Characterization of the autoantibodies. Recombinant qVEGFR and mVEGFR can be recognized by the sera isolated from mice immunized with qVEGFR (A) but negative staining from mice immunized with mVEGFR (B) or ALUM (C) in Western blot analysis. Sera from mice immunized with qVEGFR also recognized a single band in Flk-1–positive mouse SVEC4-10 endothelial cells and KDR-positive HUVECs (D). The number of anti-qVEGFR– (□) or anti-mVEGFR– (▪) producing B lymphocytes in spleens of mice immunized with qVEGFR, mVEGFR, or ALUM were counted in an ELISPOT assay (E). Mice were immunized once a week for 4 weeks, and mononuclear cells were prepared from spleens 1 week after the fourth immunization. Data presented are mean ± SEM. There was endothelial deposition of autoantibodies within tumor tissue from mice immunized with qVEGFR (F), but not from mice immunized with mVEGFR (G) or ALUM (H). Panels F-H were stained with immunofluorescence stain; original magnifications, × 200.