Probing the primary screening efficiency by multiple replicate testing: A quantitative analysis of hit confirmation and false screening results of 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 multiple replicate screening data from a different perspective and conducted a systematic data analysis in order to gain some insights into the hit-finding efficiency of a typical primary screening process. Specifically, hit confirmation, false-positive (declaration) 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 screening.