A convenient enzyme-linked immunosorbent assay for rapid screening of anti-adeno-associated virus neutralizing antibodies

This version was published on 1 November 2009

Ann Clin Biochem 2009;46:508-510
doi:10.1258/acb.2009.009077
© 2009 Association for Clinical Biochemistry

 

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Tetsuo Ito1,
Shigekazu Yamamoto1,
Tsukasa Hayashi1,
Mika Kodera1,3,
Hiroaki Mizukami2,
Keiya Ozawa2 and
Shin-ichi Muramatsu3


1 KAINOS Laboratories Inc., Tokyo;
2 Division of Genetic Therapeutics;
3 Division of Neurology, Jichi Medical University, Tochigi, Japan


Corresponding author: Shin-ichi Muramatsu, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Email: muramats{at}jichi.ac.jp


Background: Recombinant adeno-associated virus vectors based on serotype2 (AAV-2) have become leading vehicles for gene therapy. Mosthumans in the general population have anti-AAV-2 antibodiesas a result of naturally acquired infections. Pre-existing immunityto AAV-2 might affect the functional and safety consequencesof AAV-2 vector-mediated gene transfer in clinical applications.

Methods: An enzyme-linked immunosorbent assay (ELISA) method was developedusing microwell plates coated with intact particles of recombinantAAV-2 vectors, and horseradish peroxidase-conjugated anti-humanimmunoglobulin G (HRP-IgG). Neutralizing antibody titres wereanalysed by assessing the ability of serum antibody to inhibittransduction into HEK293 cells of AAV vectors that express β-galactosidase.

Results: Anti-AAV-2 antibodies were detected by ELISA in two of 20 healthysubjects. The positivity criterion (optical density >0.5)in ELISA corresponded to the cut-off value (320-fold dilutionof serum) in the AAV-2 neutralization assay. Influences of interferingsubstances were not observed.

Conclusion: This ELISA method may be useful for rapid screening of anti-AAV-2neutralizing antibodies in candidates for gene therapy.


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