How reliable are G-protein-coupled receptor antibodies?
A cluster of manuscripts in this issue of the Journal highlights a lack of selectivity of 49 antibodies against 19 subtypes of alpha(1)- and beta-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.
Michel
Martin C
MC
Wieland
Thomas
T
Tsujimoto
Gozoh
G
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D017124:Conserved Sequence / Q000276:immunology; D035081:Editorial Policies; D006801:Humans; D011941:Receptors, Adrenergic / Q000276:immunology; D011954:Receptors, Dopamine / Q000276:immunology; D043562:Receptors, G-Protein-Coupled / Q000276:immunology*; D044088:Receptors, Galanin / Q000276:immunology; D011976:Receptors, Muscarinic / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D050916:TRPV Cation Channels / Q000276:immunology; D054928:Validation Studies as Topic
D000906:Antibodies; D011941:Receptors, Adrenergic; D011954:Receptors, Dopamine; D043562:Receptors, G-Protein-Coupled; D044088:Receptors, Galanin; D011976:Receptors, Muscarinic; D050916:TRPV Cation Channels