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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

1
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
2
Paige Laboratory, Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA.
3
Kingfisher Biotech Inc., St. Paul, MN, USA.
4
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA. Electronic address: bw73@cornell.edu.

Abstract

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.

KEYWORDS:

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

PMID:
28895863
DOI:
10.1016/j.vetimm.2017.07.011
[Indexed for MEDLINE]

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