Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, that is involved in acute inflammation and is employed as a biomarker of inflammatory diseases in several species for which reliable quantification is available. We aimed to develop suitable tools to quantify TNF-α in equine samples. We generated two new mAbs against equine TNF-α (clones 48 and 292), evaluated their specificity for this cytokine, and confirmed detection of native TNF-α in stimulated equine PBMC. The TNF-α mAbs were paired in a fluorescent bead-based assay for quantification of equine TNF-α. The TNF-α assay had a wide quantification range of 12 pg/mL - 38.4 ng/mL. In addition, TNF-α mAb 48 was used for a detailed analysis of TNF-α production in PBMC by intracellular staining and flow cytometry. TNF-α was expressed by CD14⁺ monocytes after LPS stimulation and by monocytes and lymphocytes after polyclonal stimulation with PMA and ionomycin in vitro. TNF-α expressing lymphocytes consisted mainly of CD4⁺ T cells. CD8⁺ T cells and other lymphocytes also expressed TNF-α. The new mAbs evaluated here for soluble and intracellular TNF-α will enable the detailed analysis of this important pro-inflammatory cytokine during equine immune responses and inflammatory diseases of the horse.

肿瘤坏死因子-α (TNF-α) 是一种多效性细胞因子，参与急性炎症，并被用作几种物种炎症疾病的生物标志物，可对其进行可靠的量化。我们旨在开发合适的工具来量化马样本中的 TNF-α。我们生成了两种针对马 TNF-α 的新 mAb（克隆 48 和 292），评估了它们对这种细胞因子的特异性，并确认在受刺激的马 PBMC 中检测到天然 TNF-α。TNF-α mAb 在基于荧光珠的测定中配对，用于量化马 TNF-α。TNF-α 测定具有 12 pg/mL - 38.4 ng/mL 的宽量化范围。此外，TNF-α mAb 48 用于通过细胞内染色和流式细胞术详细分析 PBMC 中 TNF-α 的产生。TNF-α 由 CD14 ⁺表达LPS 刺激后的单核细胞和体外PMA 和离子霉素多克隆刺激后的单核细胞和淋巴细胞。表达 TNF-α 的淋巴细胞主要由 CD4 ⁺ T 细胞组成。CD8 ⁺ T 细胞和其他淋巴细胞也表达 TNF-α。此处针对可溶性和细胞内 TNF-α 评估的新 mAb 将能够在马免疫反应和马的炎症性疾病期间详细分析这种重要的促炎细胞因子。