Study on evaluation method of differences in human extracellular cytokine release under different incubation systems
Objective Three antibody coating methods were used to evaluate assay of various cytokines secreted by human peripheral blood cells(PBMCs)before and after OKT3 stimulation so as to provide theoretical basis for preclinical and clinical studies of immunotherapy drugs prior to marketing.Methods PBMCs were prepared by healthy human peripheral blood by density gradient centrifugation and cryopreserved in a LN tank for backup.The expression level of cytokines secreted after OKT3 stimulation at different concentrations(1 and 2 μg·well-1)and time was detected by Luminex based on the solid-phase dry,solid-phase wet(coating for 24 and 48 h),and liquid phase methods.Results In the solid phase immobilized method,OKT3 stimulated PBMC cells to release cytokines significantly.In the group where OKT3 was coated for 48 hours and stimulated for 48 hours,the levels of cytokines(IL-2,IL-6,IL-10,IFN-γ,TNF-α)were 6,2,22,214,and 51 times higher than those of the negative control group(1 μg•well-1),5,2,26,194,and 48 times higher(2 μg·well-1);in the group where OKT3 was coated for 48 hours and stimulated for 24 hours,the levels of cytokines were 3,2,2,171,and 19 times higher than those of the negative control group(1 μg·well-1),4,2,3,174,and 21 times higher(2 μg·well-1);in the group where OKT3 was coated for 24 hours and stimulated for 48 hours,the levels of cytokines were 6,2,28,356,and 50 times higher than those of the negative control group(1 μg·well-1),6,2,35,349,and 64 times higher(2 μg·well-1);in the group where OKT3 was coated for 24 hours and stimulated for 24 hours,the levels of cytokines were 3,2,1,127,and 16 times higher than those of the negative control group(1 μg·well-1),3,1,2,115,and 13 times higher(2 μg·well-1),indicating that the effects were more obvious with longer OKT3 coating time and longer time of interaction between OKT3 and PBMC.In the solid phase wet method and liquid phase method,IL-10 showed a marked decrease in content,and the two methods showed equivalent release levels,with no relationship to the group.Conclusion There was an edge of solid-phase dry method over the other two methods.Significant cytokine release in vitro was observed after OKT3 stimulation as a positive control.Therefore,solid-phase dry method can be used to assess the risk of cytokine storm triggered in GLP toxicology studies.
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