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

作者：Freer Heather; Hillegas Julia M; Wimer Christine; Baldwin Cynthia; LaBresh Joanna; Wagner Bettina^*

来源：Veterinary Immunology and Immunopathology, 2017, 191: 30-35.

DOI：10.1016/j.vetimm.2017.07.011

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

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 (46 pg/ml) and wider linear quantification range (46-100,000 pg/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-gamma(+)/IL-2(+) and IL-4(+)/IL-2(+) producing cells were observed. Testing of serum and colostrum samples froth 15 healthy horses showed that IL-2 was not detectable in these samples (< 46 pg/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.

• 出版日期2017-9