Figure 3.

Detection of Anti-VV and -B5R Antibody in Serum

(A) Whole-VV antigen-coated wells were incubated with 4-fold serial dilutions (×32, ×128, ×512) of 5 human sera (patients 221, 183, 195, 175, and 214) or control immunized rabbit IgG. Antibody response was detected by measuring optical density at 405 nm (OD₄₀₅) after incubation with horseradish peroxidase-conjugated rabbit anti-human IgG (SouthernBiotech) and substrate. (B) Purified B5R protein-coated wells were incubated with the same serial dilutions of human serum and secondary antibody shown in (A), and antibody response was detected as described above. (C) Whole-VV antigen-coated wells were incubated with 4-fold serial dilutions (×4–4,096) of serum from monkeys immunized with high (001, 002, and 003) or low (005, 006, and 008) doses of VV or from mock-immunized monkeys (004, 007, and 009). Antibody response was detected after incubation with horseradish peroxidase-conjugated goat anti-monkey IgG (Abcam) and substrate. (D) Purified B5R protein-coated wells were incubated with the same serial dilutions of monkey serum shown in (C), and antibody response was detected as described above. Data in (A)–(D) are presented as means ± SD (n = 2).