2.8. In vitro neutralizing antibody titration

Huh7 cells were seeded in white/clear 96‐well tissue culture plates at 1 × 10⁴ per well and incubated overnight. Each variant and the parental AAV containing a CMV‐Luciferase‐GFP cassette was diluted to 4.8 × 10¹⁰ vg/ml with PBS. Human Immunoglobulin for Intravenous Injection (IVIG, HUALAN BIOLOGICAL ENGINEERING CHONGQING CO., LTD, S20113011) was diluted with heat‐inactivated FBS in an adequate range and incubated with each AAV vector at 37°C for 1 h. Huh7 cells were transduced with the mixture in triplicate at an MOI of 1 × 10⁴. Luciferase activity was determined 72 h after transduction using the luciferase assay kit according to the manufacturer's instructions (Bright‐Lite Luciferase Assay System, Vazyme, DD1204). The human serum (Normal human serum [mixed], Lablead, 9193) neutralizing antibody evasion capacity detection method was the same as that for IVIGs detection. For mice serum, blood was collected from the submandibular vein and incubated at room temperature for 30 min. The clot was removed by centrifuging at 3500 rpm for 15 min at room temperature. After centrifugation, the supernatant was collected and detected.