The focus forming assay (FFA) is an immunostaining technique and a variation of the viral plaque assay. Instead of detecting the plaque formation after virus-induced cell lysis these assays detect infected host cells and infectious virus particles before a plaque is formed.
Different cell monolayer samples were infected with a serial dilution of the virus of interest. After incubating the samples overnight the host cells built clusters of infected cells which could be visualized with specific horse radish peroxidase (HRP) coupled antibodies against a viral antigen.
The HRP can oxidize with the 3-3’diaminobenzidine tetrahydrochloride (DAB) which results in brown/black insoluble precipitates. These foci were visualized with the brightfield illumination and the number of foci was quantified with the SYNENTEC YT-image analysis software®.
Extract from the software readout (for further information look at ShortNote Dark Spots)
• # of Foci
• mean Foci Size
• total Confluence
• % Foci covered Area