Sensitive detection of PrPSc by Western blot assay based on streptomycin sulphate precipitation.
Transmissible spongiform encephalopathies, also termed prion diseases, are fatal neurodegenerative disorders that affect both humans and animals, which are characterized by presences of protease-resistance disease-associated prion protein (PrP(Sc)) in brains. In the present study, we optimized the Western blot assay for PrP(Sc) with a precipitation procedure of streptomycin sulphate. After incubated with suitable amount of streptomycin sulphate, the detective sensitivity for PrP(Sc) was remarkably improved. The precipitation of PrP(Sc) was obviously influenced by pH value in the solution. Employs of PrP(Sc) stock sample into various mimic specimens, including normal hamster brain homogenate, human cerebrospinal fluid and urine, demonstrated that streptomycin precipitation markedly increased the detective sensitivity of PrP(Sc), regardless in low concentration or in large volume. In addition, the PrP(Sc) from a human brain tissue of familiar Creutzfeldt-Jakob disease (fCJD) was efficiently precipitated with streptomycin sulphate. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential, alone or combined with other techniques, to detect low levels of PrP(Sc) in the specimens not only from central nerve system, but also from peripheral organs or fluids.
Dong
C-F
CF
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention, Ying-Xin Rd 100, Beijing 100052, China.
Huang
Y-X
YX
An
R
R
Chen
J-M
JM
Wang
X-F
XF
Shan
B
B
Lei
Y-J
YJ
Han
L
L
Zhang
B-Y
BY
Han
J
J
Dong
X-P
XP
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D001921:Brain / Q000382:microbiology; D011232:Chemical Precipitation; D007562:Creutzfeldt-Jakob Syndrome / Q000175:diagnosis* / Q000473:pathology / Q000652:urine; D006224:Cricetinae; D006801:Humans; D018620:PrPSc Proteins / Q000235:genetics / Q000302:isolation & purification*; D011237:Predictive Value of Tests; D013307:Streptomycin / Q000737:chemistry
D018620:PrPSc Proteins; D013307:Streptomycin