HEp-2, L-929 and Ratec cells are responsive to the antiviral activity of interferons and yield a uniform cytopatic effect to Vesicular stomatitis virus by inhibition of cellular uptake of a colored dye (naphthol blue-black or crystal violet). The uptake of the dye is a linear function relative to cell concentration up to the maximum number of cells in a single microtiter well. Between 2.0 x 104 and 5.0 x 104 cells/well (depending on indicator cells) the function is linear with a correlation coefficient of > 0.950.
Cytopathic Indicator virus
For obtaining distinct CPE, VSV (Indiana strain) is used for the human, mouse and rat interferon antiviral bioassays. The virus is propagated on Ratec cells, a rat embryonic cell line. Virus preparations are stored at -80 °C in small aliquots containing approximately 4.0 x 107 plaque forming units (PFU) per ml. For each cell line different concentrations of viral particles are used to obtain distinct CPE values after 24h incubation at 37 °C.
Interferon reference standards
International reference standards are obtained from the National Institute for Biological Standards and Control (NIBSC) in the UK and interferons for the preparation of laboratory standards are obtained from a reputable commercial source. The laboratory standards are calibrated to International NIBSC reference standards and expressed in LU (laboratory unit)/ml in which 1.0 LU is equal to 1.0 International unit (IU). The vials with laboratory standards are stored at -80 °C in small aliquots for single use.
An example of results in a CPE-based AVA testing.
CC: cell control (100% viable cells);
standard: titration of 10 LU/ml human IFN-γ (50% CPE at approximately position F);
S1 + S2: antibody-mediated reduction in IFN-γ bioactivity (50% CPE at approximately position C);
S3 + S4: titration of samples with high amounts of human IFN-γ (>100 LU/ml, 0% CPE);
VC: virus control with >90% CPE.
Arduini R.M. et al. 2004 Protein Expr. Purif. 34: 229-242
In this study the specific anti-viral activity of rat IFN-β was determined by U-CyTech using the CPE bioassay that measures the ability of the protein to protect rat RATEC cells from cytotoxicity due to infection with VSV.