Assay design/development

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... 

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... 

SPA has been developed as a receptor binding assay using molecules prepared from a number of different sources ranging from crude tissue preparations to soluble purified proteins. The method, which is applicable to both 3H and 125I ligands, provides a number of factors and must be carefully optimized in the assay design and development. 

To reduce the likelihood of competitive inhibitors, one should run the assays with high concentrations of substrates relative to Km levels. To favor competitive inhibitors, one should run the assay below the Km values for the substrates. Thus, for making suitable conclusions for assay design, knowledge of the kinetic and binding constants of receptors and enzymes, such as Kd, kCM, Km, Bmax, is useful. Stoichiometric information, such as the number of enzyme molecules per assay, may also be useful because it can serve as a guideline to ensure that the assays are maximally sensitive to the mechanism of action one wants to discover. Problems in assay development often occur when the conditions required for sensitivity to the desired mechanism of action do not yield the best conditions for statistical reproducibility therefore, compromises and balances of these two opposing factors must be often made. 

Precision is a fundamental measure of assay performance and it should be assessed whenever possible [55]. This will allow a minimum understanding of how much confidence can be placed in the analytical data. In fully characterized assays designed to support definitive nonclinical and clinical studies, between-run and within-run assay precision of 15% or less (20% at the quantitation limit) is acceptable. Because of the shortened time scale for assay development and characterization in discovery these guidelines are excessively restrictive. Precision values of 20 to 30% are acceptable, as these are still less than intersubject variability associated with many nonclinical experiments. With this level of imprecision, data quality is sufficient to answer fundamental scientific questions such as relative levels of drug absorption, relative values of absolute bioavailability, and relative degree of drug clearance. 

Figure 6. Guideline for assay design of membrane-, cell- and tissue-based systems, depending on the development phase and expected output.

The development of methods to produce monoclonal antibodies (Kohler and Milstein, 1975 Section 5.4) not only enhanced the possibility of a more stringent standardization of EIA with higher specificity and sensitivity, but also contributed to new assay designs. 

BBB-permeability-liquid chromatography/mass spectroscopy- (LC/MS) Rapid, generic gradient liquid chromatography/ Chu et al. (2002) tandem mass spectroscopy (LC/MS/MS) assays, designed to accelerate sample analyses, have been developed to keep pace with the productivity of advanced synthetic procedures. In this study, LC/MS/MS was combined with an in vitro, cell-based, BBB model to evaluate the potential of new chemical entities (NCEs) to cross the BBB. This in vitro assay provides the permeability of discovery compounds across a monolayer of a primary culture of BBMEC in a fraction of the time that is required for in vivo studies (brain/plasma concentrations), using only 2 mg of the compound. The results are consistent with in vivo brain/plasma concentration ratio data. 

As a result of large international collaborative studies to evaluate various short-term In vitro and In vivo tests, it has become clear that both approaches are needed and that any battery of assays designed to evaluate environmental chemicals must include both In vitro and In vivo short-term tests. The use of the Ames assay in isolation has resulted in premature, and sometimes false, indictment of potentially useful chemicals. A data base developed with the use of this test alone Is inadequate for the evaluation of a chemical s mutagenic and carcinogenic potential in laboratory animals and humans. 

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