Three HTRF methods provide sensitivity for miniaturization in high-throughput screening

Homogeneous time-resolved fluorescence (HTRF®) assays are ideal for miniaturization in high-throughput screening. HTRF assays can be scaled down from 200 μL reactions to 25 to 70 μL reactions without loss of sensitivity. Three assay formats using proprietary europium cryptate, (Eu)K, and XL665 are described. An HTRF tyrosine kinase assay illustrates the indirect assay format. Inhibition can be detected with enzyme concentrations as low as 20 pM. An HTRF receptor-ligand binding assay is used to show a direct assay format. Reproducibility and stability are shown by a comparison of competitive binding curves under varying assay conditions. An HTRF reverse transcriptase assay is described to show the semidirect assay format. Enzyme concentrations were varied and compared with a typical [³²P]-labeled assay. All three assay formats have been optimized using HTRF reagents. Results, measured in the highly sensitive Discovery® HTRF microplate analyzer, show that the sensitivity of HTRF is maintained when converting from 96-well format to 384-well format, despite the decrease in volume of reagents. © 1999 John Wiley & Sons, Inc. Lab Robotics and Automation 11: 324–329, 1999