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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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Cell assay detection of plastic-bound prions.Infectivity from 10% PG127 brain homogenate was diluted either in culture medium (prion in culture medium) or in Triton-DOC lysis buffer (prion in detergents) and incubated for 2 h into plastic wells. Samples were removed, wells were thoroughly rinsed and air-dried. OvRK13 cells were then seeded in the presence (+) or in the absence (−) of dox. PrPres in the cultures was analyzed 4 weeks later by immunoblotting and compared to PrPres levels in ovRK13 cultures subjected to the standard cell assay (no coating). M are standard molecular mass marker proteins (20, 30 and 40 kDa).
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pone-0020563-g003: Cell assay detection of plastic-bound prions.Infectivity from 10% PG127 brain homogenate was diluted either in culture medium (prion in culture medium) or in Triton-DOC lysis buffer (prion in detergents) and incubated for 2 h into plastic wells. Samples were removed, wells were thoroughly rinsed and air-dried. OvRK13 cells were then seeded in the presence (+) or in the absence (−) of dox. PrPres in the cultures was analyzed 4 weeks later by immunoblotting and compared to PrPres levels in ovRK13 cultures subjected to the standard cell assay (no coating). M are standard molecular mass marker proteins (20, 30 and 40 kDa).

Mentions: Prions strongly bind to stainless steel wires and the contaminated wires efficiently transmit disease to mice [38], [39], [40] and infection to recipient cultured target cells [21]. Prion-coated wires, as models of contaminated surgical metal devices, were used to assess the effectiveness of decontamination procedures [38], [39], [41] [21] [42] [43], [44]. The propensity of prions for steel surface binding led recently to a blood-based assay of vCJD [45]. There is also suggestion that prions bound to tissue culture plastic may elicit infection of seeded cultured cells [46]. To further examine this possibility, the 10% PG127 homogenate was diluted in complete culture medium and serial 10-fold dilutions were deposited for 2 h in tissue culture plastic wells. Since it would be also useful to detect prion infectivity in detergents buffers used in cell or tissue fractionation [6], [10] or in PMCA reactions [47], [48], the 10% PG127 was solubilized, serially diluted in Triton-DOC lysis buffer and deposited in plastic wells as well. The samples were then removed, the wells thoroughly washed with PBS and air-dried for 2 h. OvRK13 cells were seeded into these wells and infection was allowed to proceed for 4 weeks. We found (representative data from 4 experiments are shown in Fig. 3) that infection of cultures seeded in prion-coated wells was as efficient as that observed with the standard procedure, i.e., cultures seeded in 10−5-coated wells were PrPres positive. Interestingly, coating and cell detection of detergent-solubilized prions were also very effective. These findings indicate that prions can be quantitatively detected by our cell assay upon binding to plastic surfaces.


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)

Cell assay detection of plastic-bound prions.Infectivity from 10% PG127 brain homogenate was diluted either in culture medium (prion in culture medium) or in Triton-DOC lysis buffer (prion in detergents) and incubated for 2 h into plastic wells. Samples were removed, wells were thoroughly rinsed and air-dried. OvRK13 cells were then seeded in the presence (+) or in the absence (−) of dox. PrPres in the cultures was analyzed 4 weeks later by immunoblotting and compared to PrPres levels in ovRK13 cultures subjected to the standard cell assay (no coating). 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-g003: Cell assay detection of plastic-bound prions.Infectivity from 10% PG127 brain homogenate was diluted either in culture medium (prion in culture medium) or in Triton-DOC lysis buffer (prion in detergents) and incubated for 2 h into plastic wells. Samples were removed, wells were thoroughly rinsed and air-dried. OvRK13 cells were then seeded in the presence (+) or in the absence (−) of dox. PrPres in the cultures was analyzed 4 weeks later by immunoblotting and compared to PrPres levels in ovRK13 cultures subjected to the standard cell assay (no coating). M are standard molecular mass marker proteins (20, 30 and 40 kDa).
Mentions: Prions strongly bind to stainless steel wires and the contaminated wires efficiently transmit disease to mice [38], [39], [40] and infection to recipient cultured target cells [21]. Prion-coated wires, as models of contaminated surgical metal devices, were used to assess the effectiveness of decontamination procedures [38], [39], [41] [21] [42] [43], [44]. The propensity of prions for steel surface binding led recently to a blood-based assay of vCJD [45]. There is also suggestion that prions bound to tissue culture plastic may elicit infection of seeded cultured cells [46]. To further examine this possibility, the 10% PG127 homogenate was diluted in complete culture medium and serial 10-fold dilutions were deposited for 2 h in tissue culture plastic wells. Since it would be also useful to detect prion infectivity in detergents buffers used in cell or tissue fractionation [6], [10] or in PMCA reactions [47], [48], the 10% PG127 was solubilized, serially diluted in Triton-DOC lysis buffer and deposited in plastic wells as well. The samples were then removed, the wells thoroughly washed with PBS and air-dried for 2 h. OvRK13 cells were seeded into these wells and infection was allowed to proceed for 4 weeks. We found (representative data from 4 experiments are shown in Fig. 3) that infection of cultures seeded in prion-coated wells was as efficient as that observed with the standard procedure, i.e., cultures seeded in 10−5-coated wells were PrPres positive. Interestingly, coating and cell detection of detergent-solubilized prions were also very effective. These findings indicate that prions can be quantitatively detected by our cell assay upon binding to plastic surfaces.

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