Six Commercially Available Angiotensin II AT1 Receptor Antibodies are Non-specific

Commercially available Angiotensin II AT 1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT 1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT 1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT 1 receptor. However, identical bands were observed in wild-type mice and in AT 1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT 1 receptors or transfected with AT 1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT 1A knock-out and wild-type mice and in 4B cells with or without AT 1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT 1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT 1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT 1 receptor physiology in the absence of full antibody characterization.