A Streamlined, Label-Free Real-Time 50% Tissue Culture Infectious Dose (TCID50) Assay using Impedance for Automated...

Measuring the viral titer, the concentration of a virus in a sample, is a fundamental procedure in virology research. While essential, traditional methods like the plaque assay and TCID50 assay can be time-consuming, require staining or labeling reagents, and often involve subjective interpretation, particularly when cytopathic effects (CPE) are subtle or difficult to quantify through imaging. For example, TCID50 assays may employ viability dyes like MTT or MTS, while plaque assays rely on imaging and staining to visualize viral plaques, both of which can introduce variability. Moreover, these traditional methods only offer a static snapshot of viral infectivity, limiting the ability to capture the dynamic interactions between viruses and permissive cells. To overcome the current limitations in measuring virus titer, a streamlined TCID50 assay was developed using impedance-based technology to objectively, noninvasively, and in real-time measure CPE, eliminating the need for labels. In this study, two virus-permissive cell models were used to validate the impedance-TCID50 assay: the GFP-labeled adenovirus (Adv-GFP) in HEK293A cells and influenza A virus (IAV) in MDCK cells. The workflow is simple, encompassing cell seeding, virus inoculation, real-time monitoring, and automatic analysis of TCID50 by the software. Throughout infection, TCID50 values were automatically calculated by the system's software using the Reed-Muench formula at all recorded time points. TCID50 values of IAV obtained via impedance readouts were comparable to those generated by the conventional crystal violet staining-based TCID50 assay. These results demonstrate that virus quantification can be precisely and efficiently achieved using impedance measurement in combination with the Virology Module of the system's software. The new assay streamlines traditional methods while providing enhanced insight into viral dynamics, thereby supporting advanced virological research and expanding potential clinical applications.