10.1177/1087057105279149ARTICLEZhang et al.Primary Screening Efficiency by Multiple Replicate Testing Efficiency by Multiple Replicate s of Hit Confirmation and False a Biochemical Assay

Despite a large body of references on assay development, assay optimization, strategies, and methodologies for high-throughput screening (HTS), there have been few reports on investigations of the efficiency of primary screening in a systematic and quantitative manner for a typical HTS process. Recently, the authors investigated the primary hit comparison and the effect of measurement variability by screening a library of approximately 25,000 random compounds in multiple replicate tests in a nuclear receptor recruitment assay with 2 different assay detection technologies. In this report, we utilized these sets of multi-ple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some in-sights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (dec-laration) rates, and false-negative rates at different hit cutoff limits were explored and calculated from the 2 different assay formats. Results and analyses provided some quantitative estimation regarding the reliability and efficiency of the primary screening process. For the 2 assay formats tested in this report, the confirmation rate (activity repeated at or above a certain hit limit) was found to be 65 % or above. It was also suggested that, at least in this case, applying some hit-selection strategies, it is possible to decrease the number of false-negative or false-positive hits without significantly increasing the efforts in primary D strum has b techn incre (“hits ally. of va ological, assays, capture of true hits from data noise is important in the early drug discovery process. The accuracy, sensitivity, and specificity of the assay are critical TS) nly ions nec-alse een esti-rom as-lity, ing process. The importance of assay format in hit identification has been probed by comparing screening results with different assay studies.4-11 We recently used a tivator binding (recruitment) as-say to screen a 25,000-member randomly selected compound li-12 in lo-se ie