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Updated: May 23, 2026
Protein Misfolding Cyclic Amplification of Prions
Published on: November 7, 2012
Highly efficient amplification of chronic wasting disease agent by protein misfolding cyclic amplification with beads
Chad J Johnson1, Judd M Aiken, Debbie McKenzie
1Environmental Chemistry and Technology Program and Department of Soil Science, University of Wisconsin, Madison, Wisconsin, United States of America.
Abstract:
Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7 × 10(-13) dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536(+/-) mice) allowed detection of CWD agent from the 10(-6) dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 10(5). Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.
Insights
Protein misfolding cyclic amplification (PMCA) with Teflon® beads and saponin significantly enhances detection of chronic wasting disease (CWD) agent. This improved method is over 100,000 times more sensitive than traditional transgenic mouse bioassays.
Area of Science:
- Neuroscience
- Biochemistry
- Veterinary Medicine
Background:
- Protein misfolding cyclic amplification (PMCA) is crucial for detecting pathogenic prion proteins in biological samples.
- The technique relies on the pathogenic prion protein's ability to convert normal prion proteins into a proteinase K-resistant form.
- Previous studies showed Teflon® beads improve PMCA sensitivity for specific prion strains.
Purpose of the Study:
- To evaluate the efficacy of Teflon® bead-included PMCA (PMCAb) combined with saponin for detecting the chronic wasting disease (CWD) agent.
- To assess the impact of PMCAb on the sensitivity, specificity, and robustness of CWD detection.
- To compare the sensitivity of PMCAb with traditional bioassays in transgenic mice.
Main Methods:
- Utilized Teflon® beads in the PMCA reaction (PMCAb) along with saponin to amplify the CWD agent.
- Conducted serial PMCAb reactions to determine the limit of detection for CWD agent in brain homogenate.
- Performed bioassays in transgenic mice (Tg(CerPrP)1536(+/-)) to quantify CWD agent in the same brain homogenate.
- Tested PMCAb on brain and lymph node samples from CWD-positive white-tailed deer.
Main Results:
- PMCAb with saponin dramatically increased CWD agent detection sensitivity without sacrificing specificity.
- Teflon® beads enhanced the robustness of the PMCA reaction, reducing result variability.
- Three rounds of serial PMCAb detected CWD agent from a 6.7 × 10(-13) dilution of brain homogenate (1.3 fg).
- Bioassay in transgenic mice detected CWD agent only up to a 10(-6) dilution.
- PMCAb demonstrated over 10^5-fold greater sensitivity than the transgenic mouse bioassay.
- CWD agent was successfully amplified from diverse tissues of white-tailed deer, including those with genetic resistance.
Conclusions:
- PMCAb with saponin is a highly sensitive and specific method for detecting the CWD agent.
- This enhanced PMCA technique surpasses the sensitivity of current transgenic mouse bioassays for CWD detection.
- PMCAb can effectively amplify CWD prions from various tissues and in animals with genetic variations influencing disease susceptibility.
