sPMCA analysis of ovine milk samples. Milk was clarified and seeded into brain homogenate from sheep unexposed to the scrapie agent. Samples underwent sPMCA, and products were digested with proteinase K before analysis of 10 μl of each sample. PrP was detected with monoclonal antibodies SHA31 and P4; molecular mass markers are indicated (kDa). Milk was sampled from animals not exposed to the scrapie agent (U), those displaying clinical signs of scrapie (C), or those exposed to a scrapie-positive farm environment but not displaying clinical disease (S). NS, non-seeded PMCA brain substrate subjected to identical sPMCA conditions at the same time as positive samples were analyzed. Samples from the four nonexposed animals were analyzed 18 to 20 times each by sPMCA. Samples from clinically affected or clinically normal scrapie-exposed animals were analyzed in triplicate. For this triplicate analysis of each sample, the sPMCA round at which samples became positive is indicated under the appropriate lane. n, negative at round 12. Each sample was PrP^Sc negative until the stated round and thereafter was positive.

Detection of protease-resistant PrP^Sc within CNS and mammary gland tissues of animals displaying clinical scrapie. Tissues were prepared as 10% or 40% (wt/vol) homogenates for spinal cord and mammary gland tissue, respectively, as described previously elsewhere (15). Native or proteinase K-digested homogenates (25 μg/ml; 1 h at 37°C) were analyzed as indicated. Protease-resistant PrP^Sc was readily detectable within spinal cord tissue (SC; lanes 1 to 2) but was not detectable within mammary gland samples (MG; lanes 3 to 6). Either 0.33 mg (0350/07) or 0.165 mg (0326/07 and 0344/07) of spinal cord tissue and 1.32 mg (lanes 3 and 4) and 6 mg (lane 5) of mammary gland tissue was analyzed per lane. Protease-resistant PrP^Sc was still undetectable from 20 mg of mammary gland tissue following precipitation with sodium phosphotungstic acid (25) prior to analysis (lane 6). PrP^Sc within scrapie-positive brain tissue (63 μg) was readily detected by this method after spiking into 20 mg of mammary gland homogenate (B, lane 7). Full-length and fragmented protease-sensitive PrP^C was readily detected within mammary gland tissue (lane 3). Animal numbers are indicated. PrP was detected with monoclonal antibody SHA31; molecular mass markers are indicated (kDa).