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.

Analysis of Histamine Receptor Knockout Mice in Models of Inflammation

“…Moreover, enhanced expression of mRNA does not necessarily lead to enhanced protein synthesis (de Sousa Abreu et al, 2009). Unfortunately, the analysis of histamine receptor protein expression is highly problematic because of the lack of reliable antibodies (Beermann et al, 2012b), and radioligand-binding studies are working well only for H 1 R and H 3 R . Therefore, the issue of compensatory mechanisms within the family of histamine receptors has still to be elucidated.…”

Challenges in Developing a Biochip for Intact Histamine Using Commercial Antibodies

“…Several authors have addressed the issue and claimed for better characterization and profound information on the specificity and cross-reactivity of the antibodies [19][20][21][22][23][24][25][26][27][28]. The antibodies in the market are often insufficiently characterized and furthermore they might possess batch-to-batch variation, usually in much larger extent with polyclonal antibodies compared to monoclonal.…”

“…The recommended applications are mainly immunohistochemistry (IHC) or immunocytochemistry (ICC), but also western blot, EIA and ELISA. Recently, several studies have pointed out the quality issues and traceability problem concerning commercial antibodies and the lack of quality in the antibody market has been widely recognized by the scientific community [19][20][21][22][23][24][25][26][27][28]. Often, the characterization and specifications provided by the manufacturers are somewhat incomplete and, furthermore, the authors often fail to identify the exact antibody used [28][29][30] for other researchers to be able to purchase the same product.…”

Naunyn-Schmiedeberg’s Archives of Pharmacology under new editorship: change and continuity

“…In fact, my most-cited publication, a review on constitutive GPCR activity (Seifert and Wenzel-Seifert 2002), has been cited 356 times so far. And several of my original publications from various areas (including papers with "negative" data) published in the journal have also been cited fairly often (see, e.g., Burde et al 1989;Seifert et al 1989;Ervens et al 1991;Appl et al 2012;Beermann et al 2012). A recently published meeting report (Heifetz et al 2015) reached more than 900 downloads within 4 months.…”

“…Papers in these sections are downloaded very frequently. In the field of validation of receptor antibodies, Naunyn-Schmiedeberg's Archives of Pharmacology has earned exceptionally high international recognition and constitutes the leading international discussion forum on this important topic (Beermann et al 2012;Böhmer et al 2014;Ashton et al 2014;Cécyre et al 2014;Cernecka et al 2014;Tripathi et al 2016).…”