Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Vet Immunol Immunopathol. 2024 Jul;273:110789. doi: 10.1016/j.vetimm.2024.110789. Epub 2024 May 23.
Cytokines are important markers for immune activation, regulation, and homeostasis. The lack of monoclonal antibodies (mAbs) and sensitive assays to evaluate cytokine secretion has hindered research of bovine inflammation and immune regulation. We recently developed a fluorescent bead-based multiplex assay (multiplex assay) for bovine IL-10, TNF-α, and IFN-γ. Although the original assay covers a broad concentration range for the 3 targets, analytical sensitivity for IL-10 and IFN-γ could be improved to facilitate detection of these cytokines in their physiological low pg/mL range. To optimize the multiplex assay, we generated a new bovine IL-10 mAb and explored its use for the detection of intracellular and secreted bovine IL-10. The new bovine IL-10 mAb 130 recognized recombinant bovine IL-10 fusion protein and did not react with the fusion protein tag, or the TNF-α and IFN-γ standards in the multiplex assay. For improving IFN-γ detection, we explored cross-reactivity of anti-equine IFN-γ mAbs by intracellular staining of bovine stimulated peripheral blood mononuclear cells (PBMC). Equine IFN-γ mAb 3 showed excellent cross-reactivity with bovine IFN-γ by intracellular detection. Adding IL-10 mAb 130 and IFN-γ mAb 3 to the bovine multiplex assay substantially improved the analytical sensitivity with lower limits of detection in the low pg/mL range for all analytes. The detection ranges for the optimized multiplex assay were determined as 2 - 134,000 pg/mL for IL-10, 8 - 127,000 pg/mL for IFN-γ, and 12 - 193,000 pg/mL for TNF-α. The assay was next used to measure cytokine concentrations in cell culture supernatants from PBMC stimulated in plasma from whole blood stimulation to confirm native IL-10, TNF-α, and IFN-γ recognition and to explore the upper detection limits of the assay. In PBMC stimulation with a mix of phorbol myristate acetate (PMA) and ionomycin resulted in highest cytokine concentrations, while in plasma from whole blood stimulation, highest concentrations were observed in samples stimulated with a mix of lipopolysaccharide (LPS), phytohemagglutinin (PHA), and the TLR-2/6 agonist Pam2Csk4. PBMC and whole blood stimulation protocols showed that the optimized multiplex assay covers a wide linear detection range for measuring cytokine concentrations in bovine samples. For whole blood stimulation, a cocktail of pathogen associated molecular patterns elicited a stronger cytokine response than a mix of PMA and ionomycin, but response varied considerably between individual cattle. In conclusion, optimizing the bovine cytokine assay with new reagents improved the lower detection limits and widened the linear detection ranges while lowering the background of the multiplex assay.
细胞因子是免疫激活、调节和动态平衡的重要标志物。缺乏单克隆抗体（mAbs）和敏感的检测方法来评估细胞因子的分泌，这阻碍了牛炎症和免疫调节的研究。我们最近开发了一种基于荧光珠的牛白细胞介素-10（IL-10）、肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）的多重检测分析方法（多重分析）。尽管原始分析涵盖了 3 种靶标的广泛浓度范围，但可以提高 IL-10 和 IFN-γ 的分析灵敏度，以促进在其生理低 pg/mL 范围内检测这些细胞因子。为了优化多重分析，我们生成了一种新的牛 IL-10 mAb，并探索了其用于检测细胞内和分泌的牛 IL-10 的用途。新的牛 IL-10 mAb 130 识别重组牛 IL-10 融合蛋白，并且不与融合蛋白标签或多重分析中的 TNF-α和 IFN-γ标准反应。为了提高 IFN-γ 的检测灵敏度，我们通过牛刺激外周血单个核细胞（PBMC）的细胞内染色探索了抗马 IFN-γ mAb 的交叉反应性。马 IFN-γ mAb 3 通过细胞内检测与牛 IFN-γ 具有极好的交叉反应性。在牛的多重分析中添加 IL-10 mAb 130 和 IFN-γ mAb 3，大大提高了分析灵敏度，所有分析物的检测下限均在低 pg/mL 范围内。优化后的多重分析的检测范围确定为 IL-10 为 2-134,000 pg/mL，IFN-γ 为 8-127,000 pg/mL，TNF-α为 12-193,000 pg/mL。该分析方法随后用于测量 PBMC 刺激全血刺激的细胞培养上清液中的细胞因子浓度，以确认天然 IL-10、TNF-α和 IFN-γ的识别，并探索分析方法的上限检测限。在用佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）和离子霉素混合物刺激 PBMC 时，细胞因子浓度最高，而在用全血刺激的血浆中，在用脂多糖（LPS）、植物血凝素（PHA）和 TLR-2/6 激动剂 Pam2Csk4 的混合物刺激的样品中观察到最高浓度。PBMC 和全血刺激方案表明，优化后的多重分析覆盖了牛样品中细胞因子浓度的宽线性检测范围。对于全血刺激，病原体相关分子模式的混合物比 PMA 和离子霉素混合物引起更强的细胞因子反应，但个体牛之间的反应差异很大。总之，用新试剂优化牛细胞因子分析提高了检测下限，拓宽了线性检测范围，同时降低了多重分析的背景。
