Problems associated with the use of commercial and non-commercial antibodies against the histamine H4 receptor

Neumann, Detlef; Beermann, Silke; Mägel, Lavinia; Jonigk, Danny; Weber-Steffens, Dorothea; Männel, Daniela N.; Seifert, Roland

doi:10.1007/s00210-012-0766-7

Cited by 10 publications

(9 citation statements)

References 29 publications

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“…Specifically, it has been shown that many antibodies against β 3 -adrenoceptors (Pradidarcheep et al 2009;Cernecka et al 2012), other β-adrenoceptor subtypes (Hamdani and van der Velden 2009) or other G-protein-coupled receptors in general (Pradidarcheep et al 2008;Michel et al 2009;Adams et al 2012;Beermann et al 2012;Neumann et al 2012;Yu and Hill 2013;Cecyre et al 2014;Talmont and Mouledous 2014;Böhmer et al 2014), in many, if not most cases, lack selectivity for their cognate receptors when tested based on stringent criteria. A well-validated monoclonal antibody against β 3 -adrenoceptors (Chamberlain et al 1999) unfortunately never became available commercially and no longer is available from the original investigators.…”

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confidence: 99%

Rat β3-adrenoceptor protein expression: antibody validation and distribution in rat gastrointestinal and urogenital tissues

Cernecka

Pradidarcheep

Lamers

et al. 2014

Naunyn-Schmiedeberg's Arch Pharmacol

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β3-Adrenoceptors play important roles in the regulation of urogenital and probably gastrointestinal function. However, despite recent progress, their detection at the protein level has remained difficult due to a lack of sufficiently validated selective antibodies. Therefore, we have explored the selectivity of two antibodies for the detection of rodent β3-adrenoceptors in immunoblots and immunohistochemistry. Of two reportedly promising candidates, antibody AB15688 did not exhibit subtype selectivity in immunoblots. In contrast, the antibody Sc1473 exhibited at least some selectivity in immunoblots and more promising results in immunocytochemical and immunohistochemical stains in cells transfected with cloned β-adrenoceptor subtypes and in rat and mouse tissues. In a systematic screening of rat gastrointestinal and urogenital tissues, Sc1473 produced selective staining in the epithelial cell lining of the stomach and the urothelium of ureter and bladder. We conclude that the two tested antibodies are inappropriate or at least insufficient for immunoblotting applications, but Sc1473 appears to be useful for immunohistochemical detection of β3-adrenoceptor protein in rodent tissues. The β3-adrenoceptor protein exhibits a distinct expression pattern in the rat gastrointestinal and urogenital tract, which is at least partly in line with previously reported functional data.

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confidence: 99%

Rat β3-adrenoceptor protein expression: antibody validation and distribution in rat gastrointestinal and urogenital tissues

Cernecka

Pradidarcheep

Lamers

et al. 2014

Naunyn-Schmiedeberg's Arch Pharmacol

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“…In response to this, Gutzmer et al disagreed with some of the conclusions since they felt that work from their laboratory using one of the antibodies, had proved its efficacy and that technical differences in the experimental designs were responsible for discrepant results (Gutzmer et al, 2012). While conceding some points, the Beermann group nonetheless strongly defended the primary conclusions (Neumann et al, 2012). This debate emphasizes the difficulty of ‘proving’ that antibodies don’t work but is worth having in an attempt to ‘raise the bar’ overall for what is generally considered satisfactory evidence of specificity.…”

Section: Discussionmentioning

confidence: 99%

Lack of specificity shown by P2Y6 receptor antibodies

Hill

2013

Naunyn-Schmiedeberg's Arch Pharmacol

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P2Y6 receptor in bladder smooth muscle responds to UDP by increasing muscle tone and augmenting bladder contractions. The exact cellular location of the receptor is however unknown. Three commercially available antibodies to P2Y6 receptor gave clean bands on Western blot which were eliminated by specific peptide competition. Two of the three also immunostained bladder smooth muscle cells while leaving adjacent interstitial cells of Cajal unstained. However, attempts to validate the specificity of these antibodies by performing the same assays on bladders from P2Y6 knockout mice were unsuccessful. In Western blots, all three antibodies bound similar proteins in both wild type and P2Y6 knockout tissue. Immunostaining of knockout tissue sections also showed no difference in staining patterns or intensity. We conclude that rigorous controls are required when using commercial reagents to this G-protein coupled receptor and perhaps to other members of the P2Y receptor family.

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“…The specificity of antibodies is a general problem, especially in case of transmembrane proteins like GPCRs . Several commercially available H 4 R antibodies lack specificity , which was extensively discussed in a scientific dispute (Gutzmer et al, 2012;Neumann et al, 2012). Lack of antibody specificity was not only reported for H 4 R, but also for several other GPCR types including dopamine receptors (Bodei et al, 2009), P2Y6 purinoceptors (Yu and Hill, 2013) or muscarinic and adrenergic receptors (Pradidarcheep et al, 2009) and the discussion is still ongoing (B€ ohmer et al, 2014;Talmont and Moul edous, 2014).…”

Section: Methodsmentioning

confidence: 99%

The histamine H4-receptor and the central and peripheral nervous system: A critical analysis of the literature

Schneider

Seifert

2016

Neuropharmacology

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Problems associated with the use of commercial and non-commercial antibodies against the histamine H4 receptor

Cited by 10 publications

References 29 publications

Rat β3-adrenoceptor protein expression: antibody validation and distribution in rat gastrointestinal and urogenital tissues

Rat β3-adrenoceptor protein expression: antibody validation and distribution in rat gastrointestinal and urogenital tissues

Lack of specificity shown by P2Y6 receptor antibodies

The histamine H4-receptor and the central and peripheral nervous system: A critical analysis of the literature

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