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Naunyn Schmiedebergs Arch Pharmacol. 2012 Feb;385(2):125-35. doi: 10.1007/s00210-011-0700-4. Epub 2011 Nov 10.

Commercially available antibodies against human and murine histamine H₄-receptor lack specificity.

Author information

1
Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

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.

PMID:
22071576
DOI:
10.1007/s00210-011-0700-4
[Indexed for MEDLINE]

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