Figure 2.

Ipilimumab and mAb146 block ligands human CD80 (a, c) and human CD86 (b, d) binding to hCTLA-4 by ELISA (a–b) and FACS (c–d), and their ability on blocking mouse CD80 (e) and mouse CD86 (f) binding to mCTLA-4 also detected by ELISA (e–f). In ELISA-based competition assays, hCTLA-4-hFc dimer or mCTLA-4-mFc dimer (0.5 μg/mL) were coated on 96-well plates, the antibodies pre-mixed with 0.25 μg/mL of hCD80-6xHis, hCD86-6xHis, or 0.5 μg/ml of mCD80-6xHis, 5 μg/mL of mCD86-6xHis were added. After blocking with 2% BSA, biotinylated anti-His tag antibody was added. The bound ligands were detected by HRP-conjugated streptavidin. In FACS-based competition assay, hCD80- or hCD86-expressing CHO cell lines were used. The details were described in the method section.