Development of a free cytokine immunoassay to maintain binding and dissociation equilibrium in vitro

Using competitive ELISA to detect free cytokines is limited as it can only reflect relative trends rather than accurately determine the real state and quantity of cytokines due to the dynamic equilibrium between dissociation and binding. This imprecise quantification adversely affects the usage of clinical medication and the validity assessment. In this study, we have developed a novel cytokine immunoassay that utilizes Rosetta molecular docking prediction technique, we screened two specific antibody pairs binding IL-1β and Durg respectively and then established the Total IL-1β and Total Drug ELISA assay. Protein A column could separate bound IL-1β and free IL-1β, and the bound IL-1β occupied for about 90% of the total. This innovative approach ensures the maintenance of equilibrium between the free cytokines and complex. We have developed a free cytokine content detection method that combines ELISA and solid phase extraction, which can detect the true concentration of free cytokines without destroying the free-binding dynamic equilibrium. It can be used to verify the accuracy of clinical PK/PD and other data, evaluate the applicability of detection methods, and guide clinical drug use and drug efficacy evaluation.