Comparison of 3 AT1 Receptor Binding Assays: Filtration Assay, ScreenReady™ Target, and WGA Flashplate®

In this article, the study of 3 different angiotensin II type 1 (AT1) receptor binding assays in terms of reproducibility, robustness, and feasibility for high-throughput screening (HTS) is described. The following methods were used: a nonhomogeneous filtration assay in a 96-well format using CHO-AT1 cell membranes and 2 homogeneous assays, which include the commercially available ScreenReady™ Target for the AT1 receptor and the wheat germ agglutinin (WGA) Flashplate®, which was coated “in-house” with the CHO-AT1 cell membranes. Receptors were labeled with [125I]-Sar1-Ile8-angiotensin II, and radioligand binding was displaced using the antagonist losartan and the natural agonist angiotensin II. Reproducible Kd, Bmax, and Ki values and good total binding/nonspecific binding (TB/NSB) ratios were obtained with both the ScreenReady™ Targets and the filtration assay, whereas the WGA Flashplates® showed unacceptably high nonspecific binding and high variation when applied as a homogeneous assay. However, when a...