Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays.
Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment.
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Ji-Hu
JH
Zhang
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Zhao B
ZB
Kang
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Ophelia
O
Ardayfio
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Pei-i
PI
Ho
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Thomas
T
Smith
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Iain
I
Wallace
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
Scott
S
Bowes
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
W Adam
WA
Hill
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA douglas.auld@novartis.com.
Douglas S
DS
Auld
D001681:Biological Assay; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005189:False Positive Reactions; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D010865:Pilot Projects; D015203:Reproducibility of Results; D012984:Software
assay statistics; false negatives; false positives; hit confirmation; pilot screening; quantitative HTS; sample preparation