Total and neutralizing antibodies to human F.IX in hemophilic CD-1 mice. Plasma samples were evaluated by (A) ELISA, (B) Western blot, and (C) Bethesda assay at indicated time points. (A) Plates were coated with purified plasma–derived hF.IX, plasma samples from treated mice were applied, and rabbit anti-mouse IgG conjugated with horseradish peroxidase served as a detection antibody. Each result represents duplicate measurements from a single animal. (B) Lanes 1 through 6 represent an immunoblot time course on a single animal, weeks 0, 1, 2, 3, 5, and 7, respectively, after Ad-F.IX injection. Lanes 7 through 9 represent three additional CD-1 hemophilic mice at week 2 postinjection. (C) The presence of antibodies that interfere with coagulation was assessed by the Bethesda assay and reported as Bethesda units. A time course in four representative animals is shown. (▧), 1; (▧), 2; (▩), 3; (▩), 4; (▩), 5; (▪), 6.