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A simple, versatile and sensitive cell-based assay for prions from various species.

Arellano-Anaya ZE, Savistchenko J, Mathey J, Huor A, Lacroux C, Andréoletti O, Vilette D - PLoS ONE (2011)

Bottom Line: However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species.Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures.We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay.

View Article: PubMed Central - PubMed

Affiliation: UMR Institut National de la Recherche Agronomique Ecole Nationale Vétérinaire de Toulouse 1225, Interactions Hôte Agent Pathogène, Toulouse, France.

ABSTRACT
Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

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Related in: MedlinePlus

Sensitivity of moRK13 cell assay for the detection of RML mouse prions.Serial 10-fold dilutions (from 10−4 to 10−8) of RML 10% brain homogenate were inoculated in duplicate to moRK13 cells and the inoculated cultures were proceeded as in figure 1D. PrPres was analyzed by western blot after one (A) or two rounds (B) of cell assay. M are standard molecular mass marker proteins in kDa.
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pone-0020563-g002: Sensitivity of moRK13 cell assay for the detection of RML mouse prions.Serial 10-fold dilutions (from 10−4 to 10−8) of RML 10% brain homogenate were inoculated in duplicate to moRK13 cells and the inoculated cultures were proceeded as in figure 1D. PrPres was analyzed by western blot after one (A) or two rounds (B) of cell assay. M are standard molecular mass marker proteins in kDa.

Mentions: To determine the sensitivity of the RK13-based assay for prion strains from other species, we chose RML, widely used in the mouse scrapie model. Serial 10-fold dilutions of RML 10% brain homogenate inoculated into tg20 mice showed that the last dilution inducing clinical disease in all mice (n = 6) was 10−6 (the incubation period was 108 days ±6 days, mean ± SD). Serial 10-fold dilutions of this RML 10% brain homogenate (from 10−4 to 10−8) were inoculated to moRK13 cells. The cultures were solubilized 4 weeks later, PK-digested and analyzed for the presence of PrPres. Out of 8 independent experiments, all cultures inoculated with 10−5 were PrPres positive (a typical result is shown in Fig. 2A). In two experiments, a PrPres signal was also detected in cells inoculated with the 10−6 dilution of RML. As observed with ovine prions, a second round of cell assay increased the sensitivity of RML detection by 100-fold (Fig. 2B), i.e., inoculated cultures that appear negative in the first round (10−6 and 10−7) contained infectivity that was detected upon a 2nd round of cell assay. Thus, 10−7 dilutions that do not transmit disease to tg20 mice lead to a detectable infection of moRK13 cultures.


A simple, versatile and sensitive cell-based assay for prions from various species.

Arellano-Anaya ZE, Savistchenko J, Mathey J, Huor A, Lacroux C, Andréoletti O, Vilette D - PLoS ONE (2011)

Sensitivity of moRK13 cell assay for the detection of RML mouse prions.Serial 10-fold dilutions (from 10−4 to 10−8) of RML 10% brain homogenate were inoculated in duplicate to moRK13 cells and the inoculated cultures were proceeded as in figure 1D. PrPres was analyzed by western blot after one (A) or two rounds (B) of cell assay. M are standard molecular mass marker proteins in kDa.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105100&req=5

pone-0020563-g002: Sensitivity of moRK13 cell assay for the detection of RML mouse prions.Serial 10-fold dilutions (from 10−4 to 10−8) of RML 10% brain homogenate were inoculated in duplicate to moRK13 cells and the inoculated cultures were proceeded as in figure 1D. PrPres was analyzed by western blot after one (A) or two rounds (B) of cell assay. M are standard molecular mass marker proteins in kDa.
Mentions: To determine the sensitivity of the RK13-based assay for prion strains from other species, we chose RML, widely used in the mouse scrapie model. Serial 10-fold dilutions of RML 10% brain homogenate inoculated into tg20 mice showed that the last dilution inducing clinical disease in all mice (n = 6) was 10−6 (the incubation period was 108 days ±6 days, mean ± SD). Serial 10-fold dilutions of this RML 10% brain homogenate (from 10−4 to 10−8) were inoculated to moRK13 cells. The cultures were solubilized 4 weeks later, PK-digested and analyzed for the presence of PrPres. Out of 8 independent experiments, all cultures inoculated with 10−5 were PrPres positive (a typical result is shown in Fig. 2A). In two experiments, a PrPres signal was also detected in cells inoculated with the 10−6 dilution of RML. As observed with ovine prions, a second round of cell assay increased the sensitivity of RML detection by 100-fold (Fig. 2B), i.e., inoculated cultures that appear negative in the first round (10−6 and 10−7) contained infectivity that was detected upon a 2nd round of cell assay. Thus, 10−7 dilutions that do not transmit disease to tg20 mice lead to a detectable infection of moRK13 cultures.

Bottom Line: However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species.Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures.We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay.

View Article: PubMed Central - PubMed

Affiliation: UMR Institut National de la Recherche Agronomique Ecole Nationale Vétérinaire de Toulouse 1225, Interactions Hôte Agent Pathogène, Toulouse, France.

ABSTRACT
Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

Show MeSH
Related in: MedlinePlus