Figure 1. Western blot analysis of virus neutralization (VN)–positive raccoon serum specimens. A/whooper swan/Mongolia/4/05 (H5N1) virus (clade 2.2) was purified through a 25% sucrose cushion and used as an antigen under nonreducing conditions in the Western blot assay. After blocking with 5% skim milk, each raccoon serum specimen (1:100 dilution) was incubated for 1 h and then reacted with horseradish peroxidase (HRP)–labeled protein A/G (Pierce Chemical Co., Rockford, IL, USA) and subjected to chemiluminescence detection (ECL Plus, GE Healthcare UK, Ltd, Chalfont St. Giles, UK). Serum from a mouse infected with A/whooper swan/Mongolia/4/05 was used as a marker. The negative control reaction when only HRP-protein A/G is used is also shown. HA, hemagglutinin; Ab, antibody. Lane 1, anti-H5N1 polyclonal Ab; lanes 2–11, VN-positive serum (2, A-1; 3, A-2; 4, A-3; 5, A-4; 6, A-5; 7, A-6; 8, B-1; 9, B-2; 10, C-1; 11, C-2); lane 12, VN-negative serum; lane 13, negative control.