4.4. Enzyme-Linked Immunosorbent Assay (ELISA)

The binding affinity analysis was performed by ELISA in 96-well plates. The CD73 protein (1 or 3 µg per well) was coated in a coating buffer (pH 9.6, 0.1 M bicarbonate) and washed in PBST (PBS + 0.1%Tween). A blocking buffer (3% BSA in PBST) was used to block nonspecific binding sites. The anti-CD73 antibody (1D7 or 3F7) or cell medium was added to each well and then incubated for 45 min with horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:5000). TMB substrates (Beyotime, Shanghai, China) were added into each well. After 30 min, 2 M sulfuric acid was added to stop the reaction and A_450nm was measured using a microplate reader (Bio-Rad, USA).