B-216 Validation and Optimization of AAV9 Total Antibody Testing in Gene Therapy

Abstract

Adeno-associated virus serotype 9 (AAV9) is widely used as a gene therapy vector. Pre-existing anti-AAV9 antibodies can reduce transduction efficiency and pose safety risks, making accurate antibody detection essential for patient selection and immune monitoring. However, despite its importance, current laboratory practice lacks standardized guidelines, leading to variability in assay performance and result interpretation. The development of standardized Standard Operating Procedures is therefore critical to ensure reliable, reproducible, and comparable results across laboratories. A total anti-AAV9 antibody enzyme-linked immunosorbent assay (ELISA) was developed and optimized using endpoint titer determination. Human serum samples were tested in serial dilutions on AAV9 capsid–coated [AAV9(+)] and uncoated [AAV9(–)] plates to control for nonspecific binding. Background-corrected signals (corrected OD450) were calculated by subtracting AAV9(–) from AAV9(+) readings, and OD450 ratios were determined by normalizing corrected OD450 values to blank AAV9(+) wells. In the absence of a positive reference standard, results were reported as endpoint titers, defined as the reciprocal of the highest dilution producing a detectable signal. An optimized capsid titration protocol was established by evaluating multiple capsid concentrations to enhance assay sensitivity while minimizing background. Comprehensive validation assessed precision, reportable range, linearity, accuracy, reference interval verification, interference, cross-reactivity, and sample stability. Capsid titration revealed substantial variability in antigen binding across vendors and between lots from the same vendor, highlighting the need for lot-specific optimization. Following optimization, lot-to-lot verification demonstrated consistent assay performance. The optimized ELISA showed robust analytical performance. Intra-assay, inter-assay, and inter-operator precision studies using samples ranging from negative (<1:25) to high titer (up to 1:3200) demonstrated 100% agreement within ±1 endpoint titer. Coefficients of variation for OD450 ratios were <20% for all positive samples, and negative samples consistently tested negative. The validated reportable range was <1:25 to 1:400, with titers up to 1:3200 reportable upon request. Linearity was confirmed across low, intermediate, and high titers, with slopes between 0.9 and 1.1, R² values >0.95, and observed titers within ±1 of expected values. The lower limit of quantitation (LLOQ) at 1:25 was statistically distinguishable from blanks (p<0.05), showed low variability (%CV =6%), and produced consistent endpoint titers. Accuracy assessment demonstrated strong inter-laboratory concordance. Comparison with the Immune Monitoring Core at Nationwide Children’s Hospital achieved 97.5% agreement within ±1 endpoint titer. Analysis of 92 healthy donors aged =8 years identified an AAV9 seroprevalence of 41.3% at a cutoff of >=1:50, consistent with published data. Interference studies revealed no significant effects from hemolysis or lipemia (<20% difference in OD450 ratio). However, marked icterus (icterus index ∼14; total bilirubin 13.9 mg/dL) produced positive interference, resulting in >20% differences in OD450 and a one-titer increase. Stability studies confirmed acceptable performance under refrigerated and frozen storage, as well as after multiple freeze–thaw cycles, with all results remaining within ±1 endpoint titer. This study establishes a validated and optimized ELISA for AAV9 total antibody detection, demonstrating that standardized capsid titration is critical for reducing variability and ensuring reliable results. The assay supports regulatory compliance, inter-laboratory consistency, and accurate patient testing, providing a strong foundation for harmonized AAV9 antibody testing in gene therapy applications.