FIG. 3.

Comparative capsid analysis of AAV serotypes 1–12 generated in 293 or Sf9 cells. (A) Immuno-dot blot analysis of native rAAV capsids of the panel of AAV serotypes 1–12. All rAAV subtypes were purified by AVB chromatography except for rAAV9 and rAAV11, which were purified by sucrose gradient centrifugation. About 10¹⁰, 10⁹, and 10⁸ gp of serotype-specific rAAVs were spotted on a nitrocellulose membrane as indicated by the triangles to the right of the panels. The membranes were incubated with a panel of mAbs as depicted to the left of the membrane. Each mAb detects specific conformational epitopes on the surface of defined AAV capsids. Polyclonal antibody VP51 detects all AAV serotypes and served as an internal loading control for 10⁹ gp of each rAAV serotype. (B) Silver-staining gel analysis of the rAAV preparations used in (A) separated on 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis with 10¹⁰ gp loaded per lane. (C) Electron microscopic analysis of negative-stained rAAV-GFP vectors of AAV serotypes 1, 2, and 8 generated from either transfected 293 cells or baculovirus-infected Sf9 cells. AAV vectors were highly purified by AVB chromatography. Titers ranged between 3×10¹² and 4×10¹³ gp/ml. Scale bar=25 nm.