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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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Amplification of minute amounts of PrPSc in PMCAb.(A) 263K brain material was serially diluted 1010-, 1012-, or 1014-fold and subjected to sPMCAb amplification in the presence of 3 large beads for 6 rounds as indicated (each round consists of 48 sonication cycles, 10-fold dilutions were used for subsequent rounds). Amplification in three independent experiments (A, B, and C) are shown. (B) Up to six rounds of PMCAb in non-seeded NBHs (reactions A and B) or in NBHs seeded with 10 µl of 10% NBH prepared from 661 days old Syrian Hamsters (reactions C and D) were performed as negative controls. Undigested 10% NBH is provided as a reference.
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ppat-1001277-g004: Amplification of minute amounts of PrPSc in PMCAb.(A) 263K brain material was serially diluted 1010-, 1012-, or 1014-fold and subjected to sPMCAb amplification in the presence of 3 large beads for 6 rounds as indicated (each round consists of 48 sonication cycles, 10-fold dilutions were used for subsequent rounds). Amplification in three independent experiments (A, B, and C) are shown. (B) Up to six rounds of PMCAb in non-seeded NBHs (reactions A and B) or in NBHs seeded with 10 µl of 10% NBH prepared from 661 days old Syrian Hamsters (reactions C and D) were performed as negative controls. Undigested 10% NBH is provided as a reference.

Mentions: In previous studies, sPMCA of serially diluted 263K brain homogenate was used to determine the last dilution that still contained PrPSc particles [2]. Three out of four reactions seeded with 1012-diluted 263K brain material were found to be positive, while five to seven sPMCA rounds, each consisting of 144 cycles, were required to amplify 1012-diluted 263K to levels detectible by Western blotting [2]. To test the effectiveness of PMCAb in amplifying minute quantities of PrPSc, 263K brain homogenate was serially diluted up to 1014-fold and then amplified in sPMCAb, where each round consisted of 48 cycles. 1012-diluted 263K brain material was detected in 4 out of 8 reactions in the third round (Fig. 4A). An increase in number of rounds to six did not increase the percentile of positive reactions seeded with 1012-diluted 263K brain nor did it reveal any positive signals in reactions seeded with 1014-diluted 263K brain (Fig. 4A). 1010-diluted 263K brains showed a positive signal in all independent reactions (Fig. 4A). Non-seeded reactions or reactions seeded with NBH from old animals showed no positive signals in PMCAb (Fig. 4B). These results are consistent with the previous studies where brain material diluted 1012-fold detected PrPSc and showed stochastic behavior [2] consistent with a limiting dilution of the signal [24]. In the current experiments, PMCAb achieved the same level of sensitivity as PMCA in 1/7th of the time and with no evidence of spontaneous conversion from NHB substrate.


Highly efficient protein misfolding cyclic amplification.

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

Amplification of minute amounts of PrPSc in PMCAb.(A) 263K brain material was serially diluted 1010-, 1012-, or 1014-fold and subjected to sPMCAb amplification in the presence of 3 large beads for 6 rounds as indicated (each round consists of 48 sonication cycles, 10-fold dilutions were used for subsequent rounds). Amplification in three independent experiments (A, B, and C) are shown. (B) Up to six rounds of PMCAb in non-seeded NBHs (reactions A and B) or in NBHs seeded with 10 µl of 10% NBH prepared from 661 days old Syrian Hamsters (reactions C and D) were performed as negative controls. Undigested 10% NBH is provided as a reference.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037363&req=5

ppat-1001277-g004: Amplification of minute amounts of PrPSc in PMCAb.(A) 263K brain material was serially diluted 1010-, 1012-, or 1014-fold and subjected to sPMCAb amplification in the presence of 3 large beads for 6 rounds as indicated (each round consists of 48 sonication cycles, 10-fold dilutions were used for subsequent rounds). Amplification in three independent experiments (A, B, and C) are shown. (B) Up to six rounds of PMCAb in non-seeded NBHs (reactions A and B) or in NBHs seeded with 10 µl of 10% NBH prepared from 661 days old Syrian Hamsters (reactions C and D) were performed as negative controls. Undigested 10% NBH is provided as a reference.
Mentions: In previous studies, sPMCA of serially diluted 263K brain homogenate was used to determine the last dilution that still contained PrPSc particles [2]. Three out of four reactions seeded with 1012-diluted 263K brain material were found to be positive, while five to seven sPMCA rounds, each consisting of 144 cycles, were required to amplify 1012-diluted 263K to levels detectible by Western blotting [2]. To test the effectiveness of PMCAb in amplifying minute quantities of PrPSc, 263K brain homogenate was serially diluted up to 1014-fold and then amplified in sPMCAb, where each round consisted of 48 cycles. 1012-diluted 263K brain material was detected in 4 out of 8 reactions in the third round (Fig. 4A). An increase in number of rounds to six did not increase the percentile of positive reactions seeded with 1012-diluted 263K brain nor did it reveal any positive signals in reactions seeded with 1014-diluted 263K brain (Fig. 4A). 1010-diluted 263K brains showed a positive signal in all independent reactions (Fig. 4A). Non-seeded reactions or reactions seeded with NBH from old animals showed no positive signals in PMCAb (Fig. 4B). These results are consistent with the previous studies where brain material diluted 1012-fold detected PrPSc and showed stochastic behavior [2] consistent with a limiting dilution of the signal [24]. In the current experiments, PMCAb achieved the same level of sensitivity as PMCA in 1/7th of the time and with no evidence of spontaneous conversion from NHB substrate.

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