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. 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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. 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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. Neverthe...

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 ...

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 ...

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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.