Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay.
The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.
Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, USA. smadsen@vetmed.wsu.edu
Sally A
SA
Madsen-Bouterse
Dongyue
D
Zhuang
Katherine I
KI
O'Rourke
David A
DA
Schneider
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000276:immunology; D019286:Endopeptidase K / Q000737:chemistry; D018604:Epitope Mapping; D015511:Goat Diseases / Q000175:diagnosis* / Q000276:immunology*; D006041:Goats; D051379:Mice; D008822:Mice, Transgenic; D018621:PrPC Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D018620:PrPSc Proteins / Q000032:analysis / Q000737:chemistry* / Q000276:immunology*; D017510:Protein Folding; D012608:Scrapie / Q000175:diagnosis* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D018621:PrPC Proteins; D018620:PrPSc Proteins; D019286:Endopeptidase K