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Quality Control

We offer the following QC release tests:

Virus Identity Testing

The identity of a virus stock can be tested by:

  • Restriction analysis of the virus genome extracted from purified virus particles or from infected cells
  • PCR, by amplifying a specific region of the virus genome
  • Sequencing the region of interest (e.g. expression cassette), or the entire virus genome.

Transgene Expression Testing

A reporter cell line is infected with various doses of the recombinant virus.Cells are harvested at various time points.  Transgene expression is assessed by:

  • mRNA detection by reverse transcription and end-point PCR
  • mRNA detection and quantification by reverse transcription and real-time PCR
  • protein detection by western blot

Purity Assessment

The purity of a virus stock is assessed by analyzing the A260/A280 and A320/A260 ratios (Vellekamp et al, 2001, Hum Gene Ther 12:1923-1936):

  • The A260/A280 ratio reflects the relationship between nucleic acid and protein in a purified virus suspension.  For CsCl-purified adenovirus, this ratio falls typically between 1.2 and 1.4.  Values outside that range indicate contamination.
  • The A320/A260 ratio is the scattering ratio.  It reflects the amount of any aggregation in the purified virus.  The typical range for the scattering ratio for purified virus is approximately 0.22-0.27 and can rise rapidly from 0.3 to 0.5-0.7 as virus aggregation is initiated. 

RCA Assay

The presence of replication-competent adenovirus (RCA) in a virus stock is assessed using a modified infectivity/PCR method, which combines the amplification of infectious RCA by cell culture with the sensitivity of detection of E1-specific sequences by PCR (Ishii-Watabe et al., 2003, Mol. Ther. 8,1009–1016). Parallel assays of the same vector doses with spikes of wild-type Ad5 are carried out to determine the sensitivity of our techniques.

Virus Particle/Infectious Unit Ratio

The VP/IU ratio is determined using the titers obtained by spectrophotometry (physical titer) and the TCID50 assay (infectious titer).  Virus preparations with a ratio VP/IU <30 are acceptable.

Endotoxin Detection

Endotoxin levels in adenovirus stocks are measured using the Endosafe-PTS system (Charles River Laboratories).  FDA-licensed cartridges with a detection limit as low as 0.01 EU/mL are used.

Mycoplasma Detection

The presence of mycoplasma in the cell cultures and purified virus suspensions is assessed using a PCR-based method.  The method requires as little as 5 fg of mycoplasma DNA corresponding to 5 mycoplasma per sample volume.

 

Pricing and Timing

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