Protocol

Prion Protein Protocols

Volume 459 of the series Methods in Molecular Biology™ pp 49-68

Assaying Prions in Cell Culture

The Standard Scrapie Cell Assay (SSCA) and the Scrapie Cell Assay in End Point Format (SCEPA)
  • Sukhvir P. MahalAffiliated withDepartment of Infectology, Scripps Florida
  • , Cheryl A. DemczykAffiliated withDepartment of Infectology, Scripps Florida
  • , Emery W. SmithJr.Affiliated withDepartment of Infectology, Scripps Florida
  • , Peter-Christian KlohnAffiliated withMRC Prion Unit, Institute of Neurology
  • , Charles WeissmannAffiliated withDepartment of Infectology, Scripps Florida

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Summary

Prions are usually quantified by bioassays based on intracerebral inoculation of animals, which are slow, imprecise, and costly. We have developed a cell-based prion assay that is based on the isolation of cell lines highly susceptible to certain strains (Rocky Mountain Laboratory and 22L) of mouse prions and a method for identifying individual, prion-infected cells and quantifying them. In the standard scrapie cell assay (SSCA), susceptible cells are exposed to prion-containing samples for 4 days, grown to confluence, passaged two or three times, and the proportion of rPrPSc-containing cells is determined with automated counting equipment. The dose response is dynamic over 2 logs of prion concentrations. The SSCA has a standard error of ±20–30%, is as sensitive as the mouse bioassay, 10 times faster, at least 2 orders of magnitude less expensive, and it is suitable for robotization. Assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar.

Keywords

22L CAD cells Elispot Me7 neuroblastoma PK1 cells prions PrPC rPrPSc R33 cells RML scrapie scrapie cell endpoint assay (SCEPA) standard scrapie cell assay (SSCA) strains