Evaluation of Western blotting methods using samples with or without sodium phosphotungstic acid precipitation for diagnosis of scrapie and chronic wasting disease

Abstract

The purpose of this study was to enhance the sensitivity of the Western blot (WB) test for use as an alternative and confirmatory method for the diagnosis of scrapie and chronic wasting disease (CWD) in Canada by comparing 2 sample preparation procedures: an abnormal prion protein (PrPSc) concentration procedure using sodium phosphotungstic acid (PTA) precipitation and a procedure using crude sample without precipitation. A total of 100 cerebrum samples (52 sheep and 48 elk), including 66 negative (31 sheep, 35 elk) and 34 positive (21 scrapie and 13 CWD positive) samples diagnosed by using immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN) and medulla oblongata at obex, were tested by using WB with the 2 sample preparation procedures. The WB using non-PTA enriched sample (crude extract) detected, on average, only 71.7% (9 of 15, 60.0% for scrapie, 5 of 6, 83.3% for CWD) of the samples that tested positive by using WB with PTA enriched samples. No case was positive by WB using crude extract but negative by WB using PTA enriched sample. No false positive was found. Serial dilution of PTA precipitated samples demonstrated that the technique increases the detection limit approximately 100 fold. Additionally, the comparison of the WB and IHC on cerebrum from all the positive cases demonstrated that WB following PTA precipitation and IHC had 100% agreement by detecting 6 positive for CWD on cerebrum; while IHC detected scrapie in only 14 out of 15 positive cerebrum samples by using WB following PTA precipitation. Phosphotungstic acid precipitation is therefore a useful adjunct to WB analysis of scrapie and CWD and tissues.

Figure 1

Effects of phosphotungstic acid (PTA) precipitation, dilution limit, using cerebral sample from a scrapie positive case. Lane 1, crude extract (PrP^Sc not detected); 2, original (100%) PTA precipitated sample; 3 to 5, serially (30%, 10%, and 1%, respectively) diluted from the original PTA enriched sample on lane 2.

Figure 2

Detection of scrapie PrP^Sc by Western blot (WB) of crude homogenates and phosphotungstic acid (PTA) precipitated samples from sheep sample 19 (lanes 1 to 3), sample 1 (lanes 4 to 6), sample 32 (lanes 7 to 9), sample 31 (lanes 10 to 12), and sample 30 (lanes 13 to 15). Samples were incubated without proteinase K (PK) (lanes 1, 4, 7, 10, 13), with PK as a crude extract (lanes 2, 5, 8, 11, 14) or with PTA precipitation after PK digestion (lanes 3, 6, 9, 12, 15). MW markers (arrows) 30 kDa and 20 kDa shown on left.

Figure 3

Demonstration of typical PrP^Sc band pattern by serial dilutions of a phosphotungstic acid (PTA) enriched scrapie positive sample. Lane 1, original (100%) PTA enriched sample; 2 to 4, PTA enriched, serially (50%, 25%, and 12.5%, respectively) diluted from the sample of lane 1. MW markers (arrows) 30 kDa and 20 kDa shown on left.

Figure 4

Detection of chronic wasting disease (CWD) PrP^Sc by Western blot (WB) of crude homogenates and phosphotungstic acid (PTA) precipitated samples from negative control elk sample 25 (lanes 2 to 4), positive sample 4 (lanes 5 to 7), and positive sample 7 (lanes 8 to 10), analyzed after proteinase K (PK) digestion and PTA precipitation (lanes 2, 5, 8), as crude homogenates (lanes 3, 6, 9) or without PK digestion or PTA precipitation (lanes 4, 7, 10). MW markers (arrows) 30 kDa and 20 kDa shown on left.