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A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay
Sebastian Bass-Stringer1, Colleen J Thomas2, Clive N May3
1Baker Heart and Diabetes Institute; Department of Physiology, Anatomy and Microbiology, La Trobe University; sebastian.bass@baker.edu.au.
Journal of Visualized Experiments : Jove
|December 27, 2021
Summary
A new colorimetric assay effectively detects neutralizing antibodies against adeno-associated virus serotype 6 (AAV6). This method is crucial for ensuring the success of AAV-based gene therapies in clinical applications.
Area of Science:
- Gene Therapy
- Virology
- Immunology
Background:
- Recombinant adeno-associated viruses (rAAV) are effective gene therapy vectors.
- Pre-existing neutralizing antibodies (NAbs) against AAV capsids limit gene therapy efficacy.
- Screening for host immunity is vital for AAV-based therapies.
Purpose of the Study:
- To develop and validate a colorimetric in vitro assay for detecting neutralizing factors against AAV serotype 6 (AAV6).
- To provide a reliable method for assessing pre-existing immunity to AAV6 in potential gene therapy recipients.
Main Methods:
- Utilized an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP.
- Incubated serum samples with AAV6-AP, followed by cell transduction.
- Quantified viral transduction via a chromogenic reaction and colorimetric analysis using free software.
Main Results:
- Demonstrated a strong positive correlation between coloration and viral concentration.
- Observed a significant increase in neutralizing activity in sheep serum post-AAV6 administration (125 to >10,000-fold).
- Assay showed high sensitivity, detecting neutralizing activity at serum dilutions greater than 1:32,000.
Conclusions:
- The developed assay is a simple, rapid, and cost-effective method for detecting NAbs against AAV6.
- This assay can aid in patient selection and optimize the efficacy of AAV6 gene therapies.
- Facilitates preliminary screening for host immunity, enhancing AAV gene therapy research and clinical application.