ustekinumab

Ustekinumab is a human monoclonal antibody that prevents human IL-12 and IL-23 from binding to the IL-12Rbeta1 receptor chain of IL-12 (IL-12Rbeta1/beta2) and IL-23 (IL-12Rbeta1/23R) receptor complexes on the surface of natural killer cells and T-cells[1]. Ustekinumab is a human IgG1 kappa (kappa) monoclonal antibody against the IL-12/IL-23 p40 subunit that prevents its interaction with IL-12Rbeta1, thereby blocking subsequent signaling. The FcRn affinities at pH 6.0 fell in a narrow range for all 11 antibodies. The equilibrium dissociation constant (KD) is calculated relative to Ustekinumab (Ustekinumab=1.0).

Cell Assay: HUVECs are grown on calibrated glass coverslips to a confluency of 80%. Subsequently, cells are incubated with 200 ug/mL Ustekinumab AlexaFluor488 and Briakinumab AlexaFluor594 for 20 min at 37C, pH 7.3, washed three times, and chased in fresh medium for an additional 20 min before fixation. Antibodies are directly labeled. FcRn binding is quality controlled by SPR analysis. Cells are then counterstained with anti-FcRn monoclonal antibody DVN22, followed by anti-mouse secondary antibodies conjugated to AlexaFluor647. Imaging is performed on a Leica SP8 confocal microscope.