[Establishment of an assay for PrP(Sc) detection based on streptomycin precipitation].
To establish a new Western blotting assay for PrP(Sc) detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrP(Sc) was recovered by centrifugation. The enrichment of PrP(Sc) by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrP(Sc), forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrP(Sc) was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrP(Sc) in the specimens from central nerve system, or from peripheral organs or body fluids.
Dong
Chen-Fang
CF
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China. dcf466@yahoo.com.cn
Huang
Yin-Xia
YX
An
Run
R
Chen
Jian-Ming
JM
Wang
Xiao-Fan
XF
Shan
Bing
B
Lei
Yan-Jun
YJ
Han
Lu
L
Zhang
Bao-Yun
BY
Han
Jun
J
Dong
Xiao-Ping
XP
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D001921:Brain / Q000378:metabolism / Q000473:pathology; D001923:Brain Chemistry; D011232:Chemical Precipitation; D006224:Cricetinae; D018620:PrPSc Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017096:Prion Diseases / Q000175:diagnosis* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013307:Streptomycin / Q000737:chemistry*
D018620:PrPSc Proteins; D013307:Streptomycin