We would like to thank Dr. Vesper Fe Marie Ramos for critical reading of the manuscript. This work was supported by the National Center for Research Resources (P20 RR0115635-6, C06 RR17417-01 and G20RR024001) and the National Institute for Neurological Disorders and Stroke (2R01 NS052609).

1. Preparing the Equipment
<ol>
	<li>Use a Misonix 3000 or Misonix 4000 sonicator (Farmingdale, NY) connected to a Thermo Electron Neslab EX-7 water bath (Newington, NH) to keep a constant temperature of 37 &deg;C. Sonicate the samples in 200 &mu;l thin-walled PCR tube strips with domed caps obtained from Thermo Scientific (Waltham, MA).</li>
	<li>A brand new sonicator requires a "break-in" period of continuous operation9. A two-month break-in period consists of a 40-sec sonication burst and 10-minute incuba...

<ol>
	<li>Ayers, J. I., Schutt, C. R., Shikiya, R. A., Aguzzi, A., Kincaid, A. E., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strain-encoded+relationship+between+PrP+replication,+stability+and+processing+in+neurons+is+predictive+of+the+incubation+period+of+disease.">The strain-encoded relationship between PrP replication, stability and processing in neurons is predictive of the incubation period of disease.</a> PLoS pathogens. 7, e1001317 (2011).</li><li>Barria, M. A., Mukherjee, A., Gonzalez-Romero, D., Morales, R., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+generation+of+infectious+prions+in+vitro+produces+a+new+disease+phenotype.">De novo generation of infectious prions in vitro produces a new disease phenotype.</a> PLoS Pathog. 5, e1000421 (2009).</li><li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+physical+properties+of+the+prion+protein+from+two+strains+of+the+transmissible+mink+encephalopathy+agent.">Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.</a> J. Virol. 66, 2096-2101 (1992).</li><li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+PrP+properties+suggest+the+molecular+basis+of+strain+variation+in+transmissible+mink+encephalopathy.">Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy.</a> J. Virol. 68, 7859-7868 (1994).</li><li>Bessen, R. A., Marsh, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+biologically+distinct+strains+of+transmissible+mink+encephalopathy+in+hamsters.">Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters.</a> J. Gen. Virol. 73, 329-334 (1992).</li><li>Castilla, J., Gonzalez-Romero, D., Saa, P., Morales, R., De Castro, J., Soto, C. <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, 757-768 (2008).</li><li>Castilla, J., Morales, R., Saa, P., Barria, M., Gambetti, P., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+propagation+of+prion+strains.">Cell-free propagation of prion strains.</a> EMBO J. 27, 2557-2566 (2008).</li><li>Caughey, B., Raymond, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scrapie-associated+form+of+PrP+is+made+from+a+cell+surface+precursor+that+is+both+protease-+and+phospholipase-sensitive.">The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive.</a> J. Biol. Chem. 266, 18217-18223 (1991).</li><li>Deleault, N. R., Harris, B. T., Rees, J. R., Supattapone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+native+prions+from+minimal+components+in+vitro.">Formation of native prions from minimal components in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9741-9746 (2007).</li><li>Dickinson, A. G., Fraser, H., Meikle, V. M., Outram, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+between+different+scrapie+agents+in+mice.">Competition between different scrapie agents in mice.</a> Nat. New Biol. 237, 244-245 (1972).</li><li>Gonzalez-Romero, D., Barria, M. A., Leon, P., Morales, R., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+infectious+prions+in+urine.">Detection of infectious prions in urine.</a> FEBS Lett. 582, 3161-3166 (2008).</li><li>Green, K. M., Castilla, J., Seward, T. S., Napier, D. L., Jewell, J. E., Soto, C., Telling, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+high+fidelity+prion+amplification+within+and+across+prion+species+barriers.">Accelerated high fidelity prion amplification within and across prion species barriers.</a> PLoS Pathog. 4, e1000139 (2008).</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=Temporal+distribution+of+transmissible+mink+encephalopathy+virus+in+mink+inoculated+subcutaneously.">Temporal distribution of transmissible mink encephalopathy virus in mink inoculated subcutaneously.</a> J. Virol. 61, 3235-3240 (1987).</li><li>Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. T., Caughey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+formation+of+protease-resistant+prion+protein.">Cell-free formation of protease-resistant prion protein.</a> Nature. 370, 471-474 (1994).</li><li>Kurt, T. D., Telling, G. C., Zabel, M. D., Hoover, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-species+amplification+of+PrP(CWD)+and+correlation+with+rigid+loop+170N.">Trans-species amplification of PrP(CWD) and correlation with rigid loop 170N.</a> Virology. 387, 3235-3240 (2009).</li><li>Maddison, B. C., Baker, C. A., Terry, L. A., Bellworthy, S. J., Thorne, L., Rees, H. C., Gough, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+sources+of+scrapie+prions.">Environmental sources of scrapie prions.</a> J. Virol. 84, 11560-11562 (2010).</li><li>Nichols, T. A., Pulford, B., Wyckoff, A. C., Meyerett, C., Michel, B., Gertig, K., Hoover, E. A., Jewell, J. E., Telling, G. C., Zabel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protease-resistant+cervid+prion+protein+in+water+from+a+CWD-endemic+area.">Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.</a> Prion. 3, 171-183 (2009).</li><li>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=Novel+proteinaceous+infectious+particles+cause+scrapie.">Novel proteinaceous infectious particles cause scrapie.</a> Science. 216, 136-144 (1982).</li><li>Saborio, G. P., Permanne, B., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+detection+of+pathological+prion+protein+by+cyclic+amplification+of+protein+misfolding.">Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.</a> Nature. 411, 810-813 (2001).</li><li>Saunders, S. E., Bartz, J. C., Vercauteren, K. C., Bartelt-Hunt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+treatment+of+soil-bound+prions+effectively+inhibits+replication.">An enzymatic treatment of soil-bound prions effectively inhibits replication.</a> Appl. Environ. Microbiol. 77, 4313-4317 (2011).</li><li>Saunders, S. E., Shikiya, R. A., Langenfeld, K., Bartelt-Hunt, S. L., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+efficiency+of+soil-bound+prions+varies+with+soil+type.">Replication efficiency of soil-bound prions varies with soil type.</a> Journal of virology. , (2011).</li><li>Schutt, C. R., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+interference+with+multiple+prion+isolates.">Prion interference with multiple prion isolates.</a> Prion. 2, 61-63 (2008).</li><li>Shikiya, R. A., Ayers, J. I., Schutt, C. R., Kincaid, A. E., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-infecting+prion+strains+compete+for+a+limiting+cellular+resource.">Co-infecting prion strains compete for a limiting cellular resource.</a> Journal of. 84, 5706-5714 (2010).</li><li>Shikiya, R. A., Bartz, J. C. <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+generation+of+high+titer+prions.">In vitro generation of high titer prions.</a> Journal of virology. , (2011).</li><li>Weber, P., Giese, A., Piening, N., Mitteregger, G., Thomzig, A., Beekes, M., Kretzschmar, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+genuine+prion+infectivity+by+serial+PMCA.">Generation of genuine prion infectivity by serial PMCA.</a> Veterinary microbiology. 123, 346-357 (2007).</li></ol>

No conflicts of interest declared.

Challenges of amplifying infectious prion proteins are the long incubation periods and the expenses of in vivo experiments. The PMCA technique is a cost effective means to amplify infectious prion agents. Several laboratories have confirmed the ability of PMCA to accurately amplify prion strains in vitro 7, 9, 12, 14, 19,24 .
Prion diseases can be transmitted between species. Bessen and Marsh have effectively inoculated hamsters with transmissible mink encephalopat...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent / Equipment</td>
 <td>Manufacturer</td>
 <td>Cat. Number</td>
 </tr>
 <tr>
 <td>Misonix 3000</td>
 <td>Misonix</td>
 <td>S-3000</td>
 </tr>
 <tr>
 <td>Misonix 4000</td>
 <td>Misonix</td>
 <td>S-4000</td>
 </tr>
 <tr>
 <td>Tenbroeck Tissue Grinder</td>
 <td>Kontes</td>
 <td>885000-0007</td>
 </tr>
 <tr>
 <td>Neslab EX-7 Water Bath</td>
 <td>Thermo Electron</td>
 <td>Neslab EX-7</td>
 </tr>
 <tr>
 <td>0.2 ml PCR Tube Strips</td>
 <td>Thermo Scientific</td>
 <td>AB-0451</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma Aldrich</td>
 <td>T9284-100ML</td>
 </tr>
 <tr>
 <td>Complete Protease Inhibitor</td>
 <td>Roche</td>
 <td>11 697 498 001</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>J.T. Baker</td>
 <td>4040-00</td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Mallinckrodt Baker Mediatech</td>
 <td>21-031-CV</td>
 </tr>
 <tr>
 <td>Versi-Dry Lab Soakers</td>
 <td>Fisher Scientific</td>
 <td>14 206 28</td>
 </tr>
 <tr>
 <td>Repti Therm Heater</td>
 <td>Zoo Med Laboratories, Inc.</td>
 <td>RH-4</td>
 </tr>
 </tbody>
</table>

protein misfolding cyclic amplification of prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is used to amplify PrPSc in vitro7, 12, 14, 19, 24. A successful PrPSc amplification is shown by an increase in band intensity on Western blots of the PK-resistant prion protein (migrating between 19 and 30 kDa for hamster-derived prion strains) as shown in Figure 3. The increase in its band intensity after PMCA indicates amplification of the PK-resistant PrPSc material. Successful amplification of the ham...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

protein-misfolding-cyclic-amplification-of-prions

Research

JoVE Journal

Immunology and Infection

19.4K visualizações.  University of Nebraska at Lincoln. Proteína de amplificação cíclica misfolding (PMCA) é um ensaio in vitro em para o estudo da conversão de prião e as barreiras de tensão e espécies. Também pode ser utilizado como um ensaio de detecção de prião.