No answer to the lack of specificity: mouse monoclonal antibody targeting the angiotensin II type 1 receptor AT1 fails to recognize its target

Numerous antibodies targeting G protein-coupled receptors (GPCRs) have been described as non-specific among the polyclonal antibodies against angiotensin II type 1 receptor (AT(1)). We have tested the newly developed AT(1) receptor mouse monoclonal antibody for its specificity. Human embryonic kidney (HEK293) cells, which do not endogenously express AT(1) receptor, were transfected in order to overexpress a fluorescently labeled enhanced green fluorescent protein (EGFP)-tagged human AT(1) receptor. Western blot and immunofluorescence assays were performed to test the specificity of the Santa Cruz monoclonal antibody sc-57036. These results were compared to the ones obtained with the polyclonal sc-1173 anti-AT(1) receptor antibodies that have already been described as non-specific. While the positive controls using GFP antibodies detected the EGFP-tagged AT(1) receptor, both polyclonal and monoclonal anti-AT(1) receptor antibodies failed to specifically recognize the corresponding band by Western blot, as similar bands were revealed in either transfected or non-transfected cells. It also failed to detect AT(1) receptor in immunofluorescence experiments. The lack of target recognition of the monoclonal AT(1) receptor antibody in our experimental conditions suggests that this antibody could give misleading results such as misidentification of the protein. To our knowledge, no specific antibodies targeting AT(1) receptors have been developed so far and the field is thus in need of new technical developments.