The signal was acquired with a Tundra™ imaging system with a cooled CCD camera. The results indicate that up to 1 X 10(—3) unit enzyme concentration, the enzyme titration curve is linear and can be used for inhibitor screening. At the selected conditions, a titration with a known HIV-1 reverse transcriptase inhibitor yields an IC50 of 72 nM, compared to the reported value of 46 nM determined under similar conditions in a 96-well plate. Although the signal obtained from the miniaturized format has a relatively low signal-to-background ratio compared to the 96-well plate format, the assay quality estimated by the z-factor (see the statistical analysis section of this chapter) shows that it is still suitable for use in HTS. Recently, a miniaturized SPA assay for MMP (matrix metalloproteinase) has also been performed in the 1536-well plate format with similar assay quality to the aforementioned SPA-RT assay.
In comparison with the regular SPA assays, the miniaturized SPA has several advantages: reagent saving, increased throughput, and reduced radioactive waste. The miniaturized assay also has the potential for improving hit confidence by performing the assay in duplicate or triplicate without consuming significantly more reagent. The miniaturized assay also displayed some disadvantages. For example, in the miniaturized format, the detection system is a low-light CCD camera instead of a photomultiplier-based system. The sensitivity of miniaturized SPA with the currently available CCD cameras is somewhat less than with a similar fluorescent method. The SPA assay will still remain a simple and efficient assay when the ligand or substrate is too small to be fluorescently labeled without loss of activity.
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