Phospho stat3 elisa

Example 6

The human cell line DB (ATCC, Manassas, Va.) responds to IL-23 stimulation through an endogenous IL-23Rcomplex (IL-23R and IL-12Rβ1) and phosphorylates STAT3 in an IL-23 dose dependent manner. An assay was developed for testing anti-IL-23p19 antibody inhibition of IL-23 induced STAT3 phosphorylation. DB cells were plated at 1×10e6 cells/well in a 96 well plate. Antibodies to be tested were serially diluted and preincubated with recombinant human IL-23 (10 ng/ml) for 1 hour at room temperature.

The antibody/IL-23 mixture was then added to the cells for 30 minutes at 37° C. Cells were harvested by centrifugation at 4° C. for 10 minutes and then lysed in ice cold buffer (Cell Signaling Technology, Beverly, Mass.). A portion of the lysate was run in a phospho-STAT3 ELISA (Invitrogen). Antibody IC₅₀ values were calculated as percent inhibition of STAT3 phosphorylation compared to control wells without antibody. Representative IC₅₀ values are shown in the table below.

Example 6

The human cell line DB (ATCC, Manassas, Va.) responds to IL-23 stimulation through an endogenous IL-23R complex (IL-23R and IL-12Rβ1) and phosphorylates STAT3 in an IL-23 dose dependent manner. An assay was developed for testing anti-IL-23p19 antibody inhibition of IL-23 induced STAT3 phosphorylation. DB cells were plated at 1×10e6 cells/well in a 96 well plate. Antibodies to be tested were serially diluted and preincubated with recombinant human IL-23 (10 ng/ml) for 1 hour at room temperature. The antibody/IL-23 mixture was then added to the cells for 30 minutes at 37° C. Cells were harvested by centrifugation at 4° C. for 10 minutes and then lysed in ice cold buffer (Cell Signaling Technology, Beverly, Mass.). A portion of the lysate was run in a phospho-STAT3 ELISA (Invitrogen). Antibody IC₅₀ values were calculated as percent inhibition of STAT3 phosphorylation compared to control wells without antibody. Representative IC₅₀ values are shown in the table below.