Measuring the Infectious Titer of Recombinant Adenovirus Using Tissue Culture Infection Dose 50% (TCID50) End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR).

Abstract

Traditionally, adenovirus and recombinant adenovirus infectious titers have been measured by plaque assay, in which the cells are infected with serially diluted adenovirus stock and then overlaid with agar; a plaque will form as the result of a single infectious event. Although this method gives a quantitative readout (number of plaques corrected for the dilution), there can be issues with sensitivity and reproducibility, especially when adenovirus serotypes are used that infect standard cell lines with poor efficiency. An alternative approach is to plate serial dilutions of the cells growing in the wells of a 96-well tissue culture plate and determine the dilution at which 50% of the wells are infected. This ancient and reliable technique known as the "tissue culture infection dose 50%" (TCID50) end-point dilution method has been used for titering a number of viruses, especially those that do not readily form plaques. Usually, infected wells are determined by direct examination for cytopathic effect (CPE) or cell viability. However, by combining a 96-well TCID50 format and the power of quantitative polymerase chain reaction (qPCR) for detection, a large increase in sensitivity-in our hands 10-fold, with a range of both transgenes and adenovirus serotypes-can be achieved. This protocol uses a 96-well TCID50 format, in conjunction with qPCR for sensitive and quantitative positive-well calling, to determine infectious titer of adenovirus vectors.