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Overcoming matrix effects in AAV neutralization assays with a constant serum concentration approach
Beatrix Kovács1,2,3, Viktória Szabó4, Domonkos Horváth1,5
1Institute of Cognitive Neuroscience and Psychology, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary.
Gene Therapy
|September 26, 2025
Summary
A new constant serum concentration (CSC) assay improves adeno-associated virus (AAV) neutralizing antibody detection. This method enhances sensitivity and reliability for gene therapy applications, offering better patient stratification and redosing strategies.
Area of Science:
- Biotechnology
- Immunology
- Gene Therapy
Background:
- Accurate quantification of adeno-associated virus (AAV) neutralizing antibodies (NAbs) is critical for successful gene therapy.
- Conventional assays face matrix-induced artifacts due to variable serum content, leading to inaccurate NAb detection.
Purpose of the Study:
- To develop and validate a novel assay format, the constant serum concentration (CSC) assay, for enhanced NAb quantification.
- To improve the sensitivity and reliability of NAb detection compared to conventional variable serum concentration (VSC) assays.
Main Methods:
- Development of the CSC assay, maintaining constant serum levels across dilutions to stabilize assay baselines.
- Testing the CSC assay with human sera across multiple AAV serotypes and validating with monoclonal antibodies and multi-species serum benchmarks.
Main Results:
- The CSC assay reclassified up to 21.7% of samples as neutralizing that were previously classified as non-neutralizing by VSC assays.
- Enhanced reliability in selecting seronegative control pools and detection of persistent seropositivity in preclinical models up to one year longer than VSC assays.
Conclusions:
- The CSC assay offers improved sensitivity and reliability for AAV NAb quantification, addressing limitations of conventional methods.
- Findings have direct implications for optimizing AAV gene therapy redosing strategies, patient stratification, and understanding antibody persistence.
