A 384-HTS for Human Factor VIIa: Comparison With 96- and 864-Well Formats

Abstract

A homogenous fluorescent HTS for recombinant human factor VIIa (FVIIa) using soluble tissue factor has been developed in 384-well microplates. In this report we discuss our experiences with assay development, liquid handling using a Tomtec Quadra and Matrix PlateMate, fluorescent detection and screening of -200,000 compounds against FVIIa in 384-well plate format. Assays using the entire Helix 864-well plate were prototyped using contact dispensing with a modified Hamilton Microlab 2200. FVIIa was used as a model assay to compare between 96-, 384-, and 864-plate formats in a total assay volume of 100, 25, and 10 μl, respectively. FVIIa was assayed in 864 to the same degree of sensitivity as 384- and 96-well assays and dose-response curves for a standard inhibitor (benzamidine) in the FVIIa assay were identical in all plate formats. Finally, we review the prospects for HTS in 864-well microplates.