How are recombinant adenoviruses purified and virus titers determined?

A. The amplified recombinant adenovirus was purified on 2 sequential cesium chloride gradients and then dialyzed with a buffer (PBS, 10% glycerol, pH7.4) to reduce the salt concentration. The titer of purified virus can be determined with (1) OD260 Assay or (2) Plaque Assay.

The OD260 assay measures the concentration of viral DNA and protein. It does not distinguish between intact, infectious viruses and damaged, non-infectious viruses. It is a physical assay measuring the concentration of total viruses, live and dead.

Based on OD260 data, the concentration of viral particles (VP) can be calculated using the following formula: Adenovirus titer in viral particles/ml = OD value x d x 1.1 x 1012; ( d= dilution factor) The plaque assay measures the concentration of infectious viruses, and therefore it is a biological assay. Basically, a mono-layer of HEK293 cells are infected with a series of virus dilutions. Viruses will propagate in infected cells, and eventually leading to the ... more.

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