title: Commercially available antibodies against human and murine histamine H₄-receptor lack specificity.
abstract: Antibodies are important tools to detect expression and localization of proteins within the living cell. However, for a series of commercially available antibodies which are supposed to recognize G-protein-coupled receptors (GPCR), lack of specificity has been described. In recent publications, antisera against the histamine H₄-receptor (H₄R), which is a member of the GPCR family, have been used to demonstrate receptor expression. However, a comprehensive characterization of these antisera has not been performed yet. Therefore, the purpose of our study was to evaluate the specificity of three commercially available H₄R antibodies. Sf9 insect cells and HEK293 cells expressing recombinant murine and human H₄R, spleen cells obtained from H₄⁻/⁻ and from wild-type mice, and human CD20⁺ and CD20⁻ peripheral blood cells were analyzed by flow cytometry and Western blot using three commercially available H₄R antibodies. Our results show that all tested H₄R antibodies bind to virtually all cells, independently of the expression of H₄R, thus in an unspecific fashion. Also in Western blot, the H₄R antibodies do not bind to the specified protein. Our data underscore the importance of stringent evaluation of antibodies using valid controls, such as cells of H₄R⁻/⁻ mice, to show true receptor expression and antigen specificity. Improved validation of commercially available antibodies prior to release to the market would avoid time-consuming and expensive validation assays by the user.
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Beermann
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
authors.forename: Detlef
authors.initials: D
authors.lastname: Neumann
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D043562:Receptors, G-Protein-Coupled / Q000172:deficiency / Q000235:genetics / Q000276:immunology*; D011968:Receptors, Histamine / Q000172:deficiency / Q000235:genetics / Q000276:immunology*; D000074040:Receptors, Histamine H4; D018411:Spodoptera; D014162:Transfection
chemical_list: D000906:Antibodies; C418607:HRH4 protein, human; C498829:Hrh4 protein, mouse; D043562:Receptors, G-Protein-Coupled; D011968:Receptors, Histamine; D000074040:Receptors, Histamine H4
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