Assaying prions in cell culture: the standard scrapie cell assay (SSCA) and the scrapie cell assay in end point format (SCEPA).
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 rPrP(Sc)-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.
Mahal
Sukhvir P
SP
Department of Infectology, Scripps Florida, Jupiter, FL, USA.
Demczyk
Cheryl A
CA
Smith
Emery W
EW
Klohn
Peter-Christian
PC
Weissmann
Charles
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002452:Cell Count; D002478:Cells, Cultured; D005615:Freezing; D008567:Membranes, Artificial; D051379:Mice; D011328:Prions / Q000032:analysis*; D012984:Software; D013997:Time Factors; D014343:Trypan Blue
D008567:Membranes, Artificial; D011328:Prions; D000255:Adenosine Triphosphate; D014343:Trypan Blue