Comparison of 3 AT1 receptor binding assays: filtration assay, ScreenReady Target, and WGA Flashplate
| Autor: | Regine M. van der Hee, Cindy C. Gerhardt, Tanja Deurholt, Els M. de Groene |
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| Rok vydání: | 2005 |
| Předmět: |
0301 basic medicine
Agonist medicine.drug_class Wheat Germ Agglutinins High-throughput screening CHO Cells Ligands 01 natural sciences Biochemistry Binding Competitive Losartan Receptor Angiotensin Type 1 Analytical Chemistry 03 medical and health sciences Cricetinae medicine Animals Receptor Chemistry Angiotensin II Ligand (biochemistry) Molecular biology Wheat germ agglutinin 0104 chemical sciences 010404 medicinal & biomolecular chemistry 030104 developmental biology Membrane Molecular Medicine Thermodynamics Filtration Biotechnology medicine.drug Protein Binding |
| Zdroj: | Journal of biomolecular screening. 10(2) |
| ISSN: | 1087-0571 |
| Popis: | In this article, the study of 3 different angiotensin II type 1 (AT(1)) 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-AT(1) cell membranes and 2 homogeneous assays, which include the commercially available ScreenReady Target for the AT(1) receptor and the wheat germ agglutinin (WGA) Flashplate, which was coated "in-house" with the CHO-AT(1) cell membranes. Receptors were labeled with [(125)I]-Sar(1)-Ile(8)-angiotensin II, and radioligand binding was displaced using the antagonist losartan and the natural agonist angiotensin II. Reproducible K(d), B(max), and K(i) 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 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 ScreenReady Target plates are most suitable for AT(1) receptor binding screening. |
| Databáze: | OpenAIRE |
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