马 IL-1β 的单克隆抗体可定量检测马体内成熟的 IL-1β。

IF 1.4 3区 农林科学 Q4 IMMUNOLOGY
Susanna Babasyan, Alicia Rollins, Bettina Wagner
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引用次数: 0

摘要

白细胞介素-1β(IL-1β)是先天性免疫反应中炎症的关键介质之一。成熟的生物活性 IL-1β 可介导重要的宿主防御机制,但也在多种自身炎症和退行性疾病中发挥机理作用。对于马来说,特异而灵敏的 IL-1β 检测方法对于炎症过程和疾病的免疫学研究至关重要。本文介绍了针对马 IL-1β 的四种单克隆抗体(mAbs)的开发情况。使用一组马重组细胞因子和趋化因子证实了新型 IL-1β mAbs 的特异性。这些 mAbs 在荧光珠检测法中用于检测原生成熟 IL-1β,以及在流式细胞术中用于对产生 IL-1β 的免疫细胞进行染色都得到了验证。基于荧光珠的马 IL-1β 检测法的线性定量范围为 60 pg/ml 至 960 ng/ml。马外周血单核细胞(PBMC)在受到脂多糖(LPS)刺激后分泌IL-1β,其分泌的时间和剂量与新型马IL-1β珠基测定法的定量结果有关。将两种商用马 IL-1β 酶联免疫吸附试剂盒与新型 IL-1β 荧光珠检测法进行比较后发现,荧光珠检测法对 LPS 刺激的 PBMC 上清液中的原生马 IL-1β 的定量效果更好,其检测强度高,线性定量范围宽,而两种酶联免疫吸附试剂盒的信号都很低,对原生 IL-1β 的识别能力差。细胞内染色和流式细胞仪分析证实,LPS 刺激后马 PBMC 中 IL-1β 的主要细胞来源是 CD14+ 单核细胞。Caspase 抑制剂抑制了 PBMC 的 IL-1β 分泌,但细胞内的蛋白翻译却没有受到抑制,这证明即使缺少激活 IL-1β 的蛋白水解裂解,原 IL-1β 也会在细胞内积累。这证实了特异性 mAbs 对分析马体内具有生物活性的成熟 IL-1β 的重要性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Monoclonal antibodies for equine IL-1β enable the quantification of mature IL-1β in horses

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.

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来源期刊
CiteScore
3.40
自引率
5.60%
发文量
79
审稿时长
70 days
期刊介绍: The journal reports basic, comparative and clinical immunology as they pertain to the animal species designated here: livestock, poultry, and fish species that are major food animals and companion animals such as cats, dogs, horses and camels, and wildlife species that act as reservoirs for food, companion or human infectious diseases, or as models for human disease. Rodent models of infectious diseases that are of importance in the animal species indicated above,when the disease requires a level of containment that is not readily available for larger animal experimentation (ABSL3), will be considered. Papers on rabbits, lizards, guinea pigs, badgers, armadillos, elephants, antelope, and buffalo will be reviewed if the research advances our fundamental understanding of immunology, or if they act as a reservoir of infectious disease for the primary animal species designated above, or for humans. Manuscripts employing other species will be reviewed if justified as fitting into the categories above. The following topics are appropriate: biology of cells and mechanisms of the immune system, immunochemistry, immunodeficiencies, immunodiagnosis, immunogenetics, immunopathology, immunology of infectious disease and tumors, immunoprophylaxis including vaccine development and delivery, immunological aspects of pregnancy including passive immunity, autoimmuity, neuroimmunology, and transplanatation immunology. Manuscripts that describe new genes and development of tools such as monoclonal antibodies are also of interest when part of a larger biological study. Studies employing extracts or constituents (plant extracts, feed additives or microbiome) must be sufficiently defined to be reproduced in other laboratories and also provide evidence for possible mechanisms and not simply show an effect on the immune system.
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