Replication Competent Retrovirus (RCR) Detection
Retrovirus vectors have a safe history of use in gene therapy protocols. Depending on the nature of the cell line, tests for murine or mouse-based retroviruses may also be needed. MDS offers a panel of sensitive tests to detect infectious murine retroviruses.
Our procedures can detect viruses from the four classes of these retroviruses originally isolated from mice that are of concern for various biological products.
- Amplification of retrovirus on Mus dunni cells (five passages) using PG4 S+L- assay (for the detection of murine xenotropic and amphotropic retroviruses) as end-point. The assay is validated to detect one RCR in 300 ml using the recommended ATCC reference standard.
Direct and extended MiCl1 S+L- assays
For the detection of xenotropic retroviruses (X-MLV), S+L- focus-forming assays utilizing mink ( MiCl1) or cat (PG4 S + L -) cell lines are used.
Direct assay: The number of foci that form in a monolayer after inoculation with a sample is proportional to the number of infectious units in the inoculum. This assay can be used to quantitate the amount of virus in a sample
The extended assay: Low levels of virus not detected in a direct assay can be detect. After passaging, the monolayers are observed. The presence of foci indicates the presence of virus in the sample.
Extended Mus dunni assay : Mus dunni cells are broadly susceptible to different classes of murine leukemia virus (MLV) includingamphotropic, ecotropic, polytropic (also called mink cell focus forming or MCF), and xenotropic viruses. M. dunni cells are inoculated with samples, then passaged to amplify low-level virus that may be present in the inoculum . As MLV does not typically cause cytopathic effect in the cells, after incubation, the cells are stained with the antibodies, using a fluorescent antibody technique. If our virologists observe any fluorescent cells present after staining, this indicates that virus was present in the sample.
Extended XC plaque assay: The extended XC plaque assay is utilized to detect infectious ecotropic murine retroviruses (E-MLV). E-MLV infects only cells of mouse and rat origin. Cells from the mouse embryo cell line, SC-1, are inoculated with the sample, and passaged to amplify any low level of virus present. Then the cells are overlaid with XC cells, which will form giant multinucleated cells (syncytia) where virus was present in the SC-1 cells.
Custom-designed assays available on request.