Ap 4510 01

Inhibition of the interaction between TIM-3 and CEACAM1 was detected using an ELISA-based assay in the presence of 10 mM CaCL₂ (Sigma, C3306-100 G). In a flat bottom 96-well MaxiSorp plate, 0.5 µg rhTIM-3-IgG1-Fc in 1xTBS (Thermo Scientific, 28358) with 10 mM CaCL₂ (Ca²⁺) was added to each well and the plate was incubated at 4°C overnight. The plate was then blocked in 2% BSA (SeraCare Life Sciences, AP-4510-01) TBS (Ca²⁺) at room temperature with shaking for 1 hour. After the supernatant was removed, M6903 or isotype control (anti-HEL IgG2) in TBS (Ca²⁺) was added to each well and the plate was incubated at room temperature for 1 hour, and then CEACAM1-poly His (ARCO, CE1-H5220) in TBS (Ca²⁺) was added to each well and the plate was incubated at room temperature for 1 hour with shaking. The plate was then washed and anti-6XHis-HRP (Biolegend, 652504) in TBS (Ca²⁺) was added to each well followed by incubation at room temperature for 1 hour with shaking. After washing, TMB was added to each well followed by shaking the plate at room temperature for 20 minutes and adding stop solution to each well. The OD (450 nm and 570 nm) was read immediately using EnVision plate reader.