Freer Heather, Hillegas Julia M, Wimer Christine, Baldwin Cynthia, LaBresh Joanna, Wagner Bettina
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.
Vet Immunol Immunopathol. 2017 Sep;191:30-35. doi: 10.1016/j.vetimm.2017.07.011. Epub 2017 Jul 31.
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.
白细胞介素-2（IL-2）是一种T细胞生长因子，也是T辅助（Th）细胞分化的主要调节因子。在此，我们研发并鉴定了一种针对马IL-2的单克隆抗体（抗IL-2 mAb，克隆158-1）。该IL-2 mAb通过酶联免疫吸附测定（ELISA）、细胞内染色、流式细胞术分析及蛋白质免疫印迹法检测重组马IL-2（rIL-2）。在ELISA和基于荧光微球的检测中，该IL-2 mAb还与一种多克隆IL-2检测抗体配对使用。当使用相同试剂对这两种检测方法进行比较时，基于荧光微球的检测在IL-2定量方面具有更高的分析灵敏度（46pg/ml）和更宽的线性定量范围（46 - 100,000pg/ml）。马rIL-2标准品在酵母细胞和哺乳动物细胞中均有表达，但发现哺乳动物细胞表达的rIL-2标准品在天然IL-2定量方面最为准确。使用该系统，我们发现用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）和离子霉素刺激马外周血单个核细胞（PBMC）能最有效地诱导IL-2分泌。商陆有丝分裂原（PWM）持续导致PBMC产生少量IL-2，而刀豆蛋白A（ConA）、植物血凝素-L（PHA-L）和脂多糖（LPS）对马PBMC的IL-2分泌要么仅有轻微刺激作用，要么无法刺激其分泌。在用PMA和离子霉素刺激马PBMC后，通过流式细胞术分析在13.0%（范围7.5 - 16.8%）的淋巴细胞中检测到IL-2产生。IL-2表达主要在CD4细胞、CD8细胞的一个亚群以及CD4⁻/CD8⁻细胞群体中受到刺激。此外，还观察到了产生IFN-γ/IL-2和IL-4/IL-2的细胞。对15匹健康马的血清和初乳样本进行检测，结果显示这些样本中未检测到IL-2（<46pg/ml）。总之，马IL-2 mAb为鉴定产生IL-2的马细胞以及在灵敏检测中定量分泌的马IL-2提供了一种新工具。
