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Fast and ultrasensitive method for quantitating prion infectivity titre
Natallia Makarava1, Regina Savtchenko, Irina Alexeeva
1Center for Biomedical Engineering and Technology, University of Maryland, 725 W. Lombard Street, Baltimore 21201, USA.
Abstract:
Bioassay by end-point dilution has been used for decades for routine determination of prion infectivity titre. Here we show that the new protein misfolding cyclic amplification with beads (PMCAb) technique can be used to estimate titres of the infection-specific forms of the prion protein with a higher level of precision and in 3-6 days as opposed to 2 years, when compared with the bioassay. For two hamster strains, 263 K and SSLOW, the median reactive doses determined by PCMAb (PMCAb(50)) were found to be 10(12.8) and 10(12.2) per gram of brain tissue, which are 160- and 4,000-fold higher than the corresponding median infectious dose (ID(50)) values measured by bioassay. The 10(2)- to 10(3)-fold differences between ID(50) and PMCAb(50) values could be due to a large excess of PMCAb-reactive prion protein seeds with little or no infectivity. Alternatively, the differences between ID(50) and PMCAb(50) could be due to higher rate of clearance of infection-specific prion protein seeds in animals versus PMCAb reactions. A well-calibrated PMCAb reaction can be an efficient and cost-effective method for the estimation of infection-specific prion protein titre.
Insights
The protein misfolding cyclic amplification with beads (PMCAb) technique precisely estimates prion protein titres in days, significantly faster than traditional bioassays. This method offers a more efficient and cost-effective approach for prion disease research.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Prion infectivity titre determination traditionally relies on end-point dilution bioassays.
- These bioassays are time-consuming, often requiring up to two years for results.
Purpose of the Study:
- To evaluate the efficacy of the protein misfolding cyclic amplification with beads (PMCAb) technique for prion titre estimation.
- To compare the precision and speed of PMCAb with conventional bioassays.
Main Methods:
- Utilized the PMCAb technique to estimate prion titres in hamster brain tissue.
- Compared PMCAb-derived median reactive doses (PMCAb(50)) with bioassay-derived median infectious doses (ID(50)).
Main Results:
- PMCAb determined prion titres with higher precision and in 3-6 days, compared to the 2-year bioassay.
- For hamster strains 263K and SSLOW, PMCAb(50) values were 10(12.8) and 10(12.2) per gram, respectively.
- PMCAb(50) values were significantly higher (160- to 4,000-fold) than corresponding ID(50) values.
Conclusions:
- PMCAb is a precise, rapid, and potentially cost-effective method for estimating infection-specific prion protein titres.
- Discrepancies between PMCAb and bioassay titres may indicate an excess of non-infectious seeds or differential clearance rates.
