We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

Animal work conducted at the USGS National Wildlife Health Center was performed in accordance with the NIH Office of Laboratory Animal Welfare guidelines and under institutional animal care and use committee protocol #EP080716. Tissues from hunter-harvested animals were gifts from the Wisconsin or Michigan Departments of Natural Resources.
1. Solution Preparation
NOTE: The solutions listed below are required for preparation of the CER assay substrate in Section 2 below. Prepare each of these solutions from higher concentration stock solutions; recommended concentrations for stock solutions will be given in parentheses after each respective chemical name listed. Prepare all solutions fresh upon substrate preparation and store at 4 &#176;C until used.
<ol>
	<li>For lysis buffer, prepare a solution of the following in 10 mM Tris, pH 7.5 (1 M Tris, pH 7.5 stock solution recommended): 100 mM sodium chloride (NaCl, 1 M stock solution), 10 mM ethylenediaminetetraacetic acid (EDTA, 500 mM EDTA, pH 8.0 stock solution), 0.5% NP-40 (10% stock solution), 0.5% deoxycholate (DOC, 10% stock solution).</li>
	<li>For conversion buffer, prepare a solution of 0.05% sodium dodecyl sulfate (SDS, 10% stock solution) and 0.5% Triton X-100 (10% stock solution) in 1 phosphate buffered saline (PBS, 10, pH 7.4 stock solution).</li>
	<li>For chaotropic solutions, prepare two 3 M solutions of guanidine hydrochloride (GdnHCl, 8 M stock) in 1x PBS (10x stock solution). Adjust the pH of one of the solutions to 3.5 &#177; 0.05 using concentrated hydrochloric acid (HCl). Check that the pH of the second solution remains at pH 7.4 &#177; 0.05 and adjust using concentrated HCl or sodium hydroxide (NaOH) if necessary.</li>
</ol>
2. Prepare CER Assay Substrate Pairs
<ol>
	<li>Obtain healthy, non-TSE-infected brain tissue from species of interest and prepare a 10% weight-per-volume (w/v) homogenate in lysis buffer using any of the following methods: dounce, bead-mill, or mortar and pestle homogenization. Further refine resulting homogenates by subjecting them to syringe-and-needle homogenization, using needles of increasing gauge until substrate can be aspirated and expelled with ease through a 27 G syringe needle.
	<ol>
		<li>For substrates prepared from rodents and other small mammals, homogenize whole brain(s); for substrates prepared from larger mammals, use obex (brainstem) tissue to prepare homogenates. When selecting the quantity of brain homogenate, prepare no more than 50% of the capacity of the centrifuge used for protein precipitation in step 2.6.</li>
		<li>If the quality of acquired brain tissue is questionable, assess PrPC levels by SDS-PAGE16 and immunoblot17 prior to substrate preparation.</li>
	</ol>
	</li>
	<li>Divide brain homogenate into two equal volumes in separate labelled plastic conical tubes. Add GdnHCl (3 M solutions in 1 PBS) at either pH 3.5 or 7.4 to each tube in a 1:1 volume ratio to brain homogenate.</li>
	<li>Check the pH values of the two solutions. Adjust the pH of the pH 3.5 substrate to pH &#177; 0.05 using concentrated HCl. Ensure the pH of the other substrate remains at pH 7.4 &#177; 0.05 and adjust pH if necessary with HCl or NaOH as needed. Avoid overshooting pH values by judicious addition of acid or base.</li>
	<li>Rotate solutions on an end-over-end mixer at room temperature (RT) for 5 hr.</li>
	<li>Precipitate protein by adding four volumes of methanol to the substrate solutions in each conical tube, vortex to mix well, and incubate at -20 &#176;C for 16-18 hr.</li>
	<li>Sediment samples by centrifugation at 13,000 &#215; g for 30 min at 4 &#176;C. Carefully decant and discard the supernatants and allow methanol to evaporate from the pellets. Use cotton-tipped applicators to assist in absorbing methanol clung to the inside of the tube. Do not allow pellets to over-dry and once methanol is no longer visible, proceed to the next step.</li>
	<li>Resuspend protein pellets in conversion buffer. Use a quantity of conversion buffer equal to the amount of brain homogenate starting material dispensed into each tube in step 2.2. 
	NOTE: It is critical that the conversion buffer used to resuspend protein pellets from both pH 3.5 and 7.4-treated substrate preparations is the solution defined in step 1.2 above (which contains low levels of detergents and a physiological pH).</li>
	<li>Briefly sonicate substrate solutions in a cuphorn sonicator (10 sec at ~30% maximal power), aliquot into 0.5 - 1.0 ml volumes in labeled 1.5 ml microcentrifuge tubes. Perform a quality control immunoblot (step 2.9) on an aliquot of each substrate. Store other aliquots at -80 &#176;C until ready to perform CER assay (Section 4).</li>
	<li>Perform quality control on CER substrate pairs prior to use (Figure 2). These steps ensure that CER substrates will provide optimal results in conversion studies.
	<ol>
		<li>Ensure comparable amounts of PrPC in the two substrates. Compare PrP levels in 10-25 &#181;l of each substrate by SDS-PAGE16 and immunoblotting17. If protein levels in the pH 7.4 and 3.5 substrates are within ~10% of each other, the substrate pairs are appropriate for use in CER studies. 
		NOTE: The PrP immunoblots should not show evidence of extensive PrPC degradation. The PrP immunoreactivity should be mainly &#62;20 kDa. Bands less than this molecular mass can be degradation products. Banding patterns should be roughly equivalent between substrate pairs.</li>
		<li>As the PrPC in both substrates should be PK sensitive, treat 10-25 &#181;l of each substrate with PK at a final concentration of 100 &#956;g/ml and digest samples at 1,000 rpm at 37 &#176;C for 1 hr in the thermo-shaker. Assess any remaining PrP signal by SDS-PAGE16 and immunoblotting17. Substrates with PrPres are not appropriate for use in CER studies.</li>
	</ol>
	</li>
</ol>
3. Prepare TSE Agent Seeds
<ol>
	<li>Obtain TSE-infected brain tissue from species of interest and prepare a 10% (w/v) homogenate in 1x PBS pH 7.4 using the methods listed in step 2.1 above. 
	NOTE: Seeds could also be produced from other TSE-infected tissues outside the central nervous system, however, efficiency of conversion of brain derived substrates by seeds derived from TSE-infected peripheral tissues has not yet been experimentally assessed in the published literature.</li>
	<li>Aliquot resulting homogenate(s) into 100 - 300 &#181;l volumes in 0.5 ml microcentrifuge tubes and store at -80 &#176;C until ready to perform the CER assay.</li>
</ol>
4. CER Assay
NOTE: The CER assay involves addition of a relatively small amount of PrPTSE (provided in the &#8220;seed&#8221;) from one species into a relative excess of PrPC (provided in the uninfected brain &#8220;substrate&#8221;) from another species that has been partially denatured at either pH 3.5 or 7.4. Following an extended shaking incubation period, template-driven conversion of PrPC by PrPTSE in each reaction is assessed by the densitometric signal of PrPres remaining after PK digestion and immunoblot detection. Comparing PrPres levels in the substrates previously denatured at pH 3.5 vs. 7.4 provides a measure of the species barrier. An experimental overview of this assay procedure is provided in Figure 1.
<ol>
	<li>Slowly thaw uninfected CER substrate pairs (assay requires equal volumes of substrates previously denatured at both pH 3.5 and 7.4) and TSE-infected seed solutions by placing them on top of a bed of ice (approximately 1 hr thaw time).</li>
	<li>Once fully thawed, aspirate and then expel each substrate solution through a 27 G syringe needle several times to re-homogenize it. Briefly sonicate each TSE-infected seed solution for 10 sec at ~30% maximal power. Keep all solutions on ice while preparing conversion reactions.</li>
	<li>Label 5 individual low binding, thin-walled PCR tubes per TSE agent being tested.</li>
	<li>Prepare conversion reactions in these tubes by adding 5 &#956;l of 10% TSE-infected seed solution to 95 &#956;l of (a) CER substrate prepared at pH 7.4 and (b) CER substrate prepared at pH 3.5.
	<ol>
		<li>For each experimental sample, prepare parallel reactions in CER substrate pairs previously denatured at both pH 3.5 and 7.4.</li>
		<li>Optimize ratios of TSE agent seed to uninfected brain substrate through empirical determination. Common seed:substrate ratios employed maintain a total reaction volume of 100 &#956;l and include 1:99 &#956;l, 2:98 &#956;l and 5:95 &#956;l. For most applications, the 5:95 &#956;l seed:substrate volume ratio is appropriate and is what is presented in this protocol.</li>
	</ol>
	</li>
	<li>Prepare three control samples per TSE agent being tested as follows: (a) add 5 &#181;l of 10% TSE-infected seed solution to 95 &#181;l conversion buffer as a control for input PrPres; add 5 &#181;l conversion buffer to 95 &#181;l substrate prepared at (b) pH 3.5 and (c) pH 7.4 as controls for non-specific, non-templated conversion.</li>
	<li>Briefly vortex each sample in its closed PCR tube to mix seed and substrate well. Follow with a momentary pulse in a low-speed bench top mini-centrifuge to remove any volume of the reaction mixture trapped in the PCR tube lid.</li>
	<li>Load samples in individually-labeled PCR tubes into a bench top thermo-shaker equipped to accept PCR tubes. Shake samples at 1,000 rpm at 37 &#176;C for 24 hr.</li>
	<li>Following shaking, add sarkosyl to a final concentration of 2% (w/v) and PK to a final concentration of 100 &#956;g/ml. Digest samples at 1,000 rpm at 37 &#176;C for 1 hr in the thermo-shaker. 
	NOTE: Addition of sarkosyl to samples post-conversion aids in PK digestion of PrPC. False positive signals in unseeded samples (Step 4.5) are virtually eliminated by addition of sarkosyl.</li>
	<li>Add SDS-PAGE sample buffer, heat samples to 95&#176;C for 5 minutes and resolve remaining PrPres in each sample by SDS-PAGE16 followed by immunoblotting17. 
	NOTE: After addition of sample buffer and heating, samples may be immediately processed or samples can be stored at -20 &#176;C or -80 &#176;C prior to immunoblotting. All data presented here were generated using the Novex NuPAGE gel and transfer systems. Individual samples from the CER assay were treated with sample buffer and reducing agent according to manufacturer&#8217;s instructions. Samples were subsequently heated at 95 &#176;C for 5 min and 30 &#181;l of each sample was loaded into a 12% bis-tris precast gels.</li>
	<li>Calculate the conversion efficiency ratio (CER) as a measure of species barrier. Measure levels of PrPres by densitometry17 and divide the total density of the pH 7.4 sample by that of the pH 3.5 sample. Multiply by 100 to produce a percentage.</li>
	<li>Compile data from independent experimental replicates to generate a mean CER &#177; standard deviation for a species with a given TSE agent.</li>
</ol>

<ol>
	<li>Colby, D. W., Prions Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cold Spring Harb Perspect Biol. 3 (1), a006833 (2011).</li><li>Hill, A. F., Collinge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+strains+and+species+barriers.">Prion strains and species barriers.</a> Contrib Microbiol. 11, 33-49 (2004).</li><li>Scott, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+expressing+hamster+prion+protein+produce+species-specific+scrapie+infectivity+and+amyloid+plaques.">Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques.</a> Cell. 59 (5), 847-857 (1989).</li><li>Prusiner, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenetic+Studies+Implicate+Interactions+between+Homologous+Prp+Isoforms+in+Scrapie+Prion+Replication.">Transgenetic Studies Implicate Interactions between Homologous Prp Isoforms in Scrapie Prion Replication.</a> Cell. 63, 673-686 (1990).</li><li>Telling, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+Propagation+in+Mice+Expressing+Human+and+Chimeric+Prp+Transgenes+Implicates+the+Interaction+of+Cellular+Prp+with+Another+Protein.">Prion Propagation in Mice Expressing Human and Chimeric Prp Transgenes Implicates the Interaction of Cellular Prp with Another Protein.</a> Cell. 83, 79-90 (1995).</li><li>Hill, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+same+prion+strain+causes+vCJD+and+BSE.">The same prion strain causes vCJD and BSE.</a> Nature. 389 (6650), 448-450 (1997).</li><li>Torres, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elements+modulating+the+prion+species+barrier+and+its+passage+consequences.">Elements modulating the prion species barrier and its passage consequences.</a> PLoS One. 9 (3), e89722 (2014).</li><li>Groschup, M. H., Buschmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+prion+diseases.">Rodent models for prion diseases.</a> Vet Res. 39 (4), 32 (2008).</li><li>Korth, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abbreviated+incubation+times+for+human+prions+in+mice+expressing+a+chimeric+mouse-human+prion+protein+transgene.">Abbreviated incubation times for human prions in mice expressing a chimeric mouse-human prion protein transgene.</a> Proc Natl Acad Sci U S A. 100 (8), 4784-4789 (2003).</li><li>Kocisko, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+specificity+in+the+cell-free+conversion+of+prion+protein+to+protease-resistant+forms:+a+model+for+the+scrapie+species+barrier.">Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.</a> Proc Natl Acad Sci U S A. 92 (9), 3923-3927 (1995).</li><li>Raymond, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+molecular+barrier+limiting+susceptibility+of+humans,+cattle+and+sheep+to+chronic+wasting+disease.">Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease.</a> EMBO J. 19 (7), 4425-4430 (2000).</li><li>Fernandez-Borges, N., de Castro, J., Castilla, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+of+the+transmission+barrier.">In vitro studies of the transmission barrier.</a> Prion. 3 (4), 220-223 (2009).</li><li>Zou, W. Q., Cashman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidic+pH+and+detergents+enhance+in+vitro+conversion+of+human+brain+PrPC+to+a+PrPSc-like+form.">Acidic pH and detergents enhance in vitro conversion of human brain PrPC to a PrPSc-like form.</a> J Biol Chem. 277 (46), 43942-43947 (2002).</li><li>Li, L., Coulthart, M. B., Balachandran, A., Chakrabartty, A., Cashman, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+barriers+for+chronic+wasting+disease+by+in+vitro+conversion+of+prion+protein.">Species barriers for chronic wasting disease by in vitro conversion of prion protein.</a> Biochem Biophys Res Commun. 364 (4), 796-800 (2007).</li><li>Morawski, A. R., Carlson, C. M., Chang, H., Johnson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+prion+protein+conversion+suggests+risk+of+bighorn+sheep+(Ovis+canadensis)+to+transmissible+spongiform+encephalopathies.">In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies.</a> BMC Vet Res. 9, 157 (2013).</li><li>Chandler, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encephalopathy+in+mice+produced+by+inoculation+with+scrapie+brain+material.">Encephalopathy in mice produced by inoculation with scrapie brain material.</a> Lancet. 1 (7191), 1378-1379 (1961).</li><li>Browning, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+prions+from+mule+deer+and+elk+with+chronic+wasting+disease+to+transgenic+mice+expressing+cervid+PrP.">Transmission of prions from mule deer and elk with chronic wasting disease to transgenic mice expressing cervid PrP.</a> J Virol. 78 (23), 13345-13350 (2004).</li><li>Hadlow, W. J., Race, R. E., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+sheep+and+goats+with+transmissible+mink+encephalopathy+virus.">Experimental infection of sheep and goats with transmissible mink encephalopathy virus.</a> Can J Vet Res. 51 (1), 135-144 (1987).</li><li>Rubenstein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+surveillance+and+antigen+conformation+determines+humoral+immune+response+to+the+prion+protein+immunogen.">Immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen.</a> J Neurovirol. 5 (4), 401-413 (1999).</li><li>Castilla, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+the+species+barrier+by+PrP(Sc)+replication+in+vitro+generates+unique+infectious+prions.">Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions.</a> Cell. 134 (5), 757-768 (2008).</li><li>Kubista, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+real-time+polymerase+chain+reaction.">The real-time polymerase chain reaction.</a> Mol Aspects Med. 27, 95-125 (2006).</li><li>Atarashi, R., Sano, K., Satoh, K., Nishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quaking-induced+conversion:+a+highly+sensitive+assay+for+prion+detection.">Real-time quaking-induced conversion: a highly sensitive assay for prion detection.</a> Prion. 5 (3), 150-153 (2011).</li><li>Ladner-Keay, C. L., Griffith, B. J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaking+Alone+Induces+De+Novo+Conversion+of+Recombinant+Prion+Proteins+to+beta-Sheet+Rich+Oligomers+and+Fibrils.">Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to beta-Sheet Rich Oligomers and Fibrils.</a> PLoS One. 9 (6), e98753 (2014).</li><li>Mathiason, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+domestic+cats+to+chronic+wasting+disease.">Susceptibility of domestic cats to chronic wasting disease.</a> J Virol. 87 (4), 1947-1956 (2013).</li><li>Seelig, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lesion+Profiling+and+Subcellular+Prion+Localization+of+Cervid+Chronic+Wasting+Disease+in+Domestic+Cats.">Lesion Profiling and Subcellular Prion Localization of Cervid Chronic Wasting Disease in Domestic Cats.</a> Vet Pathol. , (2014).</li><li>Stewart, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+predictions+of+prion+disease+susceptibility+in+carnivore+species+based+on+variability+of+the+prion+gene+coding+region.">Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.</a> PLoS One. 7 (12), e50623 (2012).</li><li>Russell, W. M., Burch, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. Metheun. , (1959).</li></ol>

The authors have nothing to disclose.

For successful completion of this protocol, attention should be paid to PrPC levels in uninfected brain tissue used for substrate preparation (step 2.1.2) and the seed to substrate ratio for conversion reactions (step 4.4.2). In our experience, brains can be extracted for use as CER substrate after a substantial period post-mortem, as long as PrPC is present by immunoblotting (Figure 4). In fact, some autolysis was observed in the brains of the bobcats used for CER studies. Nevertheless, incubation of substrates derived from these brains still resulted in the formation of PrPres. We speculate that, in part, the robustness of the CER assay is derived from the denaturation of CER substrates in 1.5 M GdnHCl, which will inactivate many proteases.
Seed to substrate ratios may need to be empirically determined to obtain acceptable PrPres signal by immunoblotting. Too much or too little PrPres to perform densitometry, high background PrPres levels in control samples lacking substrate and inconsistent conversion ratios are all reasons to optimize seed:substrate ratios. Variations maintain a total reaction volume of 100 &#956;l and include 1:99 &#956;l, 2:98 &#956;l and 5:95 &#956;l. On almost all occasions, we have found that 5:95 &#956;l seed:substrate volume ratio is optimal. No matter what ratio is employed, equal amounts of TSE agent are used and the assay is internally consistent. However, seed concentration of the prion-infected brain homogenate used to template reactions likely affects the outcome of the CER assay in that varying seed titers or dilutions could result in varying levels of PrPC-to-PrPres conversion. This effect has been demonstrated in other in vitro prion conversion assays22 and may complicate the comparison of different prion isolates. To address this in the CER assay, seeds could be titrated in each PrPC substrate prepared at pH 3.5 and 7.4 to identify a range of dilutions that template PrPC conversion. Ideal seed dilutions avoid saturating the solution with excess seed prions and yet provide insight into the sensitivity of the conversion reaction. Alternatively, PrPC-to-PrPres conversion in CER assay reactions could be measured at multiple, regular timepoints throughout the duration of the assay and plotted as rate-of-conversion curves, akin to the readouts for real-time polymerase chain reaction (qPCR)23 and real-time quaking induced conversion (RT-QuIC)24 assays. Such readouts could allow more comprehensive interpretations of species barriers and are an interesting focus of future development of this methodology.
In the protocol presented here, we use low binding, thin-walled PCR tubes, but we have also used standard 1.5 ml tubes in a thermo-shaker equipped for this type of tube. No other variations to the protocol are needed to use the larger sized tubes. In our experience, however, we have had more PrPres production and more reproducible results in low-binding PCR tubes compared to 1.5 ml tubes. While explanations for the improved performance of the assay in PCR tubes are only speculative at this time, based on results indicating that the air-water interface is critical for PrP fibrillization25, we hypothesize that the smaller tubes provide a more optimal surface tension for PrP conversion.
Perhaps the chief limitation to use of the CER assay is in understanding what conversion ratios represent in terms of in vivo species barriers determinants, such as disease penetrance or length of incubation period. While currently unknown, translation between in vitro and in vivo species barrier factors should become clearer with continued use of the CER assay in testing additional TSE species barriers that have already been established in vivo and will help to qualify results in species where bioassay data are not available. An advantage of the CER assay compared to PMCA is the presence of an internal control for PrP conversion, namely the pH 3.5 substrate, which can be converted by any TSE agent. This control is of value when it is unclear which, if any, TSE agents should cause misfolding of an exotic host&#8217;s PrPC. The inability of a given host substrate to amplify a specific TSE agent in PMCA could be interpreted as evidence of a species barrier or could be due to technical failings. Disadvantages of the CER assay compared to PMCA include limited amplification of PrPres, additional steps in CER substrate preparation and no known infectivity in the PrPres produced by CER reactions. It is to be noted that in a previous study, however, we found the CER assay resulted in a similar pattern of PrPres formation as that of PMCA15. The results presented here also indicate that the CER assay correctly predicts the species barriers for transmission of various TSEs to laboratory mice (Figure 3) and suggest that bobcats may have susceptibility to CWD (Figure 4), in agreement with reports that domestic cats (Felis catus) can acquire prion disease after experimental CWD challenge26,27. Studies using the CER assay could compliment PrP sequencing efforts to understand wildlife susceptibility to CWD28.
The CER assay enables rapid, low-cost, reliable assessment of TSE species barriers in vitro. While not a complete replacement for the &#8220;gold standard&#8221; of animal bioassay, the CER assay can be used as an initial assessment of the existence of a TSE species barrier that may justify or obviate further studies in living animals. For this reason, and because the assay relies only on brain tissue which can be acquired from non-experimental animals, the CER is in keeping with efforts to replace, reduce and refine animal experimental procedures29. In addition to assessing species barriers, the CER assay can also provide a simplified platform with which to investigate the mechanisms of the fundamental event defining prion disease biology: the conversion of normal, functional PrPC to a misfolded form.

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a group of fatal neurodegenerative diseases with extended incubation periods that affect a variety of animals and humans. The putative etiological agent of TSEs is comprised of a misfolded isomer of the host prion protein (PrP) that is capable of self-propagation by template-driven conversion of the normal cellular form of PrP (PrPC) into an infectious, disease-associated form (PrPTSE) that accumulates in central nervous system tissues of the infected host1. Infectious mammalian prions generally transmit from host-to-host in a species-specific manner, which has given rise to the concept of the &#8220;TSE species barrier&#8221; limiting interspecies transmission events2. The biological determinants of the prion species barrier are not well understood. Amino acid sequence similarity between the infectious PrPTSE and the host PrPC can strongly influence whether conversion takes place3-5, but remains insufficient to explain all prion transmission events observed in vivo6,7.
Thus, characterization of TSE species barriers has largely relied upon animal challenge studies: exposing na&#239;ve animals from a given species to prions from another and measuring the resulting incubation time to disease onset and attack rate as indicators of transmission efficiency. Mice expressing PrPC from heterologous species are also used for this type of study8. The costs associated with transgenic mouse production and protracted prion bioassays, as well as ethical considerations of animal use, are obstacles to experimental investigation of TSE species barriers. Assessment of the human species barrier to TSEs relies on mice engineered to express human PrPC. These mice require long incubation periods to succumb to human TSEs or modifications to the human PrPC molecule for rapid disease onset9. Incubation periods for non-human TSEs in these mice may extend beyond the normal mouse lifespan making interpretation of negative results challenging. Non-human primates have also been used as proxies for studying human species barriers, but these studies are fraught with the same challenges as other types of animal experimentation and non-human primates may not precisely recapitulate disease as it proceeds in the human host.
Animal bioassays remain the &#8220;gold standard&#8221; method for measuring the susceptibility of a species to a TSE, but the obstacles, costs and ethics of these live animal studies have compelled investigation into alternatives. A number of in vitro assays, based on assessing the conversion of host PrPC to a proteinase K (PK)-resistant state (PrPres) when seeded by PrPTSE, have been developed and used to investigate TSE species barriers10-12. Examples of in vitro assays include cell-free conversion assays, protein misfolding cyclic amplification (PMCA), and the conversion efficiency ratio (CER) assay10-14. While none of these assays take into account peripheral factors involved in species barriers after natural infection, all can be useful to identify potentially susceptible hosts for TSEs.
Here we present the protocol for the CER assay, in which two denatured PrPC substrates derived from normal brain homogenates are used in bench top prion conversion reactions (Figure 1)13. PrPC in the substrate denatured at pH 7.4 can only be converted to PrPres by PrPTSE seeded into the reaction in the absence of a species barrier14. In contrast, denaturation of PrPC in the other substrate at pH 3.5 allows it to be converted to PrPres following incubation with PrPTSE from any species and serves as a control for conversion. The ratio of conversion of PrPC to PrPres in pH 7.4 substrate relative to that of the pH 3.5 substrate provides a measure of the species barrier. We have found that the CER assay predicts known species barriers of laboratory mice to various TSEs and have used the assay in efforts to predict the species barrier of numerous mammalian species, including bighorn sheep, to chronic wasting disease (CWD) and other TSEs14,15. Investigators interested in a tool allowing rapid screening of TSE species barriers or assessment of PrPC-to-PrPres conversion will find this methodology useful.

<table border="0" cellpadding="0" cellspacing="0" width="944">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Name of Material/ Equipment</td>
			<td style="width:205px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:287px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">12% Bis-Tris SDS-PAGE gels</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0342</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">0.5 mm zirconium oxide beads</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">ZROB05&#160;</td>
			<td style="width:287px;">Other varieties of beads are also effective</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:296px;">Antibodies</td>
			<td style="width:205px;">various suppliers</td>
			<td style="width:157px;"></td>
			<td style="width:287px;">Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bead homogenizer</td>
			<td style="width:205px;">Next Advance</td>
			<td style="width:157px;">BBY24M</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Centrifuge&#160;</td>
			<td style="width:205px;">Beckman Coulter</td>
			<td style="width:157px;">369434</td>
			<td style="width:287px;">High speed with temperature control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Conical tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cotton-tipped applicator</td>
			<td style="width:205px;">Uline</td>
			<td style="width:157px;">S-18991</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Cuphorn sonicator</td>
			<td style="width:205px;">Heat Systems-Ultrasonics</td>
			<td style="width:157px;">W-380</td>
			<td style="width:287px;">Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">Densitometry software program</td>
			<td style="width:205px;">UVP</td>
			<td style="width:157px;">Vision Works LS Image Acquisition and Analysis software</td>
			<td style="width:287px;">Other programs, such as NIH ImageJ, will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dounce homogenizer</td>
			<td style="width:205px;">Kimble Chase</td>
			<td style="width:157px;">885300</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">End-over-end mixer</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">H5600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetra acetic acid</td>
			<td style="width:205px;">Boston Bioproducts</td>
			<td style="width:157px;">P-770</td>
			<td style="width:287px;">Hazardous chemical: eye irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine hydrochloride, 8M</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">24115</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating block</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">11-718</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">435570</td>
			<td style="width:287px;">Hazardous chemical: strong acid</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Lithium dodecyl sulfate sample buffer, 4X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0008</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; respiratory irritation, serious eye damage, flammable solid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">A454-4</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, flammable liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td style="width:205px;">any brand</td>
			<td style="width:157px;"></td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-centrifuge</td>
			<td style="width:205px;">Labnet International</td>
			<td style="width:157px;">C1301</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">N-lauroyl-sarcosine (sarkosyl)</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">L-5125</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Nonidet P-40</td>
			<td style="width:205px;">Amresco</td>
			<td style="width:157px;">M158</td>
			<td style="width:287px;">Hazardous chemical: skin irritation, eye damage</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:296px;">SDS-PAGE gel system</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NuPAGE electrophoresis system</td>
			<td style="width:287px;">Other SDS-PAGE systems will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">PCR tubes (low-binding)</td>
			<td style="width:205px;">Axygen</td>
			<td style="width:157px;">PCR-02-L-C</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Pestle homogenizer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">03-392-106</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">pH meter</td>
			<td style="width:205px;">Sentron</td>
			<td style="width:157px;">SI600</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polyvinyldifluoride membrane</td>
			<td style="width:205px;">Millipore</td>
			<td style="width:157px;">IPVH00010</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Proteinase K</td>
			<td style="width:205px;">Promega</td>
			<td style="width:157px;">V3021</td>
			<td style="width:287px;">Hazardous chemical: skin &#38; eye irritation, respiratory sensitisation, organ toxicity</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:296px;">Reducing agent for SDS-PAGE samples, 10X</td>
			<td style="width:205px;">Life Technologies</td>
			<td style="width:157px;">NP0009</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium Chloride</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">7647-14-5</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium deoxycholate</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">D6750</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Sodium dodecyl sulfate</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:157px;">28364</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium hydroxide</td>
			<td style="width:205px;">Sigma-Aldrich&#160;</td>
			<td style="width:157px;">S5881</td>
			<td style="width:287px;">Hazardous chemica; strong base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Syringe</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;">Use syringe size appropriate to volumes of substrate to be homogenized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Syringe needles</td>
			<td style="width:205px;">BD Biosciences</td>
			<td style="width:157px;">various</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Thermoshaker (PCR tube shaker)</td>
			<td style="width:205px;">Hangzhou All Sheng Instruments</td>
			<td style="width:157px;">MS-100</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Tris base</td>
			<td style="width:205px;">Bio Basic&#160;</td>
			<td style="width:157px;">77-86-1</td>
			<td style="width:287px;">Hazardous chemical: skin, eye, respiratory irritation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Triton X-100</td>
			<td style="width:205px;">Integra Chemical Company</td>
			<td style="width:157px;">T756.30.30</td>
			<td style="width:287px;">Hazardous chemical: acute toxicity, eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vortexer</td>
			<td style="width:205px;">Fisher Scientific</td>
			<td style="width:157px;">12-812</td>
			<td style="width:287px;"></td>
		</tr>
	</tbody>
</table>

assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Successful use of the CER assay is largely dependent upon the quality of the substrate pairs used in conversion reactions. For this reason, following procedures to prepare CER substrate pairs, a quality control immunoblot should be performed (Figure 2). For both substrates, assay 10-25 &#956;l by immunoblotting. PrPC should be easily detectable in each substrate and PrPC levels must be approximately the same between the two. Immunoreactivity will be mainly visible above 20 kDa, but smaller bands may also be evident. In our experience, the presence of smaller bands is species dependent. If lower molecular weight products are observed, their presence should be confirmed in both substrate pairs.
It is important to ensure that CER substrates are free from endogenous PrPres by treating substrates with PK (Figure 2). If PrPres is present, the animal used for substrate preparation may have been TSE-infected or subclinically-infected. Alternatively, the PrPC in the substrate may have misfolded to a PK-resistant state during denaturation or precipitation procedures. Independent from the reason, CER substrate containing PrPres is not acceptable for use in the conversion assay.
Laboratory mice are one of the most frequently used experimental animals in TSE research; as such, their susceptibility to most TSEs is well-characterized8, providing an in vivo benchmark against which to test the fidelity of the CER assay. Figure 3A presents results of CER assay conversion of PrPC from mouse (CD1 strain) substrate to PrPres by 1) the RML strain of mouse-passaged scrapie, an agent adapted to the mouse host18, 2) domestic sheep classical scrapie, an agent that can transmit to mice following a lengthy incubation period18, and 3) white-tailed deer (Odocoileus virginianus) CWD and 263K strain of hamster-passaged scrapie, agents to which mice are minimally or not susceptible19,20. Resulting PrPres levels were variable across mouse substrates denatured at pH 7.4 and seeded with the various TSE agents while PrPres was found in all substrates denatured at pH 3.5 and seeded with a TSE agent. Conversion ratios comparing the PrPres levels in the pH 7.4 and 3.5 substrates were independently calculated at least three times and means &#177; SD are shown in Figure 3B. For reactions seeded by RML, conversion ratios between pH 7.4 and 3.5 substrates were approximately 100%. For those seeded by scrapie, conversion in the pH 7.4 substrate was approximately 75% of that in the pH 3.5 substrate. CWD or 263K-induced conversion of the pH 7.4 substrate was minimal. A one-way analysis of variance could not distinguish a difference in the conversion ratios of RML and scrapie or CWD and 263K, however conversion ratios of RML and scrapie were significantly different from those of CWD and 263K.
The CER assay can be adapted for use with human tissues or with animal species where bioassays are too challenging or not ethical and transgenic mouse production is not desired. We have been using this assay to characterize the susceptibility of various wildlife species to CWD. As an example of the use of this assay, we present some preliminary results in Figure 4. Brains from hunter-trapped bobcats (Lynx rufus) were found to still contain detectable levels of PrPC and PrP breakdown products were not extensive, suggesting that these tissues could be an acceptable source for CER substrate (Figure 4A). Substrate pairs generated from bobcat brain showed PrP immunoreactivity of an appropriate molecular weight and did not contain endogenous PrPres (Figure 4B). When bobcat CER substrate pairs generated from two separate animals were incubated with the same white-tailed deer CWD agent used in the mouse CER assay described in Figure 3, we found substrates prepared at pH 7.4 had levels of PrPres compared to substrates prepared at pH 3.5 (Figure 4C), indicative of a minimal species barrier of bobcat PrPC to conversion by CWD. The CER for conversion of white-tailed deer PrPC by the same CWD isolate used to convert bobcat PrPC here, as a positive control, was previously reported as 95.2 &#177; 18.4% in Morawski et al. (2013)15.
<img alt="Figure 1" src="/files/ftp_upload/52522/52522fig1.jpg" width="450" /> 
Figure 1: Experimental overview of the CER assay. Two assay substrates are prepared by partially denaturing PrPC in non-prion infected brain tissue with chaotropic solutions at either pH 3.5 or pH 7.4. Substrates are seeded with PrPTSE from prion agent of interest and experimental samples are subjected to 24 hr incubation with shaking to allow PrPC to PrPTSE conversion in both pH 3.5 and 7.4 substrates. Following incubation, conversion is assessed by SDS-PAGE and immunoblotting. Signal densities are read by densitometry and the CER is calculated by the ratio of pH 7.4 to pH 3.5 signal densities for each experimental sample.
<img alt="Figure 2" src="/files/ftp_upload/52522/52522fig2.jpg" /> 
Figure 2: Quality control on CER substrates. (A) Prior to their use in the CER assay, mouse substrates prepared at either pH 7.4 or 3.5 are assayed by immunoblot in the 1) absence of PK treatment to assess PrPC content of each substrate pair (PrPC levels should be equivalent between pH 7.4 and 3.5 substrates for use in CER assay) and 2) presence of PK (100 &#181;g/ml) treatment to test for pre-existing PrPres in tissues used to prepare substrates (any substrates that display pre-existing PrPres are not suitable for use in the CER assay). (B) To test for non-specific PrPres formation during the conversion assay, mouse PrPC substrates prepared at either pH 7.4 or 3.5 are seeded with conversion buffer, shaken for 24 hr at 1,000 rpm, 37 &#176;C, and subsequently PK-treated (100 &#181;g/ml) and assayed for PrPres by immunoblotting (right two lanes). Non-shaken, non-PK treated mouse substrates represent total PrPC content in assay reactions (left two lanes). For (B), irrelevant lanes have been cropped for clarity, but each immunoblot panel derives from the same gel, membrane and exposure. Immunoblots in all panels used monoclonal antibody SAF 83.
<img alt="Figure 3" src="/files/ftp_upload/52522/52522fig3.jpg" /> 
Figure 3: CER assay using laboratory mouse substrate. (A) Mouse CER assay substrate prepared at either pH 7.4 or 3.5 was incubated with RML mouse-adapted scrapie, domestic sheep classical scrapie, white-tailed deer chronic wasting disease (CWD) or 263K strain of hamster-adapted scrapie in the CER assay. Control samples (labeled &#8220;none&#8221;) contained only an equal amount of infectious agent and no mouse substrate. Samples were analyzed for the presence of PK-resistant prion protein (PrPres) by immunoblot with monoclonal antibody SAF 83. Raw densitometric values for each sample are displayed below each lane. (B) Bar graph indicating the average ratios (&#177; standard deviation) between pH 7.4 and 3.5 mouse substrates for each infectious agent based on at least 3 independent assay runs. Lower-case letters refer to statistically homogenous subsets (analysis of variance with Tukey-Kramer minimum significance differences method; p &#60;0.05). This figure has been modified from Morawski et al. 201315.
<img alt="Figure 4" src="/files/ftp_upload/52522/52522fig4.jpg" /> 
Figure 4: Sample use of CER assay to investigate bobcat susceptibility to CWD. Bobcat brain homogenate (A) and CER assay substrate (B) was tested by immunoblotting in the absence and presence of PK (100 &#181;g/ml) to assess existing PrPC levels and the presence of pre-existing PrPres, respectively. (C) Bobcat substrates were seeded with conversion buffer and assayed in the CER assay to assess non-specific PrPres formation (left two lanes). Non-shaken, non-PK treated bobcat substrates represent total PrPC content in assay reactions (right two lanes). (D) Bobcat substrate prepared at either pH 7.4 or 3.5 was incubated with white-tailed deer CWD using the CER assay. The control sample (labeled &#8220;none&#8221;) contained an equal amount of CWD agent but no bobcat substrate. Raw densitometric values for each sample are displayed below each lane. Immunoblots in all panels used monoclonal antibody 3F4, which reacts with the feline prion protein21.

Watch this Scientific Journal Video about Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay at JoVE.com

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

assessing-transmissible-spongiform-encephalopathy-species-barriers

Nanyang Technological University

Research

JoVE Journal

Medicine

7.7K Views.  USGS National Wildlife Health Center. Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Video: Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Summary

Explore More Videos

Chapters in this video

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Assessing Specificity of Anticancer Drugs In Vitro

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research