Detection of neutralizing antibodies (NAb) against AAV vectors in serum samples is a critical step in determining eligibility of subjects for enrollment in clinical trials with AAV therapeutics and monitoring post-dose immune responses. We have developed and validated a NAb assay for AAV9 in human serum with a glycosylation-deficient cell line that is highly permissive for transduction with AAV9 expressing firefly luciferase. The assay yielded a cut point of 0.695 (5% false positive rate) at a minimum required dilution of 2. Sensitivity with a monoclonal IgG against AAV9 was 26.5 ng/mL in NAb-negative pooled human serum (NHS). A low positive control (LPC) with NAb-positive pooled serum diluted in NHS showed intra-assay precision of 7.4 to 20.0%, and inter-assay precision of 17.9% over 11 runs. The assay is suitable for screening subjects for AAV9 NAb under CLIA guidelines and assessing long-term immunogenic responses to AAV9 therapeutics under GCP/GCLP guidelines.
understand criteria for validation of neutralizing antibody assays for adeno-associated virus vectors.
evaluate immunogenicity parameters important for enrollment and post-dose analysis of subjects in clinical trials with AAV-based therapeutics.
describe methodology for cell-based assays for neutralizing antibodies against AAV vectors.