Affiliation:
1. Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products National Institutes for Food and Drug Control Beijing China
Abstract
AbstractIL‐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.