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Highly efficient protein misfolding cyclic amplification.

Gonzalez-Montalban N, Makarava N, Ostapchenko VG, Savtchenk R, Alexeeva I, Rohwer RG, Baskakov IV - PLoS Pathog. (2011)

Bottom Line: Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format.The increase in the amplification efficiency did not come at the expense of prion replication specificity.The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America.

ABSTRACT
Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP(C) into PrP(Sc) from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP(Sc) by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 10¹²-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP(C) susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.

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Effect of bead material on efficiency of amplification.(A) 263K scrapie brain material was serially diluted 105-fold into 10% hamster NBH and subjected to 48 PMCA cycles in the presence of two beads made from Teflon (purchase from McMaster-Carr – lane 2, or Small Parts – lane 3), stainless steel 440C, neoprene, nylon, EPDM, nitrile, stainless still 302, or acetal, as indicated. Prior to electrophoresis, samples in lanes 2–10 were digested with PK. Undigested 10% hamster NBH (lane 1) was loaded as a reference. Without PMCA, 105- fold diluted 263K brain material was not detectable by Western blotting (not shown). (B) RML scrapie brain material was serially diluted 104-fold into 10% mouse NBH and subjected to 48 PMCA cycles in the absence of beads (lane 3) or presence of two beads made from Teflon (purchase from Small Parts – lane 4), neoprene, nylon, EPDM, nitrile, stainless still 302, or acetal, as indicated. Prior to electrophoresis, samples in lanes 2–10 were digested with PK. Undigested 10% mouse NBH (lane 1) was loaded as a reference.
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ppat-1001277-g008: Effect of bead material on efficiency of amplification.(A) 263K scrapie brain material was serially diluted 105-fold into 10% hamster NBH and subjected to 48 PMCA cycles in the presence of two beads made from Teflon (purchase from McMaster-Carr – lane 2, or Small Parts – lane 3), stainless steel 440C, neoprene, nylon, EPDM, nitrile, stainless still 302, or acetal, as indicated. Prior to electrophoresis, samples in lanes 2–10 were digested with PK. Undigested 10% hamster NBH (lane 1) was loaded as a reference. Without PMCA, 105- fold diluted 263K brain material was not detectable by Western blotting (not shown). (B) RML scrapie brain material was serially diluted 104-fold into 10% mouse NBH and subjected to 48 PMCA cycles in the absence of beads (lane 3) or presence of two beads made from Teflon (purchase from Small Parts – lane 4), neoprene, nylon, EPDM, nitrile, stainless still 302, or acetal, as indicated. Prior to electrophoresis, samples in lanes 2–10 were digested with PK. Undigested 10% mouse NBH (lane 1) was loaded as a reference.

Mentions: Prion amplification in PMCA was previously shown to exhibit species specificity that faithfully reflects the transmission barrier observed in animals [17], [18]. Considering that beads were found to improve significantly the amplification efficiency, we were interested in testing whether the species specificity was preserved in PMCAb. To address this question, two hamster strains, 263K and SSLOW were used to seed PMCA reactions in mouse NBHs. Consistent with the previous results, beads improved the conversion yield for both strains when they were amplified in Syrian hamster NBH (Fig. 7A,B). However, when 263K or SSLOW were diluted with mouse NBH no detectible amplification was observed for at least three serial PMCA rounds in the presence or absence of beads (Fig. 7A,B). A control experiment revealed that mouse RML strain could be amplified in mouse NBH (data not shown, and Fig. 8B). Therefore, the lack of detectible amplification of hamster strains in serial PMCA in mouse NBH confirmed that the presence of beads does not eliminate the species barrier. Taken together, these results illustrate that significant improvements in amplification efficiency do not come at the expense of amplification specificity.


Highly efficient protein misfolding cyclic amplification.

Gonzalez-Montalban N, Makarava N, Ostapchenko VG, Savtchenk R, Alexeeva I, Rohwer RG, Baskakov IV - PLoS Pathog. (2011)

Effect of bead material on efficiency of amplification.(A) 263K scrapie brain material was serially diluted 105-fold into 10% hamster NBH and subjected to 48 PMCA cycles in the presence of two beads made from Teflon (purchase from McMaster-Carr – lane 2, or Small Parts – lane 3), stainless steel 440C, neoprene, nylon, EPDM, nitrile, stainless still 302, or acetal, as indicated. Prior to electrophoresis, samples in lanes 2–10 were digested with PK. Undigested 10% hamster NBH (lane 1) was loaded as a reference. Without PMCA, 105- fold diluted 263K brain material was not detectable by Western blotting (not shown). (B) RML scrapie brain material was serially diluted 104-fold into 10% mouse NBH and subjected to 48 PMCA cycles in the absence of beads (lane 3) or presence of two beads made from Teflon (purchase from Small Parts – lane 4), neoprene, nylon, EPDM, nitrile, stainless still 302, or acetal, as indicated. Prior to electrophoresis, samples in lanes 2–10 were digested with PK. Undigested 10% mouse NBH (lane 1) was loaded as a reference.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037363&req=5

ppat-1001277-g008: Effect of bead material on efficiency of amplification.(A) 263K scrapie brain material was serially diluted 105-fold into 10% hamster NBH and subjected to 48 PMCA cycles in the presence of two beads made from Teflon (purchase from McMaster-Carr – lane 2, or Small Parts – lane 3), stainless steel 440C, neoprene, nylon, EPDM, nitrile, stainless still 302, or acetal, as indicated. Prior to electrophoresis, samples in lanes 2–10 were digested with PK. Undigested 10% hamster NBH (lane 1) was loaded as a reference. Without PMCA, 105- fold diluted 263K brain material was not detectable by Western blotting (not shown). (B) RML scrapie brain material was serially diluted 104-fold into 10% mouse NBH and subjected to 48 PMCA cycles in the absence of beads (lane 3) or presence of two beads made from Teflon (purchase from Small Parts – lane 4), neoprene, nylon, EPDM, nitrile, stainless still 302, or acetal, as indicated. Prior to electrophoresis, samples in lanes 2–10 were digested with PK. Undigested 10% mouse NBH (lane 1) was loaded as a reference.
Mentions: Prion amplification in PMCA was previously shown to exhibit species specificity that faithfully reflects the transmission barrier observed in animals [17], [18]. Considering that beads were found to improve significantly the amplification efficiency, we were interested in testing whether the species specificity was preserved in PMCAb. To address this question, two hamster strains, 263K and SSLOW were used to seed PMCA reactions in mouse NBHs. Consistent with the previous results, beads improved the conversion yield for both strains when they were amplified in Syrian hamster NBH (Fig. 7A,B). However, when 263K or SSLOW were diluted with mouse NBH no detectible amplification was observed for at least three serial PMCA rounds in the presence or absence of beads (Fig. 7A,B). A control experiment revealed that mouse RML strain could be amplified in mouse NBH (data not shown, and Fig. 8B). Therefore, the lack of detectible amplification of hamster strains in serial PMCA in mouse NBH confirmed that the presence of beads does not eliminate the species barrier. Taken together, these results illustrate that significant improvements in amplification efficiency do not come at the expense of amplification specificity.

Bottom Line: Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format.The increase in the amplification efficiency did not come at the expense of prion replication specificity.The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America.

ABSTRACT
Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP(C) into PrP(Sc) from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP(Sc) by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 10¹²-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP(C) susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.

Show MeSH
Related in: MedlinePlus