A field-deployable diagnostic assay for the visual detection of misfolded prions

Peter R. Christenson; Manci Li; Gage Rowden; Marc D. Schwabenlander; Tiffany M. Wolf; Sang Hyun Oh; Peter A. Larsen

doi:10.1038/s41598-022-16323-y

A field-deployable diagnostic assay for the visual detection of misfolded prions

Peter R. Christenson, Manci Li, Gage Rowden, Marc D. Schwabenlander, Tiffany M. Wolf, Sang Hyun Oh, Peter A. Larsen

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

Diagnostic tools for the detection of protein-misfolding diseases (i.e., proteopathies) are limited. Gold nanoparticles (AuNPs) facilitate sensitive diagnostic techniques via visual color change for the identification of a variety of targets. In parallel, recently developed quaking-induced conversion (QuIC) assays leverage protein-amplification and fluorescent signaling for the accurate detection of misfolded proteins. Here, we combine AuNP and QuIC technologies for the visual detection of amplified misfolded prion proteins from tissues of wild white-tailed deer infected with chronic wasting disease (CWD), a prion disease of cervids. Our newly developed assay, MN-QuIC, enables both naked-eye and light-absorbance measurements for detection of misfolded prions. MN-QuIC leverages basic laboratory equipment that is cost-effective and portable, thus facilitating real-time prion diagnostics across a variety of settings. In addition to laboratory-based tests, we deployed to a rural field-station in southeastern Minnesota and tested for CWD on site. We successfully demonstrated that MN-QuIC is functional in a non-traditional laboratory setting by performing a blinded analysis in the field and correctly identifying all CWD positive and CWD not-detected deer at the field site in 24 h, thus documenting the portability of the assay. White-tailed deer tissues used to validate MN-QuIC included medial retropharyngeal lymph nodes, parotid lymph nodes, and palatine tonsils. Importantly, all of the white-tailed deer (n = 63) were independently tested using ELISA, IHC, and/or RT-QuIC technologies and results secured with MN-QuIC were 95.7% and 100% consistent with these tests for positive and non-detected animals, respectively. We hypothesize that electrostatic forces help govern the AuNP/prion interactions and conclude that MN-QuIC has great potential for sensitive, field-deployable diagnostics for CWD, with future potential diagnostic applications for a variety of proteopathies.

Original language	English (US)
Article number	12246
Journal	Scientific reports
Volume	12
Issue number	1
DOIs	https://doi.org/10.1038/s41598-022-16323-y
State	Published - Dec 2022

Bibliographical note

Funding Information:
Funding for research performed herein was provided by the Interdisciplinary Doctoral Fellowship from the University of Minnesota to P.R.C., the Minnesota State Legislature through the Minnesota Legislative-Citizen Commission on Minnesota Resources (LCCMR), Minnesota Agricultural Experiment Station Rapid Agricultural Response Fund, the Sanford P. Bordeau Chair in Electrical Engineering at the University of Minnesota to S.-H.O., and start-up funds awarded to P.A.L. through the Minnesota Agricultural, Research, Education, Extension and Technology Transfer (AGREETT) program. We thank C. Ertsgaard and D. J. Lee for helpful discussions on experimental results and protocols. Portions of this work were conducted in the Minnesota Nano Center, which is supported by the National Science Foundation through the National Nano Coordinated Infrastructure Network (NNCI) under Award Number ECCS-2025124. W. Zhang provided the expertise for TEM studies. Portions of this work were carried out in the University of Minnesota Characterization Facility, which receives partial support from the NSF through the MRSEC (Award Number DMR-2011401) and the NNCI (Award Number ECCS-2025124) programs. F. Schendel, T. Douville, and staff of the University of Minnesota Biotechnology Resource Center provided critical support concerning the large-scale production of recombinant proteins. We thank the Minnesota Department of Natural Resources, especially M. Carstensen, L. Cornicelli, E. Hildebrand, P. Hagen, and K. LaSharr, for providing the white-tailed deer tissues used for our analyses and logistical assistance for MN-QuIC field deployment. K. Wilson of the Colorado State University Veterinary Diagnostic Laboratory provided assistance with ELISA and IHC testing of samples reported herein. S. Stone provided valuable logistical assistance with our molecular work. We thank NIH Rocky Mountain Labs, especially B. Caughey, A. Hughson, and C. Orru for training and assistance with the implementation of RT-QuIC and for supplying the original rPrP clone. Figure and parts of Fig. were created using BioRender (BioRender.com).

Funding Information:
Funding for research performed herein was provided by the Interdisciplinary Doctoral Fellowship from the University of Minnesota to P.R.C., the Minnesota State Legislature through the Minnesota Legislative-Citizen Commission on Minnesota Resources (LCCMR), Minnesota Agricultural Experiment Station Rapid Agricultural Response Fund, the Sanford P. Bordeau Chair in Electrical Engineering at the University of Minnesota to S.-H.O., and start-up funds awarded to P.A.L. through the Minnesota Agricultural, Research, Education, Extension and Technology Transfer (AGREETT) program. We thank C. Ertsgaard and D. J. Lee for helpful discussions on experimental results and protocols. Portions of this work were conducted in the Minnesota Nano Center, which is supported by the National Science Foundation through the National Nano Coordinated Infrastructure Network (NNCI) under Award Number ECCS-2025124. W. Zhang provided the expertise for TEM studies. Portions of this work were carried out in the University of Minnesota Characterization Facility, which receives partial support from the NSF through the MRSEC (Award Number DMR-2011401) and the NNCI (Award Number ECCS-2025124) programs. F. Schendel, T. Douville, and staff of the University of Minnesota Biotechnology Resource Center provided critical support concerning the large-scale production of recombinant proteins. We thank the Minnesota Department of Natural Resources, especially M. Carstensen, L. Cornicelli, E. Hildebrand, P. Hagen, and K. LaSharr, for providing the white-tailed deer tissues used for our analyses and logistical assistance for MN-QuIC field deployment. K. Wilson of the Colorado State University Veterinary Diagnostic Laboratory provided assistance with ELISA and IHC testing of samples reported herein. S. Stone provided valuable logistical assistance with our molecular work. We thank NIH Rocky Mountain Labs, especially B. Caughey, A. Hughson, and C. Orru for training and assistance with the implementation of RT-QuIC and for supplying the original rPrP clone. Figure 1 and parts of Fig. 2 were created using BioRender (BioRender.com).

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© 2022, The Author(s).

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Research Support, U.S. Gov't, Non-P.H.S.

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title = "A field-deployable diagnostic assay for the visual detection of misfolded prions",

abstract = "Diagnostic tools for the detection of protein-misfolding diseases (i.e., proteopathies) are limited. Gold nanoparticles (AuNPs) facilitate sensitive diagnostic techniques via visual color change for the identification of a variety of targets. In parallel, recently developed quaking-induced conversion (QuIC) assays leverage protein-amplification and fluorescent signaling for the accurate detection of misfolded proteins. Here, we combine AuNP and QuIC technologies for the visual detection of amplified misfolded prion proteins from tissues of wild white-tailed deer infected with chronic wasting disease (CWD), a prion disease of cervids. Our newly developed assay, MN-QuIC, enables both naked-eye and light-absorbance measurements for detection of misfolded prions. MN-QuIC leverages basic laboratory equipment that is cost-effective and portable, thus facilitating real-time prion diagnostics across a variety of settings. In addition to laboratory-based tests, we deployed to a rural field-station in southeastern Minnesota and tested for CWD on site. We successfully demonstrated that MN-QuIC is functional in a non-traditional laboratory setting by performing a blinded analysis in the field and correctly identifying all CWD positive and CWD not-detected deer at the field site in 24 h, thus documenting the portability of the assay. White-tailed deer tissues used to validate MN-QuIC included medial retropharyngeal lymph nodes, parotid lymph nodes, and palatine tonsils. Importantly, all of the white-tailed deer (n = 63) were independently tested using ELISA, IHC, and/or RT-QuIC technologies and results secured with MN-QuIC were 95.7% and 100% consistent with these tests for positive and non-detected animals, respectively. We hypothesize that electrostatic forces help govern the AuNP/prion interactions and conclude that MN-QuIC has great potential for sensitive, field-deployable diagnostics for CWD, with future potential diagnostic applications for a variety of proteopathies.",

author = "Christenson, {Peter R.} and Manci Li and Gage Rowden and Schwabenlander, {Marc D.} and Wolf, {Tiffany M.} and Oh, {Sang Hyun} and Larsen, {Peter A.}",

note = "Funding Information: Funding for research performed herein was provided by the Interdisciplinary Doctoral Fellowship from the University of Minnesota to P.R.C., the Minnesota State Legislature through the Minnesota Legislative-Citizen Commission on Minnesota Resources (LCCMR), Minnesota Agricultural Experiment Station Rapid Agricultural Response Fund, the Sanford P. Bordeau Chair in Electrical Engineering at the University of Minnesota to S.-H.O., and start-up funds awarded to P.A.L. through the Minnesota Agricultural, Research, Education, Extension and Technology Transfer (AGREETT) program. We thank C. Ertsgaard and D. J. Lee for helpful discussions on experimental results and protocols. Portions of this work were conducted in the Minnesota Nano Center, which is supported by the National Science Foundation through the National Nano Coordinated Infrastructure Network (NNCI) under Award Number ECCS-2025124. W. Zhang provided the expertise for TEM studies. Portions of this work were carried out in the University of Minnesota Characterization Facility, which receives partial support from the NSF through the MRSEC (Award Number DMR-2011401) and the NNCI (Award Number ECCS-2025124) programs. F. Schendel, T. Douville, and staff of the University of Minnesota Biotechnology Resource Center provided critical support concerning the large-scale production of recombinant proteins. We thank the Minnesota Department of Natural Resources, especially M. Carstensen, L. Cornicelli, E. Hildebrand, P. Hagen, and K. LaSharr, for providing the white-tailed deer tissues used for our analyses and logistical assistance for MN-QuIC field deployment. K. Wilson of the Colorado State University Veterinary Diagnostic Laboratory provided assistance with ELISA and IHC testing of samples reported herein. S. Stone provided valuable logistical assistance with our molecular work. We thank NIH Rocky Mountain Labs, especially B. Caughey, A. Hughson, and C. Orru for training and assistance with the implementation of RT-QuIC and for supplying the original rPrP clone. Figure and parts of Fig. were created using BioRender (BioRender.com). Funding Information: Funding for research performed herein was provided by the Interdisciplinary Doctoral Fellowship from the University of Minnesota to P.R.C., the Minnesota State Legislature through the Minnesota Legislative-Citizen Commission on Minnesota Resources (LCCMR), Minnesota Agricultural Experiment Station Rapid Agricultural Response Fund, the Sanford P. Bordeau Chair in Electrical Engineering at the University of Minnesota to S.-H.O., and start-up funds awarded to P.A.L. through the Minnesota Agricultural, Research, Education, Extension and Technology Transfer (AGREETT) program. We thank C. Ertsgaard and D. J. Lee for helpful discussions on experimental results and protocols. Portions of this work were conducted in the Minnesota Nano Center, which is supported by the National Science Foundation through the National Nano Coordinated Infrastructure Network (NNCI) under Award Number ECCS-2025124. W. Zhang provided the expertise for TEM studies. Portions of this work were carried out in the University of Minnesota Characterization Facility, which receives partial support from the NSF through the MRSEC (Award Number DMR-2011401) and the NNCI (Award Number ECCS-2025124) programs. F. Schendel, T. Douville, and staff of the University of Minnesota Biotechnology Resource Center provided critical support concerning the large-scale production of recombinant proteins. We thank the Minnesota Department of Natural Resources, especially M. Carstensen, L. Cornicelli, E. Hildebrand, P. Hagen, and K. LaSharr, for providing the white-tailed deer tissues used for our analyses and logistical assistance for MN-QuIC field deployment. K. Wilson of the Colorado State University Veterinary Diagnostic Laboratory provided assistance with ELISA and IHC testing of samples reported herein. S. Stone provided valuable logistical assistance with our molecular work. We thank NIH Rocky Mountain Labs, especially B. Caughey, A. Hughson, and C. Orru for training and assistance with the implementation of RT-QuIC and for supplying the original rPrP clone. Figure 1 and parts of Fig. 2 were created using BioRender (BioRender.com). Publisher Copyright: {\textcopyright} 2022, The Author(s).",

year = "2022",

month = dec,

doi = "10.1038/s41598-022-16323-y",

language = "English (US)",

volume = "12",

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T1 - A field-deployable diagnostic assay for the visual detection of misfolded prions

AU - Christenson, Peter R.

AU - Li, Manci

AU - Rowden, Gage

AU - Schwabenlander, Marc D.

AU - Wolf, Tiffany M.

AU - Oh, Sang Hyun

AU - Larsen, Peter A.

N1 - Funding Information: Funding for research performed herein was provided by the Interdisciplinary Doctoral Fellowship from the University of Minnesota to P.R.C., the Minnesota State Legislature through the Minnesota Legislative-Citizen Commission on Minnesota Resources (LCCMR), Minnesota Agricultural Experiment Station Rapid Agricultural Response Fund, the Sanford P. Bordeau Chair in Electrical Engineering at the University of Minnesota to S.-H.O., and start-up funds awarded to P.A.L. through the Minnesota Agricultural, Research, Education, Extension and Technology Transfer (AGREETT) program. We thank C. Ertsgaard and D. J. Lee for helpful discussions on experimental results and protocols. Portions of this work were conducted in the Minnesota Nano Center, which is supported by the National Science Foundation through the National Nano Coordinated Infrastructure Network (NNCI) under Award Number ECCS-2025124. W. Zhang provided the expertise for TEM studies. Portions of this work were carried out in the University of Minnesota Characterization Facility, which receives partial support from the NSF through the MRSEC (Award Number DMR-2011401) and the NNCI (Award Number ECCS-2025124) programs. F. Schendel, T. Douville, and staff of the University of Minnesota Biotechnology Resource Center provided critical support concerning the large-scale production of recombinant proteins. We thank the Minnesota Department of Natural Resources, especially M. Carstensen, L. Cornicelli, E. Hildebrand, P. Hagen, and K. LaSharr, for providing the white-tailed deer tissues used for our analyses and logistical assistance for MN-QuIC field deployment. K. Wilson of the Colorado State University Veterinary Diagnostic Laboratory provided assistance with ELISA and IHC testing of samples reported herein. S. Stone provided valuable logistical assistance with our molecular work. We thank NIH Rocky Mountain Labs, especially B. Caughey, A. Hughson, and C. Orru for training and assistance with the implementation of RT-QuIC and for supplying the original rPrP clone. Figure and parts of Fig. were created using BioRender (BioRender.com). Funding Information: Funding for research performed herein was provided by the Interdisciplinary Doctoral Fellowship from the University of Minnesota to P.R.C., the Minnesota State Legislature through the Minnesota Legislative-Citizen Commission on Minnesota Resources (LCCMR), Minnesota Agricultural Experiment Station Rapid Agricultural Response Fund, the Sanford P. Bordeau Chair in Electrical Engineering at the University of Minnesota to S.-H.O., and start-up funds awarded to P.A.L. through the Minnesota Agricultural, Research, Education, Extension and Technology Transfer (AGREETT) program. We thank C. Ertsgaard and D. J. Lee for helpful discussions on experimental results and protocols. Portions of this work were conducted in the Minnesota Nano Center, which is supported by the National Science Foundation through the National Nano Coordinated Infrastructure Network (NNCI) under Award Number ECCS-2025124. W. Zhang provided the expertise for TEM studies. Portions of this work were carried out in the University of Minnesota Characterization Facility, which receives partial support from the NSF through the MRSEC (Award Number DMR-2011401) and the NNCI (Award Number ECCS-2025124) programs. F. Schendel, T. Douville, and staff of the University of Minnesota Biotechnology Resource Center provided critical support concerning the large-scale production of recombinant proteins. We thank the Minnesota Department of Natural Resources, especially M. Carstensen, L. Cornicelli, E. Hildebrand, P. Hagen, and K. LaSharr, for providing the white-tailed deer tissues used for our analyses and logistical assistance for MN-QuIC field deployment. K. Wilson of the Colorado State University Veterinary Diagnostic Laboratory provided assistance with ELISA and IHC testing of samples reported herein. S. Stone provided valuable logistical assistance with our molecular work. We thank NIH Rocky Mountain Labs, especially B. Caughey, A. Hughson, and C. Orru for training and assistance with the implementation of RT-QuIC and for supplying the original rPrP clone. Figure 1 and parts of Fig. 2 were created using BioRender (BioRender.com). Publisher Copyright: © 2022, The Author(s).

PY - 2022/12

Y1 - 2022/12

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AB - Diagnostic tools for the detection of protein-misfolding diseases (i.e., proteopathies) are limited. Gold nanoparticles (AuNPs) facilitate sensitive diagnostic techniques via visual color change for the identification of a variety of targets. In parallel, recently developed quaking-induced conversion (QuIC) assays leverage protein-amplification and fluorescent signaling for the accurate detection of misfolded proteins. Here, we combine AuNP and QuIC technologies for the visual detection of amplified misfolded prion proteins from tissues of wild white-tailed deer infected with chronic wasting disease (CWD), a prion disease of cervids. Our newly developed assay, MN-QuIC, enables both naked-eye and light-absorbance measurements for detection of misfolded prions. MN-QuIC leverages basic laboratory equipment that is cost-effective and portable, thus facilitating real-time prion diagnostics across a variety of settings. In addition to laboratory-based tests, we deployed to a rural field-station in southeastern Minnesota and tested for CWD on site. We successfully demonstrated that MN-QuIC is functional in a non-traditional laboratory setting by performing a blinded analysis in the field and correctly identifying all CWD positive and CWD not-detected deer at the field site in 24 h, thus documenting the portability of the assay. White-tailed deer tissues used to validate MN-QuIC included medial retropharyngeal lymph nodes, parotid lymph nodes, and palatine tonsils. Importantly, all of the white-tailed deer (n = 63) were independently tested using ELISA, IHC, and/or RT-QuIC technologies and results secured with MN-QuIC were 95.7% and 100% consistent with these tests for positive and non-detected animals, respectively. We hypothesize that electrostatic forces help govern the AuNP/prion interactions and conclude that MN-QuIC has great potential for sensitive, field-deployable diagnostics for CWD, with future potential diagnostic applications for a variety of proteopathies.

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A field-deployable diagnostic assay for the visual detection of misfolded prions

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