Development And Characterization Of Sphingosine 1-Phosphate Receptor 1 Monoclonal Antibody Suitable For Cell Imaging And Biochemical Studies Of Endogenous Receptors

Although sphingosine-1-phosphate receptor 1 (S1P(1)) has been shown to trigger several P targeted functions such as immune cell trafficking, cell proliferation, migration, or angiogenesis, tools that allow the accurate detection of endogenous S1P(1) localization and trafficking remain to be obtained and validated. In this study, we developed and characterized a novel monoclonal S1P(1) antibody. Mice were immunized with S1P(1) produced in the yeast Pichia pastoris and nine hybridoma clones producing monoclonal antibodies were created. Using different technical approaches including Western blot, immunoprecipitation and immunocytochemistry, we show that a selected clone, hereinafter referred to as 2B9, recognizes human and mouse S1P(1) in various cell lineages. The interaction between 2B9 and S1P(1) is specific over receptor subtypes, as the antibody does not binds to S1P(2) or Si P5 receptors. Using cell-imaging methods, we demonstrate that 2B9 binds to an epitope located at the intracellular domain of S1P(1); reveals cytosolic and membrane localization of the endogenous S1P(1); and receptor internalization upon S1 P or FTY720-P stimulation. Finally, loss of 2B9 signal upon knockdown of endogenous S1P(1) by specific small interference RNAs further confirms its specificity. 2B9 was also able to detect S1P(1) in human kidney and spinal cord tissue by immunohistochemistry. Altogether, our results suggest that 2B9 could be a useful tool to detect, quantify or localize low amounts of endogenous S1P(1) in various physiological and pathological processes.