The immunized mouse serum antibody recognizing VEGF and blocking VEGF binding with VEFGR. (A) Wells of 96-well microtitre plates were coated with the recombinant VEGF (50 ng/well) and, after blocking, were incubated with the mouse serum: 4% pre-immune serum; 4% serum after immunization or 2 μg/ml purified antibody of immunized mice serum. The antiserum elicited by 12P-KLH could bind to VEGF. This result is representative of three independent experiments. (B) Wells of 96-well microtitre plates were coated with the recombinant VEGFR2 (10 ng/well) and then incubated with VEGF with or without purified serum. Bound human VEGF was detected with a rabbit anti-human VEGF and following anti-rabbit IgG conjugated with HRP. All assays were carried out in triplicate and the error bars indicate standard deviation. Statistical analysis was performed using the Student’s t test (*p < 0.05).