Due to the lack of a defined immune response or nucleic acid component of the infectious agent, approaches for transmissible spongiform encephalopathy (TSE) diagnosis rely upon methods of immunodetection including immunohistochemistry (IHC), Western blotting and enzyme-linked immunosorbent assay (ELISA)-based approaches for detection of the infectious agent. [1–3] Generally speaking, IHC relies upon formalin fixed paraffin embedded tissues, while Western blotting and ELISA utilize fresh or frozen tissues. Recently, methods have been reported that allow detection of PrPSc in formalin fixed tissues by Western blot [4–6]. Here we report an extension of this approach to allow ELISA-based detection of PrPSc in formalin-fixed paraffin-embedded tissues.
Tissue samples
This study utilized archived paraffin-embedded tissue samples from studies of scrapie in sheep, chronic wasting disease (CWD) in white-tailed deer (WTD) and transmissible mink encephalopathy (TME) in cattle as part of TSE research conducted at the National Animal Disease Center-USDA-ARS (Ames, IA). Animals were cared for and euthanized under National Animal Disease Center approved institutional animal care and use protocols. Samples were collected in 10% neutral buffered formalin prior to standard processing into paraffin blocks, with time in formalin ranging from 7 days to ~450 days. Previous studies of formalin fixed tissues report a marked sensitivity decrease for Western blots on tissues left in formalin for 2 or more years [6]. Based on this observation we limited our analysis to samples with fixation times less than 2 years.
Sample preparation
The method described here is an extension of previously published methods for Western blotting of formalin-fixed paraffin embedded samples differing only in the method for detecting PrPSc[4, 5]. As previously described, four 5 μm thick tissue sections from each paraffin block were collected into a 1.5 ml centrifuge. To each tube, 150 μl of 0.05 M Tris (pH 7.5), 1 mM EDTA, and 0.5% Tween 20 was added. The tube was placed at 100°C for 10 min and immediately placed into a dry ice ethanol slurry until frozen. The 10 min boil/freeze cycle was repeated once. The sample was brought to 100°C for an additional 10 min and immediately centrifuged at 3,000 × g for 10 min to separate the paraffin from the aqueous phase while also pelleting the tissue. In the event that the separation of paraffin was incomplete, the tube was reboiled for 10 min and the centrifugation step repeated. The aqueous layer including the tissue pellet was transferred to a clean 1.5 ml tube. At this point, the sample volumes were approximately 120 μl. Tissue disruption by sonication was done in 30 intervals of 40 sec with brief vortex mixing between sonication steps in a bath sonicator filled with ice water.
Sample Analysis
Following tissue disruption, 100 μL was removed and placed in a clean tube. From this 100 μl sample, a 20 μL sample was used to detect the presence of PrPSc using the IDEXX HerdChek Bovine Spongiform Encephalopathy-Scrapie Antigen Test Kit ELISA by incorporating the 20 μl sample in place of the 120 μl of tissue homogenate called for in the manufacturer's instructions. The remaining 80 μl was enriched by centrifugation at 186,000 × g for 55 minutes. The supernatant was removed and 20 μl analyzed using the HerdChek kit as described previously. The pellet was resuspended in 20 μl of 0.05 M Tris (pH 7.5), 1 mM EDTA, and 0.5% Tween 20 and analyzed as described above for both the supernatant and the unenriched sample.