Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection

Autores
Llauger, Gabriela; Monti, Demian Esteban; Aduriz Guerrero, Matías; Romão, Ema; Dumon, Analia Delina; Mattio, Maria Fernanda; Wigdorovitz, Andres; Muyldermans, Serge; Vincke, Cécile; Parreño, Viviana; Del Vas, Mariana
Año de publicación
2021
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.
Instituto de Biotecnología
Fil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Monti, Demian Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Monti, Demian Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Aduriz Guerrero, Matí­as. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina
Fil: Aduriz Guerrero, Matí­as. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Romão, Ema. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica
Fil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina
Fil: Dumon, Analia Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina
Fil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; Argentina
Fil: Wigdorovitz, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Muyldermans, Serge. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica
Fil: Muyldermans, Serge. Dalian University of Technology. School of Bioengineering. Liaoning Key Laboratory of Molecular Recognition and Imaging; China
Fil: Vincke, Cécile. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica
Fil: Vincke, Cécile. VIB Center for Inflammation Research. Myeloid Cell Immunology Lab; Bélgica
Fil: Parreño, Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; Argentina
Fil: Parreño, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fuente
Scientific Reports 11 : 20013 (Octubre 2021)
Materia
Nanobiotechnology
ELISA
Immunodiagnosis
Nanobiotecnología
Inmunodiagnóstico
Mal de Río Cuarto virus
Virus del Mal de Río Cuarto
Viroplasma
Viroplasm
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
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spelling Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetectionLlauger, GabrielaMonti, Demian EstebanAduriz Guerrero, MatíasRomão, EmaDumon, Analia DelinaMattio, Maria FernandaWigdorovitz, AndresMuyldermans, SergeVincke, CécileParreño, VivianaDel Vas, MarianaNanobiotechnologyELISAImmunodiagnosisNanobiotecnologíaInmunodiagnósticoMal de Río Cuarto virusVirus del Mal de Río CuartoViroplasmaViroplasmMal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.Instituto de BiotecnologíaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Monti, Demian Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Monti, Demian Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romão, Ema. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Dumon, Analia Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Wigdorovitz, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Muyldermans, Serge. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Muyldermans, Serge. Dalian University of Technology. School of Bioengineering. Liaoning Key Laboratory of Molecular Recognition and Imaging; ChinaFil: Vincke, Cécile. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Vincke, Cécile. VIB Center for Inflammation Research. Myeloid Cell Immunology Lab; BélgicaFil: Parreño, Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Parreño, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaSpringer Nature2022-08-24T15:41:02Z2022-08-24T15:41:02Z2021-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/12670https://www.nature.com/articles/s41598-021-99275-z2045-2322https://doi.org/10.1038/s41598-021-99275-zScientific Reports 11 : 20013 (Octubre 2021)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repograntAgreement/INTA/2019-PD-E4-I081-001/2019-PD-E4-I081-001/AR./Generación de reactivos, desarrollo de metodologías, validación y acreditación de ensayos para el diagnóstico de patógenos vegetales.info:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2025-09-29T13:45:41Zoai:localhost:20.500.12123/12670instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-29 13:45:42.08INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
title Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
spellingShingle Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
Llauger, Gabriela
Nanobiotechnology
ELISA
Immunodiagnosis
Nanobiotecnología
Inmunodiagnóstico
Mal de Río Cuarto virus
Virus del Mal de Río Cuarto
Viroplasma
Viroplasm
title_short Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
title_full Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
title_fullStr Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
title_full_unstemmed Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
title_sort Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
dc.creator.none.fl_str_mv Llauger, Gabriela
Monti, Demian Esteban
Aduriz Guerrero, Matías
Romão, Ema
Dumon, Analia Delina
Mattio, Maria Fernanda
Wigdorovitz, Andres
Muyldermans, Serge
Vincke, Cécile
Parreño, Viviana
Del Vas, Mariana
author Llauger, Gabriela
author_facet Llauger, Gabriela
Monti, Demian Esteban
Aduriz Guerrero, Matías
Romão, Ema
Dumon, Analia Delina
Mattio, Maria Fernanda
Wigdorovitz, Andres
Muyldermans, Serge
Vincke, Cécile
Parreño, Viviana
Del Vas, Mariana
author_role author
author2 Monti, Demian Esteban
Aduriz Guerrero, Matías
Romão, Ema
Dumon, Analia Delina
Mattio, Maria Fernanda
Wigdorovitz, Andres
Muyldermans, Serge
Vincke, Cécile
Parreño, Viviana
Del Vas, Mariana
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Nanobiotechnology
ELISA
Immunodiagnosis
Nanobiotecnología
Inmunodiagnóstico
Mal de Río Cuarto virus
Virus del Mal de Río Cuarto
Viroplasma
Viroplasm
topic Nanobiotechnology
ELISA
Immunodiagnosis
Nanobiotecnología
Inmunodiagnóstico
Mal de Río Cuarto virus
Virus del Mal de Río Cuarto
Viroplasma
Viroplasm
dc.description.none.fl_txt_mv Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.
Instituto de Biotecnología
Fil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Monti, Demian Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Monti, Demian Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Aduriz Guerrero, Matí­as. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina
Fil: Aduriz Guerrero, Matí­as. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Romão, Ema. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica
Fil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina
Fil: Dumon, Analia Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina
Fil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; Argentina
Fil: Wigdorovitz, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Muyldermans, Serge. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica
Fil: Muyldermans, Serge. Dalian University of Technology. School of Bioengineering. Liaoning Key Laboratory of Molecular Recognition and Imaging; China
Fil: Vincke, Cécile. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica
Fil: Vincke, Cécile. VIB Center for Inflammation Research. Myeloid Cell Immunology Lab; Bélgica
Fil: Parreño, Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; Argentina
Fil: Parreño, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.
publishDate 2021
dc.date.none.fl_str_mv 2021-10
2022-08-24T15:41:02Z
2022-08-24T15:41:02Z
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/12670
https://www.nature.com/articles/s41598-021-99275-z
2045-2322
https://doi.org/10.1038/s41598-021-99275-z
url http://hdl.handle.net/20.500.12123/12670
https://www.nature.com/articles/s41598-021-99275-z
https://doi.org/10.1038/s41598-021-99275-z
identifier_str_mv 2045-2322
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repograntAgreement/INTA/2019-PD-E4-I081-001/2019-PD-E4-I081-001/AR./Generación de reactivos, desarrollo de metodologías, validación y acreditación de ensayos para el diagnóstico de patógenos vegetales.
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer Nature
publisher.none.fl_str_mv Springer Nature
dc.source.none.fl_str_mv Scientific Reports 11 : 20013 (Octubre 2021)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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