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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 ovRK13 cell assay for the detection of PG127 ovine prion.A) Morphology of inoculated ovRK13 cultures kept in the same wells during the whole cell assay procedure (d0 is the time of inoculation and d28 is 4 weeks later). B) Sensitivity of ovRK13 cell assay as assessed by immunoblotting. Right panel: Serial 10-fold dilutions (from 10−4 to 10−6) of infectious PG127 10% brain homogenate were inoculated to single wells of ovRK13 cells. Four weeks later, inoculated cultures were analyzed for PrPres by immunoblotting. Positive transmission was detected for dilutions up to 10−5. No PrPres was observed when inoculated ovRK13 did not express the ovine PrP (dox-). Left panel: total PrP from infected cells was analyzed before (−) or after (+) PK digestion to illustrate band shift upon PK proteolysis. M are standard molecular mass marker proteins (20, 30 and 40 kDa). C) Sensitivity of ovRK13 cell assay as assessed by Elispot. Replicate wells from the same experiment shown in Fig. 1B were analyzed. Left: representative wells of an Elispot plate showing spots given by ovRK13 cells exposed to the indicated dilutions of PG127. Right: double-logarithmic plot of spot number versus PG127 dilution shown for inoculations in the presence (triangle) or in the absence (square) of dox. For each dilution, the mean value ± SD of 8 measurements is shown. Background values for ovRK13 cells inoculated in the absence of dox are less than 4 spots per 50,000 cells. D) Sensitivity is strongly improved by 2 successive rounds of cell assay. Serial 10-fold dilutions (from 10−5 to 10−7) of infectious PG127 10% brain homogenate were inoculated to duplicate wells of ovRK13 cells. Four weeks later, PrPres in a 1st set of inoculated cultures was isolated (1st round) while cultures of the 2nd set were homogenized to inoculate new ovRK13 cells. Four weeks later, PrPres was isolated (2nd round) and all the samples were analyzed by immunoblotting. M are standard molecular mass marker proteins (20, 30 and 40 kDa).
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pone-0020563-g001: Sensitivity of ovRK13 cell assay for the detection of PG127 ovine prion.A) Morphology of inoculated ovRK13 cultures kept in the same wells during the whole cell assay procedure (d0 is the time of inoculation and d28 is 4 weeks later). B) Sensitivity of ovRK13 cell assay as assessed by immunoblotting. Right panel: Serial 10-fold dilutions (from 10−4 to 10−6) of infectious PG127 10% brain homogenate were inoculated to single wells of ovRK13 cells. Four weeks later, inoculated cultures were analyzed for PrPres by immunoblotting. Positive transmission was detected for dilutions up to 10−5. No PrPres was observed when inoculated ovRK13 did not express the ovine PrP (dox-). Left panel: total PrP from infected cells was analyzed before (−) or after (+) PK digestion to illustrate band shift upon PK proteolysis. M are standard molecular mass marker proteins (20, 30 and 40 kDa). C) Sensitivity of ovRK13 cell assay as assessed by Elispot. Replicate wells from the same experiment shown in Fig. 1B were analyzed. Left: representative wells of an Elispot plate showing spots given by ovRK13 cells exposed to the indicated dilutions of PG127. Right: double-logarithmic plot of spot number versus PG127 dilution shown for inoculations in the presence (triangle) or in the absence (square) of dox. For each dilution, the mean value ± SD of 8 measurements is shown. Background values for ovRK13 cells inoculated in the absence of dox are less than 4 spots per 50,000 cells. D) Sensitivity is strongly improved by 2 successive rounds of cell assay. Serial 10-fold dilutions (from 10−5 to 10−7) of infectious PG127 10% brain homogenate were inoculated to duplicate wells of ovRK13 cells. Four weeks later, PrPres in a 1st set of inoculated cultures was isolated (1st round) while cultures of the 2nd set were homogenized to inoculate new ovRK13 cells. Four weeks later, PrPres was isolated (2nd round) and all the samples were analyzed by immunoblotting. M are standard molecular mass marker proteins (20, 30 and 40 kDa).

Mentions: This PG127 10% brain homogenate was serially diluted in cell culture medium and 10-fold dilutions (from 10−4 to 10−6) were applied to confluent ovRK13 cells in 6-well plates (one well per dilution). One week later, the infected media were removed and replaced by fresh medium. RK13 cells, like many other epithelial cell lines [35] can be kept for weeks as viable, confluent monolayers of polarized cells ([36]–[37] and Fig. 1A). The inoculated ovRK13 cultures were kept for 3 more weeks in the same wells (the medium being changed once a week) and detergent-solubilized, digested with proteinase K (PK) and assayed for PrPres by immunoblotting. Transmission was scored positive when a typical PrPres signal (Fig. 1B, left) was observed. Data from 16 independent transmissions carried out by 4 different operators over a 6 month-period demonstrated positive transmission for all cultures inoculated with PG127 dilutions down to 10−5. No detectable PrPres was observed in cultures inoculated with 10−6 dilutions. A representative immunoblot is shown in Fig. 1B, right. When the experiments were performed in the absence of dox, the inoculated cultures were PrPres negative. This important control demonstrates that the detected PrPres indeed results from de novo infection of the cells and not from residual PrPres from the diluted inocula. In addition, immunoblotting was not sensitive enough to detect the very low amounts of PrPres in the 10−5 dilutions (data not shown).


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 ovRK13 cell assay for the detection of PG127 ovine prion.A) Morphology of inoculated ovRK13 cultures kept in the same wells during the whole cell assay procedure (d0 is the time of inoculation and d28 is 4 weeks later). B) Sensitivity of ovRK13 cell assay as assessed by immunoblotting. Right panel: Serial 10-fold dilutions (from 10−4 to 10−6) of infectious PG127 10% brain homogenate were inoculated to single wells of ovRK13 cells. Four weeks later, inoculated cultures were analyzed for PrPres by immunoblotting. Positive transmission was detected for dilutions up to 10−5. No PrPres was observed when inoculated ovRK13 did not express the ovine PrP (dox-). Left panel: total PrP from infected cells was analyzed before (−) or after (+) PK digestion to illustrate band shift upon PK proteolysis. M are standard molecular mass marker proteins (20, 30 and 40 kDa). C) Sensitivity of ovRK13 cell assay as assessed by Elispot. Replicate wells from the same experiment shown in Fig. 1B were analyzed. Left: representative wells of an Elispot plate showing spots given by ovRK13 cells exposed to the indicated dilutions of PG127. Right: double-logarithmic plot of spot number versus PG127 dilution shown for inoculations in the presence (triangle) or in the absence (square) of dox. For each dilution, the mean value ± SD of 8 measurements is shown. Background values for ovRK13 cells inoculated in the absence of dox are less than 4 spots per 50,000 cells. D) Sensitivity is strongly improved by 2 successive rounds of cell assay. Serial 10-fold dilutions (from 10−5 to 10−7) of infectious PG127 10% brain homogenate were inoculated to duplicate wells of ovRK13 cells. Four weeks later, PrPres in a 1st set of inoculated cultures was isolated (1st round) while cultures of the 2nd set were homogenized to inoculate new ovRK13 cells. Four weeks later, PrPres was isolated (2nd round) and all the samples were analyzed by immunoblotting. M are standard molecular mass marker proteins (20, 30 and 40 kDa).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020563-g001: Sensitivity of ovRK13 cell assay for the detection of PG127 ovine prion.A) Morphology of inoculated ovRK13 cultures kept in the same wells during the whole cell assay procedure (d0 is the time of inoculation and d28 is 4 weeks later). B) Sensitivity of ovRK13 cell assay as assessed by immunoblotting. Right panel: Serial 10-fold dilutions (from 10−4 to 10−6) of infectious PG127 10% brain homogenate were inoculated to single wells of ovRK13 cells. Four weeks later, inoculated cultures were analyzed for PrPres by immunoblotting. Positive transmission was detected for dilutions up to 10−5. No PrPres was observed when inoculated ovRK13 did not express the ovine PrP (dox-). Left panel: total PrP from infected cells was analyzed before (−) or after (+) PK digestion to illustrate band shift upon PK proteolysis. M are standard molecular mass marker proteins (20, 30 and 40 kDa). C) Sensitivity of ovRK13 cell assay as assessed by Elispot. Replicate wells from the same experiment shown in Fig. 1B were analyzed. Left: representative wells of an Elispot plate showing spots given by ovRK13 cells exposed to the indicated dilutions of PG127. Right: double-logarithmic plot of spot number versus PG127 dilution shown for inoculations in the presence (triangle) or in the absence (square) of dox. For each dilution, the mean value ± SD of 8 measurements is shown. Background values for ovRK13 cells inoculated in the absence of dox are less than 4 spots per 50,000 cells. D) Sensitivity is strongly improved by 2 successive rounds of cell assay. Serial 10-fold dilutions (from 10−5 to 10−7) of infectious PG127 10% brain homogenate were inoculated to duplicate wells of ovRK13 cells. Four weeks later, PrPres in a 1st set of inoculated cultures was isolated (1st round) while cultures of the 2nd set were homogenized to inoculate new ovRK13 cells. Four weeks later, PrPres was isolated (2nd round) and all the samples were analyzed by immunoblotting. M are standard molecular mass marker proteins (20, 30 and 40 kDa).
Mentions: This PG127 10% brain homogenate was serially diluted in cell culture medium and 10-fold dilutions (from 10−4 to 10−6) were applied to confluent ovRK13 cells in 6-well plates (one well per dilution). One week later, the infected media were removed and replaced by fresh medium. RK13 cells, like many other epithelial cell lines [35] can be kept for weeks as viable, confluent monolayers of polarized cells ([36]–[37] and Fig. 1A). The inoculated ovRK13 cultures were kept for 3 more weeks in the same wells (the medium being changed once a week) and detergent-solubilized, digested with proteinase K (PK) and assayed for PrPres by immunoblotting. Transmission was scored positive when a typical PrPres signal (Fig. 1B, left) was observed. Data from 16 independent transmissions carried out by 4 different operators over a 6 month-period demonstrated positive transmission for all cultures inoculated with PG127 dilutions down to 10−5. No detectable PrPres was observed in cultures inoculated with 10−6 dilutions. A representative immunoblot is shown in Fig. 1B, right. When the experiments were performed in the absence of dox, the inoculated cultures were PrPres negative. This important control demonstrates that the detected PrPres indeed results from de novo infection of the cells and not from residual PrPres from the diluted inocula. In addition, immunoblotting was not sensitive enough to detect the very low amounts of PrPres in the 10−5 dilutions (data not shown).

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