Screening assays precision

A. Wald NJ, Hackshaw AK, George LM. Assay precision of serum alpha fetoprotein in antenatal screening for neural tube defects and Down s syndrome. J Med Screen 2000 7 74-7. Erratum in J Med Screen 2000 7 168. 

Screen validation phase tests the screening assay in a more production-like environment. For example, the lab bench results are replicated on the HTS robotic system. Critical quality control experiments are performed at this stage in the process. This involves screen rehearsal with a small number of compounds (i.e., a few thousand), thus validating the screening process. Process precision is measured by repeating the mini-screen on a different day. This procedure allows definition of all the quality control parameters. Typical values include the following ... 

If known pharmacological agents are available, then the screening assay is validated across multiple assays. This is more difficult if the target of interest is novel and no pharmacological tools are available. It requires rigorous attention to detail to achieve inter-laboratory precision even down to the way the compounds are solubilized and diluted, the types of equipment, and the detection technology. 

Precision is a quantitative measure of the random variation between a series of measurements from a method. Approaches for determining precision of data from the screening assay (assay response), specificity confirmation assay (percent inhibition), and titration assay (titers) are described below. 

Protein arrays may offer advantages over libraries. The array format provides a precise spatially oriented grid that allows side-by-side comparison of assay results for all of the proteins on the array. The spatial arrangement also permits immediate identification of a clone that tests positive in an assay based on its location on the array. Therefore, less effort is required to identify the protein responsible for an interaction than with a library screen. A disadvantage of a protein array, however, is that fewer proteins can be efficiently arrayed and screened ( 104) versus a library ( 109). Therefore, the number of elements that can be effectively arrayed and assayed currently limits protein arrays. 

We have developed chemiluminescent immunoenzymatic assays for (3-ago-nist drugs in the 96-well-microtiter-plate format. Such competitive assays have been used for determination of clenbuterol and of the overall content of p-agonist drugs in the sample. They matched the standard requirements of precision and accuracy, and were more sensitive compared to the conventional colorimetric methods. Moreover, CL detection was very rapid, making these assays suitable for screening analysis. 

After primary screenings, which are less quantitative than biological assays giving a positive result or HIT , another more precise secondary screening is conducted, by calculations of IC50 values. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

Several other UV/Vis-based ee assays have been developed, but their general application in directed evolution remains to be demonstrated 66-70). One of them is a well- designed screening system based on enzyme immunoassays (85). The success of the ee assay depends upon the availability of specific antibodies that are easily raised for almost any chiral product of interest. In a given reaction, two antibodies are needed, one that measures the product concentration and the other that measures the amount of one of the enantiomers. About 1000 ee determinations are possible per day, the precision amounting to + 9% (85). 

There are proficiency testing programs that are geared toward clinical sensitivity or specificity by seeking to determine whether a disease can be detected versus other types of controls that are use to test sensitivity, selectivity, and most importantly, reproducibility and precision. With mass spectrometry, the controls are and should be no different than those used for other assays, with one interesting exception. Quality assurance materials prepared for MS/MS may not be useful in other assays that are less selective. The example is newborn screening where quality assurance/control QA/QC materials have a mixture of compounds present in the blood specimens. However, in less selective immunoassays, the mixture creates interferences. In addition, material is used to spike a blood sample is key and one should ensure there is no enzyme activity. We have encountered such a problem with a d/1 mixture of metabolites where one form was degraded in the prepared blood. 

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