Assay development time line

Unfortunately, few analytical methods are developed in a perfect world. A more likely situation has method development and validation scheduled on a tight time line. Using an HPLC assay and related compounds method for a novel DS as an example,Table 5 outlines a realistic development scenario that would provide for a method that could be successfully validated and utilized. 

Fig. 9.2 Typical progress curve obtained in the isomer-specific proteolysis (ISP) assay developed by C. Fischer in 1984 [34]. Extrapolation of the slow kinetic phase (black solid line) allows the calculation of the zero time (t0) absorbance value A0 and the infinite reaction time absorbance A,. The difference gives...

The use of a reporter line has multiple benefits for the high content screening process (a) assay development is facilitated as reporter expression can be followed in real time, (b) Assay controls are immediately visible before processing the plates as described, (c) Antibody-based read onts are costly and can be prohibitive when running larger screens. 

Mammalian cells in culture are exposed to the test substance. Established cell lines are treated both with and without metabolic activation. Cells are incubated for an appropriate length of time, then rinsed, fixed, and dried. Slides are developed, stained, and exposed silver grains are counted. The endpoint of UDS is measured by determining the uptake of labeled nucleosides in cells that are not undergoing scheduled (S-phase) DNA synthesis. The most widely used technique is the determination of the uptake of H-TdR by autoradiography. Primary cultures (e.g., rat hepatocytes), human lymphocytes, or established cell lines (e.g., human diploid fibroblasts) may be used in the assay. Multiple concentrations of the test substance over a range adequate to define the response, should be used. 

The development of this type of nuclear translocation assay can be relatively simple because the manufacturers have identified conditions to maximize the response. While a cell titration coupled with agonist and/or antagonist titrations is still required, the optimal conditions for the chemistry involved in the detection are generally provided by the assay reagents. In addition, time and temperature of incubations should be optimized for each receptor and cell line. A sample protocol of a 1536-well plate assay in which the detection of the nuclear translocation of GR is measured is listed below ... 

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