Ultra-efficient Amplification of Abnormal Prion Protein by Modified Protein Misfolding Cyclic Amplification with Electric Current.

Prion diseases are clinically diagnosed and confirmed upon post-mortem histopathological examination of brain tissue. The only reliable molecular marker for prion diseases is abnormal prion protein (PrPSc), a pathologically conformed prion protein that primarily accumulates in the central nervous system and to a lesser extent in lymphoreticular tissues. However, the use of PrPSc as a marker for preclinical diagnoses is limited because the concentration of PrPSc in easily accessible body fluids is extremely low. Hence, one of the most promising approaches would be the development of an efficient in vitro amplification method for PrPSc. Indeed, protein misfolding cyclic amplification (PMCA) has become an important diagnostic tool for prion diseases. Here, we first describe a new superior PMCA device that employs electricity (referred to as ePMCA) to amplify PrPSc. The ePMCA device markedly improved the detection limit for PrPSc by amplifying trace amounts of pathogenic prion protein by applying electricity to improve PMCA. To increase the cavitation of sonication, a glass sample tube was used, and the upper side of the horn was shaped such that it had a curved cross-section. The ePMCA device enabled PrPSc to be amplified even from a sample seeded with 10-28-fold diluted 263K scrapie-infected brain homogenates with recombinant hamster prion protein (rHaPrP). In addition, the efficiency of prion amplification was best when 50 mM HEPES and 1% Triton X-100 were used as a PMCA conversion buffer in the various conditions that we applied. These results indicate that ePMCA would be very valuable for the rapid and specific diagnosis of human prion diseases and, thus, may provide a practically improved method for antemortem diagnoses using the body fluids of patients and animals with prion disease.