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Specificity evaluation of antibodies against human β3-adrenoceptors

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Abstract

β3-Adrenoceptors are a promising drug target for the treatment of urinary bladder dysfunction, but knowledge about their expression at the protein level and their functional role is limited, partly due to a lack of well validated tools. As many antibodies against G-protein-coupled receptors, including those against β3- and other β-adrenoceptor subtypes, lack selectivity for their target, we have evaluated the specificity of five antibodies raised against the full-length protein of the human β3-adrenoceptor (H155-B01), its N-terminus (LSA4198 and TA303277) and its C-terminus (AB5122, Sc1472) in immunoblotting and immunocytochemistry. Our primary test system were Chinese hamster ovary cells stably transfected to express each of the three human β-adrenoceptor subtypes at near physiological levels (100–200 fmol/mg protein). None of the five antibodies exhibited convincing target specificity in immunoblotting with Sc1472 apparently being least unsuitable. In immunocytochemistry, LSA4198 and Sc1472 appeared most promising, exhibiting at least some degree of specificity. As these two antibodies have been raised against different epitopes (N- and C-terminus of the receptor, respectively), we propose that concordant staining by both antibodies provides the most convincing evidence for β3-adrenoceptor labelling in cyto- or histochemistry studies.

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Acknowledgements

We thank Dr. Dave Speijer (Dept. Biochemistry, AMC) and Dr. Maurice van den Hoff (Dept. Anatomy, AMC) for their help and advice. This work was funded in part by a grant from Astellas Europe BV and through Coordination Theme 1 (Health) of the European Community’s FP7, grant agreement number HEALTH-F2-2008-223234. M.C. Michel has been a paid consultant to AltheRx and Astellas in the β3-adrenoceptor field and is an employee of Boehringer Ingelheim Pharma GmbH & Co KG.

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Authors and Affiliations

  1. Department of Urology, University of Amsterdam, Amsterdam, The Netherlands

    Hana Cernecka

  2. Department of Pharmacology and Pharmacotherapy, University of Amsterdam, Amsterdam, The Netherlands

    Peter Ochodnicky

  3. Tytgat Institute of Liver and Intestinal Research, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

    Wouter H. Lamers

  4. Department of Pharmacology, Johannes Gutenberg University Mainz, Mainz, Germany

    Martin C. Michel

  5. Institut für Pharmakologie, Johannes Gutenberg Universität, Obere Zahlbacher Str. 67, 55101, Mainz, Germany

    Martin C. Michel

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  1. Hana Cernecka
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  2. Peter Ochodnicky
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  4. Martin C. Michel
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Correspondence to Martin C. Michel.

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Cernecka, H., Ochodnicky, P., Lamers, W.H. et al. Specificity evaluation of antibodies against human β3-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 385, 875–882 (2012). https://doi.org/10.1007/s00210-012-0767-6

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