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Vet Immunol Immunopathol. 2017 Sep;191:30-35. doi: 10.1016/j.vetimm.2017.07.011. Epub 2017 Jul 31.

A monoclonal antibody for detection of intracellular and secreted interleukin-2 in horses.

Author information

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Paige Laboratory, Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA.
Kingfisher Biotech Inc., St. Paul, MN, USA.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA. Electronic address:


Interleukin-2 (IL-2) is a T cell growth factor and major modulator of T helper (Th) cell differentiation. Here, we have developed and characterized a monoclonal antibody to equine IL-2 (anti-IL-2 mAb, clone 158-1). The IL-2 mAb detected rIL-2 by ELISA, intracellular staining and flow cytometry analysis and Western blotting. The IL-2 mAb was also paired with a polyclonal IL-2 detection antibody in both ELISA and a fluorescent bead-based assay. When these two assays were compared using identical reagents there was an improved analytical sensitivity (46pg/ml) and wider linear quantification range (46-100,000pg/ml) of IL-2 quantification using the fluorescent bead assay. Equine rIL-2 standards were expressed in both yeast and mammalian cells but the mammalian cell-expressed rIL-2 standard was found to be most accurate for native IL-2 quantification. Using this system we found that stimulation of equine peripheral blood mononuclear cells (PBMC) with phorbol 12-myristate 13-acetate (PMA) and ionomycin induced IL-2 secretion most potently. Pokeweed mitogen (PWM) consistently resulted in low amounts of IL-2 from PBMC, while concanavalin A (ConA), phytohemagglutinin-L (PHA-L) and lipopolysaccharide (LPS) either marginally stimulated or failed to stimulate IL-2 secretion from equine PBMC. After stimulation of equine PBMC with PMA and ionomycin, IL-2 production was detected in 13.0% (range 7.5-16.8%) of the lymphocytes by flow cytometric analysis. IL-2 expression was mainly stimulated in CD4+ cells, in a sub-population of CD8+ cells, and also in CD4-/CD8- cell population. In addition, both IFN-γ+/IL-2+ and IL-4+/IL-2+ producing cells were observed. Testing of serum and colostrum samples from 15 healthy horses showed that IL-2 was not detectable in these samples (<46pg/ml). In summary, the equine IL-2 mAb provides a new tool for the characterization of IL-2 producing equine cells and the quantification of secreted equine IL-2 in sensitive assays.


Flow cytometry; Fluorescent bead assay; Horse; Interleukin-2; Monoclonal antibody

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