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Comparative Study
. 2008 Oct;10(10):1092-101.
doi: 10.1002/jgm.1236.

Characterization of high-capacity adenovirus production by the quantitative real-time polymerase chain reaction: a comparative study of different titration methods

Julien Crettaz  1 , Cristina Olague, Africa Vales, Igor Aurrekoetxea, Pedro Berraondo, Itziar Otano, Stephan Kochanek, Jesús Prieto, Gloria González-Aseguinolaza
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Free article
Comparative Study

Characterization of high-capacity adenovirus production by the quantitative real-time polymerase chain reaction: a comparative study of different titration methods

Julien Crettaz et al. J Gene Med. 2008 Oct.
Free article

Abstract

Background: High-capacity adenoviruses (HC-Ad) hold great promise for the treatment of many diseases. The major drawbacks for the clinical application of this vector concern difficulties with respect to large-scale production, and the absence of standardized methods for production and titration. In the present study, we compare the different methods found in the literature for characterizing HC-Ad production.

Methods: Two productions of the HC-Ad carrying murine IL-12 gene were obtained. The viral titer and adenovirus-helper contamination as well as viral particle concentration of both productions were determined using different methods: (i) quantification of total viral particles by spectrophotometry and plaque assay to estimate first-generation (FG)-helper-Ad contamination; (ii) quantification of HC-Ad and FG-helper-Ad genomes by the quantitative polymerase chain reaction (qPCR) directly from viral stock; (iii) quantification of viral genomes after cell infection by the slot-blot hybridization assay and (iv) qPCR.

Results: Dramatic differences with respect to viral titer were found depending on the method used. The first method overestimates HC-Ad titer and underestimates FG-helper-Ad contamination and no information on the infectivity of the HC-Ad is obtained. qPCR analysis of viral stock is more sensitive and accurate, but information about infectivity remains unknown and FG-helper-Ad contamination is overestimated. Quantification of HC-Ad and FG-helper-Ad infectious units by-slot blot DNA hybridization and qPCR assay are found to be equally sensitive and accurate.

Conclusions: The results of the present study demonstrate that a standardized method should be developed for HC-Ad characterization for future clinical applications of this vector. Quantification of HC-Ad production by qPCR is a fast, safe and reliable method for determining HC-Ad and FG-helper-Ad particles and infectious units.

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