(W1230-02-08) Multiplexed Assays for Detection of IgG Antibodies against AAV Serotypes

Purpose: Adeno-associated virus (AAV) vectors are commonly used in gene therapy (GT). However, even with careful design of the vector, it may not be possible to completely avoid interference due to existing cross-reactive antibodies, and the risk increases with more frequent use of these vectors. For this reason, serological analysis is an essential tool for understanding the pre-immune correlates for efficacy against future AAV treatment following natural infection or gene therapy. To ensure that only functional antibody activity is measured, the gold-standard approach for serology is the virus neutralization assay. While this approach can be highly specific, it is also labor-intensive and time-consuming, and the drug development field would benefit from improved workflows. Here we present a quantitative, multiplexed serology assay that can be used to measure circulating antibodies against AAV serotypes.

Methods: The assay utilizes 10-spot, 96-well assay plates directly coated with optimized concentrations of empty-capsid AAVs along with an electrochemiluminescent (ECL) detection system to simultaneously detect IgG antibodies to AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAV DJ, AAV.rh10 and AAV.PHP.eB. A monoclonal antibody that reacts with viral proteins VP1, VP2, and VP3 of AAV serotypes and a mid-level serum-based calibrator standard were used to assess signal reproducibility across wells and plates. A set of 100 normal adult serum samples was purchased and screened for positivity (at least 10-fold above background level) for the AAV serotypes.

Results: The assay simultaneously detects IgG antibodies to AAV serotypes (AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9, AAV DJ, AAV.rh10 and AAV.PHP.eB) in serum. Sensitivity, specificity, and signal reproducibility were assessed for individual serotypes using reference serum and monoclonal and/or polyclonal antibodies.

Conclusion: The multiplex AAV ECL serology assay allows for sensitive, high throughput, and simultaneous measurement of antibody levels to multiple AAVs, supporting its use in gene therapy applications and in developing safe and efficacious AAV-based products.