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Journal of Biomolecular Screening
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Comparison of 3 AT1 Receptor Binding Assays: Filtration Assay, ScreenReadyTM Target, and WGA Flashplate®

Regine M. van der Hee

Unilever Research & Development Vlaardingen, Unilever Health Institute (UHI), Vlaardingen, the Netherlands

Tanja Deurholt

Unilever Research & Development Vlaardingen, Unilever Health Institute (UHI), Vlaardingen, the Netherlands

Cindy C. Gerhardt

Unilever Research & Development Vlaardingen, Unilever Health Institute (UHI), Vlaardingen, the Netherlands

Els M. de Groene

Unilever Research & Development Vlaardingen, Unilever Health Institute (UHI), Vlaardingen, the Netherlands

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 ScreenReadyTM 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 ScreenReadyTM 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 applied as a heterogeneous assay (i.e., when a wash step at the end of the assay is included), the results were significantly better. Interestingly, ligand affinities were consistently lower in Flashplate®-based assays than in the filtration assay. This may be due to the immobilization of the receptors onto the solid surface of the plate, affecting their conformation. In terms of reproducibility, robustness, and feasibility for HTS, the authors conclude that the ScreenReadyTM Target plates are most suitable for AT1 receptor binding screening.

Key Words: ScreenReadyTM Target • Flashplates® • angiotensin II • high-throughput screening • receptor binding assays

Journal of Biomolecular Screening, Vol. 10, No. 2, 118-126 (2005)
DOI: 10.1177/1087057104271330
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