Focus Forming Assay – Virus Titer Quantification

We have observed a steady increase in interest for the ‘Focus Forming Assay – Virus Titer Quantitation’ app note, supplied by SYNENTEC and shared by BPES over a year ago!

As this topic is so popular, and critically important due to current events, we thought a refreshed version was in order.

Similar to the Viral Plaque Assay from SYNENTEC (the benefits of which I have detailed in a previous article) – the Focus Forming Assay (FFA) is closely related, except instead of detecting the plaque formation after virus-induced cell lysis, this assay detects infected host cells and infectious virus particles before a plaque is formed.

Different cell monolayer samples are infected with a serial dilution of the virus of interest. After incubating the samples over night, the host cells build clusters of infected cells which can be visualized via immunostaining 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 (also called ‘dark spots’). These foci are visualized with brightfield illumination on the NyONE or Cellavista imagers, and the number of infected foci are quantified with the inbuilt YT-image analysis software – thus providing virus titer quantification.

Extract from the software readout (for further information look at ShortNote BF Dark Spots)

• # of Foci
• mean Foci Size
• total Confluence
• % Foci covered Area

To discuss this assay, please contact me (Ellen Harrington) for more information.

Read the original article