Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Vet Immunol Immunopathol. 2024 Aug;274:110805. doi: 10.1016/j.vetimm.2024.110805. Epub 2024 Jul 3.
Interleukin-1β (IL-1β) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1β mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1β are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1β. The specificity of the new IL-1β mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1β in a fluorescent bead-based assay and for staining of IL-1β-producing immune cells by flow cytometry. The bead-based assay for equine IL-1β had a linear quantification range between 60 pg/ml to 960 ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1β after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1β bead-based assay. A comparison of two commercial equine IL-1β ELISA kits with the new IL-1β fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1β in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1β recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1β in equine PBMC after LPS stimulation were CD14 monocytes. IL-1β secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1β within the cells even when proteolytic cleavage for IL-1β activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1β in horses.
白细胞介素-1β（IL-1β）是天然免疫反应中炎症的关键介质之一。成熟的生物活性 IL-1β 介导重要的宿主防御机制，但在几种自身炎症性和退行性疾病中也具有机制作用。在马中，针对 IL-1β 的特异性和敏感检测对于炎症过程和疾病的免疫学研究至关重要。在本文中，我们描述了针对马 IL-1β 的四种单克隆抗体（mAb）的开发。使用马重组细胞因子和趋化因子的面板证实了新的 IL-1β mAb 的特异性。该 mAb 用于通过荧光珠基测定法检测天然成熟的 IL-1β，并通过流式细胞术检测产生 IL-1β 的免疫细胞的染色。用于马 IL-1β 的珠基测定法具有 60pg/ml 至 960ng/ml 的线性定量范围。马外周血单核细胞（PBMC）在脂多糖（LPS）刺激下，按照时间和剂量依赖性分泌 IL-1β，如新型马 IL-1β 珠基测定法所定量的那样。将两种新型马 IL-1β 荧光珠基测定法与两种商业马 IL-1β ELISA 试剂盒进行比较，发现珠基测定法通过高强度和广泛的线性定量范围检测，提高了 LPS 刺激的 PBMC 上清液中天然马 IL-1β 的定量，而两种 ELISA 均导致低信号和较差的天然 IL-1β 识别。细胞内染色和流式细胞术分析证实，LPS 刺激后马 PBMC 中 IL-1β 的主要细胞来源是 CD14 单核细胞。PBMC 中 IL-1β 的分泌被半胱天冬酶抑制剂抑制，但细胞内的蛋白质翻译没有被抑制，这支持了即使缺乏 IL-1β 激活的蛋白水解切割，细胞内 pro-IL-1β 的积累。这证实了特异性 mAb 对于分析马中具有生物活性的成熟 IL-1β 的重要性。
