PDF(Pubmed)
Abstract：
IL-1β is a essential molecule in inflammatory signalling pathways and plays an essential role in inflammatory diseases. Accordingly, the development of monoclonal antibodies (mAbs) that target IL-1β has become the focus of developing new anti-inflammatory drugs. The successful clinical application of therapeutic antibodies is dependent on good quality control, which is based on accurate bioactivity determination. The aim of this work was to develop an elegant and accurate reporter gene assay to determine the bioactivity of anti-IL-1β antibody drugs. The D10-G4-1 cell line with a naturally high expression of IL-1 receptor was selected as the effector cell, and the plasmid containing luciferase reporter gene with NF-κB as a regulatory element was transfected into D10-G4-1 cells. After a period of pressure screening, a monoclonal cell line with good reactivity and stable expression of reporter gene was finally screened out. Stimulation of this cell line via IL-1β addition increased the expression of the luciferase gene by activating the NF-κB signalling pathway, with the addition of luciferase substrate, which can be quantified by relative luminescence units. When anti-IL-1β antibodies are present in the system, the expression of luciferase gene is inhibited, which demonstrates the bioactivity of anti-IL-1β antibodies. Detailed methodological optimization and comprehensive methodological validation were followed to establish a reporter gene assay for the bioactivity of anti-IL-1β antibodies.
摘要：
IL-1β是炎症信号通路中的重要分子,在炎性疾病中发挥重要作用。因此，靶向IL-1β的单克隆抗体(mAb)的开发已成为开发新型抗炎药的重点。治疗性抗体的成功临床应用依赖于良好的质量控制,这是基于准确的生物活性测定。这项工作的目的是开发一种优雅而准确的报告基因测定法，以确定抗IL-1β抗体药物的生物活性。选择IL-1受体天然高表达的D10-G4-1细胞系作为效应细胞,并将含有以NF-κB为调控元件的荧光素酶报告基因的质粒转染到D10-G4-1细胞中。经过一段时间的压力筛选，最终筛选出具有良好反应性和稳定表达报告基因的单克隆细胞系。通过添加IL-1β刺激该细胞系通过激活NF-κB信号通路增加了荧光素酶基因的表达，加入荧光素酶底物，可以通过相对发光单位来量化。当系统中存在抗IL-1β抗体时，荧光素酶基因的表达受到抑制，这证明了抗IL-1β抗体的生物活性。随后进行了详细的方法学优化和全面的方法学验证，以建立用于抗IL-1β抗体生物活性的报告基因测定。
