A Quantitative, Highly Sensitive Cell-Based Infectivity Assay for Mouse Scrapie Prions

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 100; no. 20; pp. 11666 - 11671
• Main Authors: P. -C. Klöhn, Stoltze, L., Flechsig, E., Enari, M., Weissmann, C.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 30.09.2003; National Acad Sciences
• Subjects: Animals; Antibodies; Bioassay; Biological Sciences; Brain; Cell lines; Cells; Cricetinae; Cultured cells; Enzyme linked immunospot assay; Inoculation; Lethal dose 50; Mesocricetus; Mice; Mice, Inbred C57BL; Microbiology; Prions; Prions - pathogenicity; Rodents; Scrapie - metabolism; Sensitivity and Specificity; Titration
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1834432100

Prions are usually quantified by bioassays based on intracerebral inoculation of mice that are slow, imprecise, and costly. We have isolated neuroblastoma N2a sublines highly susceptible to mouse prions, as evidenced by accumulation of infectivity and the scrapie form of prion protein (PrPSc), and developed quantitative in vitro assays for prion infectivity. In the scrapie cell (SC) assay, susceptible N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split 1:10 three times, and the proportion of PrPSc-containing cells is determined with automated counting equipment. In a log/log plot, the dose-response is linear over two logs of prion concentrations. The SC assay is about as sensitive as the mouse bioassay, 10 times faster, >2 orders of magnitude less expensive, and suitable for robotization. SC 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.