Lack of specificity of antibodies directed against human beta-adrenergic receptors

Hamdani, Nazha; Velden, Jolanda van der

doi:10.1007/s00210-009-0392-1

Cited by 74 publications

(40 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

mentioning

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

View full text Add to dashboard Cite

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.

show abstract

mentioning

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

View full text Add to dashboard Cite

show abstract

“…A greatly variable selectivity has been demonstrated for several commercially available antibodies raised against β1-, β2-, or β3-ARs (Hamdani and van der Velden 2009;Pradidarcheep et al 2009;Cernecka et al 2014b). In the present study, we tried to validate the use of the antibodies against the β-AR subtypes using KO mice and a siRNA-based approach according to recommendations on how to validate receptor antibodies ).…”

Section: Selectivity Of β-Ar Antibodiesmentioning

confidence: 97%

Role of host β1- and β2-adrenergic receptors in a murine model of B16 melanoma: functional involvement of β3-adrenergic receptors

Sereni

Monte

Filippi

et al. 2015

Naunyn-Schmiedeberg's Arch Pharmacol

View full text Add to dashboard Cite

Complex interactions between tumor cells and their surrounding compartment are strongly influenced by the host in which the tumor grows. In melanoma, for instance, stress-associated norephinephrine (NE), acting at β-adrenergic receptors (β-ARs), stimulates melanoma cell proliferation and tumor angiogenesis. Among β-ARs, β3-ARs play a role acting not only at tumor cells but also at non-neoplastic stromal cells within the melanoma. In the present study, we used a murine model of B16 melanoma to evaluate the role of the host β1- and β2-ARs in melanoma growth and we determined whether the role of β3-ARs can be influenced by the absence of stromal β1- and β2-ARs. As compared to wild-type mice, β1/2-AR knockout mice displayed (i) increased intratumoral levels of both NE and β3-ARs, as evidentiated at both messenger and protein levels; (ii) increased tumor vascularization; (iii) decreased tumor cell proliferation but increased tumor cell apoptosis; and (iv) increased responsiveness to intratumoral injection of the β3-AR blocker L-748,337 in terms of decrease in tumor growth, tumor vascular response, tumor cell proliferation, and increase in tumor cell death. These findings together validate the role of β-AR signaling in melanoma microenvironment suggesting that non-neoplastic stromal cells may be targeted by β-AR-related drugs. The additional fact that β3-ARs play an important role in melanoma growth suggests selective β3-AR antagonists as important proapoptotic agents.

show abstract

“…A second soft criterion used in the past is that antibodies against different subtypes of a given receptor exhibit distinct staining patters in tissues. However, studies with both knock-out mice (Everaerts et al 2009;Jensen et al 2009;Jositsch et al 2009;Lu and Bartfai 2009;Pradidarcheep et al 2008) or immunoblots comparing staining to cloned receptor subtypes (Bodei et al 2009;Hamdani and van der Velden 2009;Pradidarcheep et al 2009) demonstrate that this also is insufficient to prove selectivity. Rather we propose that at least one of the following four conditions must be met to consider an antiserum to indeed be selective.…”

Section: Criteria To Demonstrate Receptor Antibody Specificitymentioning

confidence: 99%

“…siRNA; if that leads to a major reduction of staining by a given antibody, this also constitutes reasonable evidence for its specificity. Thirdly, studies with transfection of multiple subtypes of a given receptor into the same host cell line in which the target receptor but not the related subtypes yield positive staining also can provide strong evidence for selectivity (Bodei et al 2009;Hamdani and van der Velden 2009;Pradidarcheep et al 2009). Finally, in a more classical immunological approach, it can be considered as reasonable evidence of selectivity if multiple antibodies raised against different epitopes of a GPCR, e.g.…”

Section: Criteria To Demonstrate Receptor Antibody Specificitymentioning

confidence: 99%

“…In this issue of the Journal, seven groups of investigators report a lack of selectivity of a large number of receptor antibodies for the claimed target receptor. This includes antibodies against subtypes of α 1 -adrenoceptors (Jensen et al 2009), β-adrenoceptors (Hamdani and van der Velden 2009;Pradidarcheep et al 2009), dopamine receptors (Bodei et al 2009), muscarinic receptors (Jositsch et al 2009;Pradidarcheep et al 2009), and galanin receptors (Lu and Bartfai 2009). An additional report shows similar problems with antibodies against receptors from the family of ligand-gated ion channels, specifically the TRPV1 receptors (Everaerts et al 2009), although other antibodies directed against these receptors apparently are selective by hard criteria (Charrua et al 2009).…”

mentioning

confidence: 99%

See 1 more Smart Citation

How reliable are G-protein-coupled receptor antibodies?

Michel

Wieland

Tsujimoto

2009

Naunyn-Schmied Arch Pharmacol

281

207

View full text Add to dashboard Cite

A cluster of manuscripts in this issue of the Journal highlights a lack of selectivity of 49 antibodies against 19 subtypes of α 1 -and β-adrenoceptors, muscarinic, dopamine and galanin receptors as well as vanilloid (TRPV1) receptors. Taken together these data demonstrate that lack of selectivity appears to be the rule rather than the exception for antibodies against G-protein-coupled and perhaps also other receptors. Thus, the previously often applied validation of such antibodies by the disappearance of staining in the presence of blocking peptide, i.e. the antigen against which the antibody was raised, alone is insufficient to demonstrate specificity. We propose that receptor antibodies should be validated by at least one of the following techniques: a) disappearance of staining in knock-out animals of the target receptor, b) reduction of staining upon knock-down approaches such as siRNA treatment, c) selectivity of staining in immunoblots or immunocytochemistry for the target receptor vs. related subtypes when expressed in the same cell line and/or d) antibodies raised against multiple distinct epitopes of a receptor yielding very similar staining patterns. Other issues of consideration to obtain reliable results based on receptor antibodies in applications such as immunohistochemistry or immunoblotting are also being discussed.

show abstract

Lack of specificity of antibodies directed against human beta-adrenergic receptors

Cited by 74 publications

References 14 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

Role of host β1- and β2-adrenergic receptors in a murine model of B16 melanoma: functional involvement of β3-adrenergic receptors

How reliable are G-protein-coupled receptor antibodies?

Contact Info

Product

Resources

About