thorough knowledge of enzyme kinetics is desirable. Several excellent reviews on this subject are available (C4, S22, W4). 

It has been shown that the relationship between substrate concentration and reaction velocity is hyperbolic and that the parameters of this curve are given by two fiictors, and (see Fig. 3). is the concentration at which half-maximal reaction velocity is achieved. The smaller value is for 

At any given concentration, the initial velocity rate (Vg) is proportional to the enzyme concentration so the term Vg can be used as a measure of the enzyme concentration provided certain precautions are taken. One should be aware of the variability of these reaction constants with pH, temperature, and ionic strength. Likewise, conditions for substrate concentration should be selected to guarantee no interference with the reaction rate. Usually, the concentration of the substrate is chosen to be well above the value to ensure acceptable conditions (H3). As a side issue, it should be noted that substrates with a free N-terminal are 10 to 100 times more soluble than the corresponding benzoyl derivatives CTIO). Finally, one should always report the conditions under which the assay was run to allow comparison with other laboratories. 

In Fig. 4, one can see the familiar UV absorbance curve for an intact chromogenic substrate and of the free pNA at the same concentration. By monitoring the formation of pNA at 405 nm one is ensured minimal interference from the reactants (F5). A linear relationship exists between the concentration of the enzyme in the assay system and the release of either the 

The basic equipment needed to perform the substrate assays is listed below  

Once an ADA-positive sample has been shown to be specific in the confirmatory assay, typically the next step is to determine the titer of the sample. Often, the titer is determined in the screening assay, using the same approach used initially to determine positive samples. Serial dilutions of the sample are made prior to analysis. 

ELISAs are the most prevalent methods used for detecting antibodies. These assays can be conducted using a variety of approaches, which include direct, indirect, bridging, and competitive inhibition formats (Fig. 8.1). These assay formats offer high throughput, are easily automated, use simple technology, require low capital investment, and enjoy comparatively short method development times. A major 

1 Direct or Sandwich ELISA The direct format represents the most simplified ELISA system. In this assay, serum or plasma samples are incubated with the antigen (usually the drug), which has been previously immobilized directly onto well surfaces of microtiter plates. The bound antibody is detected using an enzyme-labeled anti-immunoglobulin reagent of appropriate specificity. 

Cons Immobilization of the antigen can alterits conformation and/or mask epitopes such that these may not be recognized by the host s antibody response [18]. 

Pros Improved and controlled presentation of available epitopes on the drug molecule to antibodies in the sample. 

If no test compound is available, RNAi is an option for perturbation [77]. Once the assay has been established, a small library of compounds with known biological activities should be screened as a test for the viability of the assay. 

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Careful attention to the details of enzyme assay design for HTS can ensure that one will capture the full richness of inhibitors in a chemical library. 

In Chapter 2 we described the typical product progress curve for a well-behaved enzyme and introduced the concept of initial velocity. In assays designed to quantify the ability of a test compound to inhibit the target enzyme, it is critical to restrict... 

The discussion above was concerned with the effects of solution conditions on enzyme activity, hence reaction velocity. Equally important for the purpose of assay design is the influence of specific solution conditions on the detection method being used. This latter topic is beyond the scope of the present text. Nevertheless, this is an important issue for screening scientists whose job is often to balance the needs of biochemical rigor and assay practicality in development of an HTS assay. An... 

In most assays designed to study antioxidant action of carotenoids, the effects of carotenoids were followed for a relatively short periods of time, while carotenoids were still present at substantial concentrations. Carotenoids, such as [1-carotene, lutein, and zeaxanthin, undergo rapid degradation upon exposure to oxidants or irradiation with ultraviolet and visible light (Ojima et al 1993 Siems et al 1999, 2005). 

The first ee-assay designed to handle a reasonably large number of samples (400-700 ee-determi-nations per day) was a rather crude UV/Vis-based screening system for the lipase-catalyzed... 

Several assay designs that use the enhanced sensitivity afforded through biotinylated antibodies have been developed. Most of these systems use conjugates of avidin or streptavidin... 

The end-products of liposome technology are used in retail markets, for the diagnosis of disease, as therapeutic agents, as vaccines, and as important components in assays designed to either detect or quantify certain analytes. 

Hormones Androgens Testosterone Testosterone EIA kit Salivary assay test Colorimetric EIA kit Testosterone ELISA kit Immunometric Ltd. Salim etric Assay Designs Inc. Oxford Biomedical Research http //www.oxfordbiomed.com/... 

Corticosterone Corticosteone EIA kit Assay Designs Inc. http //www.assaydesigns.com... 

This experimental setup was applied for characterization of the interaction between WGA as a targeter and isolated Caco-2 cells, as well as monolayers. Accordingly, all the carbohydrate-combining sites accessible at the cell membrane were occupied within 10 min. This step was followed by internalization of 80% of membrane-bound lectin after 90 min in the case of isolated cells, and 240 min in the case of monolayers [25], The principle of this assay design also works for characterization of the interaction between targeted nanoparticles and monolayers (unpublished data). 

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