Figure 6.
The influences of some epitope hot spots to the capability of antibody cross-reactivity. The hot spots located on hPD-1 were changed to the corresponding mPD-1 amino acids and the binding activity of (A) R11 and (B) R9 were tested by ELISA. M1: V64M; M2: 83PEDRSQPGQDC93 to 83CNGLSQPVQDA93; M3: A129H, Q133K, K135E. 2 μg/ml of each antibody was coated at 96-well plate overnight and incubated with (A) 10 ng/ml and (B) 150 ng/ml mutants, then HRP-anti-His antibody were added for detection. The relative binding changes of the PD-1 mutants were normalized to the original binding of antibody and hPD-1 (unpaired, 2-tailed t test; * p< 0.05, ** p< 0.01, *** p< 0.001).