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Characterization of two polyclonal peptide antibodies that recognize the carboxy terminus of angiotensin II AT 1A and AT 1B receptors
Author(s) -
Hoffmann Andrea,
Cool David R
Publication year - 2005
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/j.1440-1681.2005.04288.x
Subject(s) - polyclonal antibodies , receptor , microbiology and biotechnology , antibody , angiotensin ii , biology , angiotensin ii receptor type 1 , western blot , peptide , biochemistry , immunology , gene
SUMMARY 1. The differential identification of the angiotensin AT 1A and AT 1B receptor subtypes is impaired by the existing > 96% homology of both receptors. In the present study, we characterized two polyclonal rabbit peptide antibodies, namely α‐AT 1A and α‐AT 1B , that recognize the C‐terminal region of mouse AT 1A and AT 1B receptors, respectively. 2. In immunoblotting, both antibodies detected two major AT 1 receptor‐specific bands at sizes of 72.5 and 87.6 kDa in mouse tissues and in Neuro‐2a cell lysates. In immunohistochemistry, antibodies demonstrated AT 1 receptor‐specific staining in renal proximal and distal tubules, as well as in kidney glomeruli. In addition, both antibodies stained AT 1 receptors in Neuro‐2a cells with G‐protein receptor typical distribution. Dot‐blot and ELISA analysis of the α‐AT 1A antibody showed 2.5‐ to fourfold higher selectivity for its AT 1A receptor target peptide (1A‐PEP) compared with the non‐specific AT 1B receptor peptide (1B‐PEP). In contrast, the α‐AT 1B antibody showed high binding affinity towards its target peptide 1B‐PEP, but also demonstrated high cross‐reactivity for the non‐specific peptide 1A‐PEP (1.4‐ to twofold in ELISA and dot‐blot analysis). In contrast with the lack of recognition by the α‐AT 1B antibody, the α‐AT 1A antibody selectively recognized the AT 1A receptor fused to red fluorescence protein in transiently transfected Chinese hamster ovary cells. 3. In summary, we have generated two new peptide antibodies to the mouse AT 1A and AT 1B receptors (α‐AT 1A and α‐AT 1B ), of which the α‐AT 1A antibody has the capability to distinguish AT 1A receptor types in immunological approaches.