Characterization of hFVIII expressed in vivo. (A) Immunoprecipitation from plasma. At 6 weeks after vector injection, plasma from Hem-A/Rag1 mice was harvested and pooled. Of each plasma pool, 10 μL was brought up to 500 μL with PBS. Immunoprecipitation was performed as described in “Materials and methods.” Lanes 1-5: plasma from LC- and HC-treated animals. Lane 6: plasma from AAV-hFVIII-LC-alone–treated animals. Lane 7: plasma from AAV-hFVIII-HC-alone–treated animals. The immunoprecipitating antibodies used were as follows: lane 1, LC and HC antibodies; lane 2, LC antibody; lane 3, HC antibody; lane 4, LC antibody and 20 mM EDTA; lane 5, HC antibody and 20 mM EDTA; lane 6-7, HC and LC antibodies. (B) Immunoprecipitation from liver lysates. At 18 weeks after vector infusion, liver lysates were prepared from 50 mg tissue harvested from Hem-A/Rag1 mice and FVIII was immunoprecipitated as described in “Materials and methods.” Mice 1.2, 2.0, 2.2, and 3.0 were coinjected with 3 × 10¹¹ vg/mouse of AAV-hFVIII-HC and AAV-hFVIII-LC; mice 3.2 and 4.2 were injected with 3 × 10¹¹ vg/mouse of AAV-hFVIII-LC and AAV-hFVIII-HC, respectively. Samples in lanes 1, 3, 5, 7, 9, and 11 were immunoprecipitated with an anti–hFVIII-HC antibody, while lanes 2, 4, 6, 8, 10, and 12 were immunoprecipitated with an anti–hFVIII-LC antibody.