Integration of fluorescence detection and image-based automated counting increases the sensitivity, robustness and speed of plaque assays

Abstract Plaque assays are used to measure the infectious titer of viral samples. These assays are multi-day, low-throughput and may be subject to analyst variability from biased or subjective manual plaque counting. Typically, on day 1, cells are adhered to plates overnight. On day 2, cells are infected with virus. After three additional days, plaques are fixed, stained with a HRP-conjugated antibody, and a HRP substrate, and counted by eye. Manual-based visual counting of plaques is time-consuming, laborious, and may be subject to variability between analysts. Also, the assay must proceed for several days to allow the plaques to increase to sufficiently large sizes for manual identification. Here, we integrate fluorescent detection and automated plaque counting to increase the sensitivity and speed of the assay. First, we stain plaques with a fluorescent-labeled antibody. Second, we implement a plate-based cell imager to perform non-biased, non-subjective plaque counting. The integration of these two technologies decreases the assay length by 40% from 5 days to 3 days because plaque size, plaque signal to noise, and manual visualization are no longer limiting. This optimized plaque assay is sensitive, fast and robust and expands the throughput and usage of this method for measuring plaque formation.