Improvement of PrPSc-detection in mouse spleen early at the preclinical stage of scrapie with collagenase-completed tissue homogenization and Sarkosyl-NaCl extraction of PrPSc.
Scrapie in sheep has recently become again a target of control measures and eradication programs. Crucial for the effectiveness of these measures is the detection of infected sheep during the long and potentially hazardous incubation period. However, routine-diagnosis is mostly limited to clinical examinations when disease becomes apparent, and to postmortem investigations. Through the detection of the scrapie-specific isoform of the prion protein (PrPSc) by Western blot in the spleen and lymph nodes from scrapie-infected mice and sheep, we have shown previously that diagnosis during the preclinical stage is possible. We introduce here an improved method for the diagnosis of mouse scrapie shortly after infection. Through a homogenization procedure that includes a collagenase digestion step, and through extraction and salting-out of PrPSc by Sarkosyl and NaCl, respectively, we were able to detect PrPSc in spleen tissue of intraperitoneally infected mice seven days postinfection. Moreover, the new protocol makes sample-handling easier and reduces the hands-on time. We also successfully enriched PrPSc from spleen tissue through immobilized metal affinity chromatography (IMAC); however, for the diagnosis at the earliest stage of infection, extraction of PrPSc by Sarkosyl and NaCl was more effective.
Grathwohl
K U
KU
Department of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
Horiuchi
M
M
Ishiguro
N
N
Shinagawa
M
M
D000818:Animals; D002846:Chromatography, Affinity; D017364:Collagenases / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D051379:Mice; D008813:Mice, Inbred ICR; D017830:Octoxynol / Q000737:chemistry; D018620:PrPSc Proteins / Q000302:isolation & purification*; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000737:chemistry; D012608:Scrapie / Q000209:etiology*; D012965:Sodium Chloride / Q000737:chemistry*; D013154:Spleen
D003902:Detergents; D018620:PrPSc Proteins; D012965:Sodium Chloride; C025231:sarkosyl; D017830:Octoxynol; D017364:Collagenases; D012521:Sarcosine