Methodological Validation and Inter-Laboratory Comparison of Microneutralization Assay for Detecting Anti-AAV9 Neutralizing Antibody in Human

Abstract

Anti-AAV neutralizing Abs (NAbs) titer is usually measured by cell-based microneutralization (MN) assay and is crucial for patient screening in AAV-based gene therapy clinical trials. However, achieving uniform operation and comparable results among different laboratories remains challenging. Here, we established a standardized MN assay for anti-AAV9 NAbs in human sera or plasma and transferred the method to the other two research teams. Then, we validated its parameters and tested a set of eight human samples in blind across all laboratories. The end-point titer, defined by a transduction inhibition of 50% (IC50), was calculated using curve-fit modelling. A mouse neutralizing monoclonal antibody in human negative serum was used for system quality control (QC), requiring inter-assay titer variation of <4-fold difference or geometric coefficient of variation (%GCV) of <50%. The assay demonstrated a sensitivity of 54 ng/mL and no cross-reactivity to 20 μg/mL anti-AAV8 MoAb. The intra-assay and inter-assay variation for the low positive QC were 7–35% and 22–41%, respectively. The titers of the blind samples showed excellent reproducibility within and among laboratories, with a %GCV of 18–59% and 23–46%, respectively. This study provides a commonly transferrable MN assay for evaluating anti-AAV9 NAbs in humans, supporting its application in clinical trials.

Keywords

• adeno-associated virus
• neutralizing antibody
• microneutralization assay
• sensitivity
• specificity
• precision