Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors

Abstract

Commercially available antisera against five subtypes of muscarinic receptors and nine subtypes of adrenoceptors showed highly distinct immunohistochemical staining patterns in rat ureter and stomach. However, using the M(1-4) muscarinic receptor subtypes and alpha(2B)-, beta(2)-, and beta(3)-adrenoceptors as examples, Western blots with membranes prepared from cell lines stably expressing various subtypes of muscarinic receptors or adrenoceptors revealed that each of the antisera recognized a set of proteins that differed between the cell lines used but lacked specificity for the claimed target receptor. We propose that receptor antibodies need better validation before they can reliably be used.

How reliable are G-protein-coupled receptor antibodies?

“…Indeed, one study reported here shows that antibodies against either β 2 -or β 3 -adrenoceptors recognize rather similar band patterns on immunoblots from cells expressing any of the nine adrenoceptors subtypes (Pradidarcheep et al 2009). However, the problem apparently goes well beyond the homology of closely related GPCR subtypes.…”

“…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.…”

“…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.…”

“…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).…”

β3-Adrenoceptors: a drug target in ophthalmology?

“…These findings suggest that conjunctival β 3 -adrenoceptor expression is species dependent. However, the expression data obtained with "selective" antibodies have to be interpreted with caution, since a variety of antibodies raised against G protein-coupled membrane receptors, including β 3 -adrenoceptors, was shown to exhibit only low target selectivity (Pradidarcheep et al 2009;Cernecka et al 2012). Moreover, some of these antibodies exhibit specificity between rodent and human β 3 -adrenoceptors, which may lead to false-positive conclusions about species differences.…”