Biomedical Engineering Reference
In-Depth Information
FIGURE 12.2
Most frequently used plates come in 96-, 384- and 1536-well densities.
Courtesy of Maggie Bartlett, NHGRI/NIH
marker. Second, wells exemplifying the desired inducible effect sought after with the
screened compounds provide
positive control wells
; they are usually mimicked by
the addition of a known modulator establishing a maximal-efficacy marker. Finally,
the majority of the wells contain the complete assay mixture and the tested chemical
compounds at a desired concentration. Control well compositions are selected such
that the compound wells are expected to fall in the range between the values of the
positive and negative controls.
One of the major challenges in HTS is selection of an compound appropriate
concentration for primary screening. On the one hand, too high a concentration
would lead to the assay being plagued by undesirable effects of the compounds
on the assay. For example, the number of compounds precipitating out of solution
increases dramatically with increasing compound concentration. On the other hand,
setting the concentration too low is also detrimental, leading to a decreased rate of
finding active compounds. To establish the optimal concentration, a limited set of
compound plates is usually tested at different compound concentrations during HTS
assay implementation stage.
12.2.2 Assay Performance Measures
The cost of HTS is directly proportional to the number of screened plates. Therefore,
plates that have failed and have to be retested incur an additional and avoidable
cost to the overall process. Clearly, a decrease in the number of retested plates or,
preferably, their complete elimination is desired. Thus, the screening process needs
to be extremely reliable and robust. Certain parameters obtained from the screening
data, termed
assay performance measures
, are utilized to gauge the precision and
reliability of the screening data. In HTS, these parameters are usually calculated
from the data of positive and negative control wells. The same parameters are also
