Neutralizing Antibody Assay

Ten-week-old female BALB/c mice were injected intravenously through the tail vein with 1×10¹¹VG of AAV2, Provector, or Scrambled vectors. Three weeks post-injection, mice were euthanized and cardiac puncture was immediately performed to collect blood. Blood was incubated at room temperature for 20 min to allow for coagulation then centrifuged at 1000g for 20 min at 4°C to separate serum. Sera were stored at −20°C until further processing. HEK293T cells were seeded at 75,000 cell/well of a 96 well plate. The following day, serial dilutions of serum in serum-free media were prepared and vectors at MOI 1000 were added to a total volume of 50μl. Virus and serum were incubated on ice for 2 h and then added to cells. At 4 h post-transduction, virus and serum were removed and replaced with complete media. At 24 h post- transduction, cells were harvested for flow cytometry.