Preparation of mouse monoclonal antibodies against AAV2 capsids

Three AAV vectors were produced by co-transfection of HEK-293 cells using AAV packaging plasmids together with an adenovirus helper, followed by purification by CsCl ultracentrifugation [58]. The AAV2-G548 capsid gene was modified from AAV2-E548 by PCR mutagenesis and used for the production of a recombinant AAV encompassing the lacZ reporter gene. The AAV2.1 and AAV1.2 capsid genes were synthesized for the production of wild-type AAVs based on comparisons of the AAV2 and AAV1 capsid sequences, respectively, with the A20 antibody binding footprint as a reference [33]. AAV2.1 was engineered based on the AAV2 scaffold with its A20 contact residues removed, and AAV1.2 was engineered based on the AAV1 scaffold with replacements introduced for the plausible binding of the A20 antibody (S1 Fig). Female BALB/c mice were intramuscularly injected with 25 μg of recombinant AAV2-G548 vector together with 50 μl of QuickAntibody Adjuvant (Biodragon). After two boosts in the following 5 weeks, the mice were sacrificed, and their splenocytes were collected for fusion with SP2/0 cells at a ratio of 5:1. The hybridoma cells were cultured in microplates in RPMI 1640/HAT medium after limited dilution. The culture supernatants were first collected for ELISA screening with effective AAV2-G548 capsid binding. Hybridoma clones positive in this screening were further negatively screened for AAV2.1 capsid binding with the A20 contact residues removed and then positively screened for AAV1.2 capsid binding with the A20 interaction regions introduced (Fig 5A). The hybridoma clones surviving the three rounds of screening were subjected to western blotting to validate the interaction of their secreted antibodies with the AAV2-G548 capsid. The hybridoma clones with the highest antibody production were injected into the abdominal cavities of BALB/c mice for ascites collection and IgG purification by affinity chromatography using a HiTrap rProtein A FF column (Cytiva).