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Validation of a monoclonal antibody directed against the human sphingosine 1-phosphate receptor type 1.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2021 Mar; Vol. 490, pp. 112953. Date of Electronic Publication: 2020 Dec 30. - Publication Year :
- 2021
-
Abstract
- The sphingosine 1-phosphate receptor type 1 (S1PR1) has several important functions, including stabilizing endothelial barrier and maintaining lymphocyte circulation. These functions are critically dependent on the regulation of S1PR1 cell surface expression. Currently available antibodies against human S1PR1 are not able to pick up cell surface expression on living cells by flow cytometry due to intracellular epitopes or unspecific binding. Here we describe the generation of a mouse monoclonal antibody specific for the N-terminal region of human S1PR1. It has an immunoglobulin M (IgM) kappa isotype and detects cell surface expression of recombinant human S1PR1 on overexpressing cells. Due to unspecific intracellular cell staining, it cannot be used for staining of dead cells and tissue slides or in microscopic analyses. It is also not suitable for Western blot analysis and immunoprecipitation. However, the antibody can stain for endogenous S1PR1 on human endothelial cell lines and primary human umbilical vein endothelial cells (HUVEC). Incubation of these cells with various S1PR1 agonists revealed potent S1PR1 internalization, which was not the case with the specific antagonist W146. Surprisingly, human T and B cells isolated from blood and palatine tonsils did not show specific staining, demonstrating significantly lower endogenous S1PR1 surface expression on lymphocytes than on endothelial cells.<br /> (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Subjects :
- Anilides pharmacology
Animals
Burkitt Lymphoma pathology
Gene Expression Regulation
Human Umbilical Vein Endothelial Cells
Humans
Mice
Organ Specificity
Organophosphonates pharmacology
Palatine Tonsil cytology
Sphingosine-1-Phosphate Receptors immunology
Antibodies, Monoclonal isolation & purification
B-Lymphocytes metabolism
Burkitt Lymphoma metabolism
Endothelial Cells metabolism
Immunoglobulin M isolation & purification
Sphingosine-1-Phosphate Receptors metabolism
T-Lymphocytes metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1872-7905
- Volume :
- 490
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 33359172
- Full Text :
- https://doi.org/10.1016/j.jim.2020.112953