Fig. 2

The binding and blocking ability of an established anti-CTLA-4 mAb. a The binding of anti-CTLA-4 mAb against CTLA-4-EGFP expressing CHO cells and CD28-EGFP expressing Cos-7 cells was measured using flow cytometry. b A dose-dependent blocking effect of 4G2-A3 on CTLA-4/CD80 and CTLA-4/CD86 binding. CTLA-4-EGFP cells were preincubated with 4G2-A3 or isotype Ab at various concentrations (1.25, 2.5, 5, 10, and 20 μg/mL), and the Ig binding was detected using flow cytometry. Each point indicates the average value of relative MFI obtained from three independent experiments (compared to no antibody control, error bar; SEM). Tukey’s test was used for the statistical analysis (* p < 0.05, between the group with 0 and 2.5 μg/ml of anti-CTLA-4 antibody and the group with 2.5 and 20 of anti-CTLA-4 antibody. † p < 0.05, between the anti-CTLA-4 antibody treated group and isotype antibody treated group of each concentration.). c PBMCs (n = 11) were isolated from healthy cattle and cultured with 20 μg/ml of control Ab or anti-CTLA-4 mAb in the presence of SEB and 10 nM CTLA-4-Ig. The culture medium was harvested after 7 days, and an ELISA was used to measure the IFN-γ concentration. The bars indicate the average of each group. Statistical comparisons between each group were made using the Wilcoxon matched-pairs test. Differences were considered statistically significant at p < 0.01