[Establishment of a protein misfolding cyclic amplification for PrPSc].

OBJECTIVE

To establish a methodology of protein misfolding cyclic amplification (PMCA) and utilize in the detection of PrP(Sc) in brain tissues from prion diseases.

METHODS

Different amounts of Scrapie 263K agent bulk were mixed with brain homogenates of health hamsters and treated with repeated incubation/sonication for 10 to 15 cycles. The proteinase K-resistant PrP(Sc) was evaluated with Western Blot.

RESULTS

In this experimental situation, 263K agent replicated rapidly in vitro, utilizing hamsters' brains as the medium. With the established PrP(Sc)-PMCA technique, PrP(Sc) signals in the preparations containing less than 10(-5) diluted 263K bulk could be detected. Compared with conveniently used immuno-blot assay, the sensitivity of PrP(Sc)-PMCA for PrP(Sc) was 10(5) to 10(6)-fold increased. It has been also shown that homogenates of cerebellar and brain stem could be used as the medium for PrP(Sc) replication.

CONCLUSION

A rapidly replicating method for PrP(Sc), PrP(Sc)-PMCA, was successfully established, providing a new approach for early diagnosis of prion diseases and research on the biological features of prion.