A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

作者： Sebastian Bass-Stringer1,2, Colleen J. Thomas2,3, Clive N. May3, Paul Gregorevic4, Kate L. Weeks1,5,6, Julie R. McMullen1,2,5,6,7 1Baker Heart and Diabetes Institute, 2Department of Physiology, Anatomy and Microbiology,La Trobe University, 3Florey Institute of Neuroscience and Mental Health,University of Melbourne, 4Department of Physiology, Centre for Muscle Research (CMR),The University of Melbourne, 5Department of Diabetes, Central Clinical School,Monash University, 6Baker Department of Cardiometabolic Health,The University of Melbourne, 7Department of Physiology and Department of Medicine Alfred Hospital,Monash University

简介：

Overview

This study addresses the challenge posed by preexisting antibodies that neutralize AAV, which hinder the advancement of AAV gene therapies. A cost-effective and straightforward colorimetric assay is developed to detect neutralizing antibodies against AAV6, using HT1080 cells as the model system.

Key Study Components

Research Area

• AAV gene therapy
• Neutralizing antibodies
• Cell-based assays

Background

• Neutralizing antibodies against AAV can limit the efficacy of gene therapies.
• A rapid and efficient screening method is needed for detecting these neutralizing elements.
• The developed assay requires minimal resources and technical skills.

Methods Used

• Plating HT1080 cells for assay implementation
• Colorimetric detection through a series of serial dilutions and incubations
• Image analysis using ImageJ to quantify assay results

Main Results

• The assay successfully correlates coloration to viral genome concentrations, determining optimal dosing.
• Neutralization titers varied significantly pre- and post-AAV administration, demonstrating the assay's efficacy.
• Assessments revealed significant differences in neutralizing activity across naive sample populations.

Conclusions

• The study presents an effective tool for identifying neutralizing antibodies, critical for AAV gene therapy research.
• The assay's simplicity and cost-efficiency make it valuable for widespread use in biological research.

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