| With the rapid development of biotechnology in recent years,biopharmaceuticals have been widely used for the prevention and treatment of various diseases.Among these,antibody drugs are of great significance in the treatment of tumours,autoimmune diseases and inflammation,and have become an important pillar of the biopharmaceutical industry,which has led to an upsurge in research on antibody drugs both at home and abroad.Advanced structure defines the biological function of recombinant antibody medicines,but even advanced physicochemical analysis cannot fully characterise advanced structure,and biological activity can only be used as a guide.Furthermore,the measurement of biological activity is an important indicator of the potency and drug content of antibody drugs.Currently,the main analytical methods for determining the biological activity of antibody drugs are cell-based in vitro assays,in which in vitro cell models are created to simulate mechanisms of action such as cell growth inhibition,cytotoxicity,complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity.There are also activity analyses based on novel technologies such as surface plasmon potential analysis,time-resolved uniform fluorescence,alpha technology and fluorescent dye labelling.Gene reporter assays,on the other hand,differ from other existing traditional methods in that they directly reflect the mechanism of action of the drug,as they bind the reporter gene to a specific regulatory sequence and utilise the signalling pathway of the drug activity in question.With advantages such as accuracy,efficiency,low variability and time and labour savings,this method has attracted much attention in quality control and offers new ideas for the development and quality control of antibody drugs.As important components,monoclonal antibody drugs have been used successfully in many areas,including suppression of organ transplant rejection,immunodiagnosis of tumours,targeted treatment of tumours,asthma,psoriasis,rheumatoid arthritis and acute myocardial infarction.Biological activity is an important quality characteristic in the development of monoclonal antibodies,and the development of efficient and stable methods for analysing biological activity is an important research issue.With the rapid development of biopharmaceuticals,cell-based gene reporter methods will have a larger market.Studies have shown that the IL-33/ST2 pathway is closely associated with many diseases and plays an important role in heart failure,tumourigenesis,atherosclerosis and various inflammatory responses.As a biomarker for diagnosis and prognosis,it is an important topic for research and development.Interleukin-33,a newly identified cytokine of the IL-1 family,is expressed in a variety of human and mouse cells and can mediate a variety of immune effects by binding to its natural receptor ST2.Another subtype is the soluble s ST2 receptor,which competes with both ST2 L subtypes for IL-33 binding and thereby blocks the IL-33/ST2 pathway,also called decoy receptors,although IL-33 binds to ST2 receptors on the cell surface and activates the NF-κB pathway,Anti-ST2 antibodies targeting the ST2 receptor block the interaction between IL-33 and ST2,thus inhibiting the activation of the NF-κB pathway The development of antibodies against ST2 as therapeutic targets is of great importance in the treatment of various diseases.In this study,a reporter gene assay for anti-ST2 monoclonal antibodies was established using a monoclonal cell line based on the NF-κB pathway to rapidly measure the biological activity of anti-ST2 antibodies and expression of fluorescent proteins upon IL-33 stimulation.Based on the signal-to-noise ratio,a dose-response curve was optimised in terms of cell density and incubation time,IL-33 stimulation concentration,initial concentration of anti-ST2 antibody and dilution factor,and the accuracy,linearity,precision,specificity and stability of the method were validated according to ICH-Q2 guidelines.A gene reporter method was developed to measure the biological activity of anti-ST2 monoclonal antibodies. |
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