Evaluation of Assay Quality

Before an expensive, large-scale screening campaign is started, certain quality parameters must be met during the development of the assay. One of the basic parameters is to outline the controls used to define assay performance, and to calculate activities. Whereas uninhibited samples typically are used to serve as the positive control, the negative or blank control is often more difficult to define. In the best case, a pharmacologically relevant standard can be used to define the level of maximal effect or the level of nonspecific signal blank (NSB). During assay development, it must be determined if there is sufficient faith that these control values are not too "artificial" to be reached by a pharmacologically active compound.

In some cases, the positive controls might require the addition of an agonistic compound to stimulate the system. Assays searching for agonistic activities might have accordingly reversed controls - for example, the blank value might represent the unperturbed control.

Assay performance and its suitability for HTS is usually calculated using the mean (M) and standard deviation (SD) of such control wells. The screening window coefficient is the most commonly used statistical parameters to describe the quality of an assay [33]:

jivp0sitive controls NSB controls

Mpositive controls MNSB controls

Z is a statistical tool that reflects the assay's dynamic range and the variability associated with the measurements. In recent years, the z'-value has been accepted as the most relevant parameter describing the assay robustness, and z'-values above 0.5 are considered as sufficient for screening campaigns. However, all of these parameters describe to a certain level only the robustness of the assay, and not whether the assay can detect the correct pharmacology and/or is capable of detecting hits with the desired sensitivity. In addition to the difficult quest for the correct pharmacology of novel targets, target-unrelated effects have been recognized as a major challenge for HTS operation [40]. Such undesired assay interference leads to so-called "false positive" or negative results, and these can create a significant burden on resources during the hit validation phase. Phenomena such as the "inner filter effect", which is caused by colored compounds, the quenching of fluorescence by various mechanisms, autofluorescence of compounds, light scattering resulting from particles, and photo bleaching are common mechanisms of assay interference.

Many compound-related assay interferences are concentration-dependent, and this creates a barrier for the selection of high compound concentrations for screening. Another major disturbing effect is caused by compound precipitation; hence, exceeding a compound's solubility limits in HTS is not recommended but, due to the variety within the compound collection, this cannot always be avoided [41]. However, the most important parameter - the relevance of the screening approach for the pathophysiological in vivo situation is the most difficult to appreciate a priori. One strategy to improve the level of confidence in the physiological nature of the screening is to present the target of choice within its cellular environment.

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