Table 1.
Prion decontamination procedures tested in this study.
Fig 1.
Optimization of steel wire contamination in 96-well microplates.
Wires were contaminated with either 1% 127S-scrapie or 10% human vCJD brain homogenates at different times (1 min, 10 min or 1 hr) and subjected to one Surf-PMCA round. Protease-resistant prion protein was detected with 6D11 (127S-scrapie) or 3F4 (human vCJD) monoclonal antibodies. NEG: Negative control (wires mock-contaminated in normal brain homogenate). M: molecular mass marker.
Fig 2.
Determination of Surf-PMCA sensitivity.
(A and B) Representative Western blot results of serial Surf-PMCA on wires contaminated with serial 10-fold dilutions of 127S (A) or vCJD (B) brain material. (C and D) Individual wires scored positive by serial Surf-PMCA as a function of infectious brain dilution (C, 127S scrapie; D, vCJD) and PMCA round. Three independent Surf-PMCA series were performed with 4 wires per brain dilution (Black cross (Χ) Series 1, Red square (■) Series 2 and Green circle (●) Series 3). Protease-resistant prion protein was detected with 6D11 (127S-scrapie) or 3F4 (human vCJD) monoclonal antibodies. NEG: Negative control (wires mock-contaminated in normal brain homogenate). M: molecular mass marker. Bars to the left of Western blot panels indicate the 30 and 20 kilo Dalton marker positions. * Positive wires were not systematically subjected to an additional PMCA round.
Table 2.
Summary of PrPTSE detection data on wires contaminated with serial dilutions of infectious brain material by Surf-PMCA.
Fig 3.
Evaluation of “standard” decontamination procedures on 127S scrapie prions.
Steel wires contaminated with 1% 127S scrapie brain homogenate were treated with “standard” prion decontamination procedures and subjected to three serial PMCA rounds. Three independent series of PMCA were performed on 4 wires per treatment (Black cross (Χ) Series 1, Red square (■) Series 2 and Green circle (●) Series 3). PAA: Per acetic acid 1.2%—60 min; NaOH: sodium hydroxide 0.1N—15 min or 1N—60 min; Auto: steam sterilization 121°C -20 min or 134°C -20 min; NaOCl: Sodium hypochlorite 0.2% (2000ppm)– 15 min or 2% (20000ppm)– 60 min; H2O: untreated positive control; NEG: negative control (wire mock-contaminated with normal brain homogenate).
Fig 4.
Evaluation of “standard” and commercially available decontamination procedures on human vCJD prions.
Steel wires contaminated with 10% human vCJD brain homogenate were treated with “standard” (A) and commercially available (B) prion decontamination procedures and subjected to 4 serial PMCA rounds. Two independent series of PMCA were performed on 4 wires per treatment (Black cross (Χ) Series 1 and Red square (■) Series 2). PAA: Per acetic acid 1.2%—60 min; NaOH: sodium hydroxide 0.1N—15 min or 1N—60 min; Auto: steam sterilization 121°C -20 min or 134°C -20 min; NaOCl: Sodium hypochlorite 0.2% (2000ppm)– 15 min or 2% (20000ppm)– 60 min; H2O: untreated positive control; NEG: negative control (wire mock-contaminated with normal brain homogenate). (C) Representative Surf-PMCA results from two individual wires after both reference and marketed decontamination procedures (3F4 monoclonal antibody). Bars to the left of Western blot panels indicate the 30 and 20 kilo Dalton marker positions.