Automated baculovirus titration assay based on viable cell growth monitoring using a colorimetric indicator

The fastest methods reported are based on viral DNA quantitation using either flow cytometry (10) or real-time PCR (11), which allow titer determination within two hours. The drawbacks of these approaches are the costs of equipment and staining reagents, and the fact that the total number of particles and not the number of infectious particles is determined. An alternative approach is based on the lytic nature of the viral system (12), and more specifically on the fact that cell growth is attenuated upon virus infection. This growth reduction is dose-dependent and can be estimated by measuring the viable cell concentration and subsequently correlating this to the virus titer. Indeed, a new method was recently developed for virus titration by spectrophotometrically monitoring the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) (4). The accuracy of the method was clearly demonstrated; however, the number of preparation steps and the overall duration (6 days) are not compatible with a fast and automated high-throughput process. In this work, we demonstrate that