title: A convenient enzyme-linked immunosorbent assay for rapid screening of anti-adeno-associated virus neutralizing antibodies.
abstract: BACKGROUND
  Recombinant adeno-associated virus vectors based on serotype 2 (AAV-2) have become leading vehicles for gene therapy. Most humans in the general population have anti-AAV-2 antibodies as a result of naturally acquired infections. Pre-existing immunity to AAV-2 might affect the functional and safety consequences of AAV-2 vector-mediated gene transfer in clinical applications.
  
  
  METHODS
  An enzyme-linked immunosorbent assay (ELISA) method was developed using microwell plates coated with intact particles of recombinant AAV-2 vectors, and horseradish peroxidase-conjugated anti-human immunoglobulin G (HRP-IgG). Neutralizing antibody titres were analysed by assessing the ability of serum antibody to inhibit transduction into HEK293 cells of AAV vectors that express beta-galactosidase.
  
  
  RESULTS
  Anti-AAV-2 antibodies were detected by ELISA in two of 20 healthy subjects. The positivity criterion (optical density >0.5) in ELISA corresponded to the cut-off value (320-fold dilution of serum) in the AAV-2 neutralization assay. Influences of interfering substances were not observed.
  
  
  CONCLUSIONS
  This ELISA method may be useful for rapid screening of anti-AAV-2 neutralizing antibodies in candidates for gene therapy.
authors.affiliation: KAINOS Laboratories Inc., Tokyo, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Ito
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Hayashi
authors.forename: Mika
authors.initials: M
authors.lastname: Kodera
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mizukami
authors.forename: Keiya
authors.initials: K
authors.lastname: Ozawa
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Muramatsu
mesh_terms: D057134:Antibodies, Neutralizing / Q000737:chemistry*; D002460:Cell Line; D002624:Chemistry, Clinical / Q000379:methods*; D000229:Dependovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006735:Horseradish Peroxidase / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D009500:Neutralization Tests; D012016:Reference Values; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D057134:Antibodies, Neutralizing; D007074:Immunoglobulin G; D006735:Horseradish Peroxidase; D001616:beta-Galactosidase