Introduction: A new non-radioactive method based on competitive ELISA has been developed for binding studies on angiotensin II (Ang II) receptors.
Method: Rat liver membrane was used as the source of angiotensin receptors and FITC-angiotensin II (FITC-Ang II) was used as the labeled ligand with an affinity similar to unlabeled Ang II. The effects of different concentrations of Ang II, losartan, CGP-42112A and saralasin were studied on FITC-Ang II binding.
Results: The Ki values for Ang II, losartan and CGP-42112A were calculated as 0.52+/-0.22 nM, 6+/-3 nM and 0.15+/-0.07 nM, respectively. Saralasin inhibited the binding of labeled ligand biphasically, revealing two different populations of Ang receptor with different affinities for saralasin. About 74% of the binding sites were more sensitive to saralasin with a Ki value of 0.32+/-0.04 nM while saralasin showed a Ki value of 2.7+/-0.8 nM for the remaining binding sites.
Discussion: The competitive ELISA method developed in this work yields Ki values for angiotensin antagonists similar to those obtained by others using radiolabeled ligands. The simplicity of this method makes it a suitable alternative to radioligand studies for routine analysis of interaction of drugs with angiotensin receptors.
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