Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection
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. Th...
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| Format: | Artículo |
| Language: | Inglés |
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Springer Nature
2022
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| Online Access: | 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 |
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| author | 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_browse | Aduriz Guerrero, Matías Del Vas, Mariana Dumon, Analia Delina Llauger, Gabriela Mattio, Maria Fernanda Monti, Demian Esteban Muyldermans, Serge Parreño, Viviana Romão, Ema Vincke, Cécile Wigdorovitz, Andres |
| 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_sort | Llauger, Gabriela |
| collection | INTA Digital |
| 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. |
| format | Artículo |
| id | INTA12670 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Springer Nature |
| publisherStr | Springer Nature |
| record_format | dspace |
| spelling | INTA126702022-08-24T15:44:43Z Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection 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 Nanobiotechnology ELISA Immunodiagnosis Nanobiotecnología Inmunodiagnóstico Mal de Río Cuarto virus Virus del Mal de Río Cuarto Viroplasma Viroplasm 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 2022-08-24T15:41:02Z 2022-08-24T15:41:02Z 2021-10 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 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 eng 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. 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) application/pdf Springer Nature Scientific Reports 11 : 20013 (Octubre 2021) |
| spellingShingle | Nanobiotechnology ELISA Immunodiagnosis Nanobiotecnología Inmunodiagnóstico Mal de Río Cuarto virus Virus del Mal de Río Cuarto Viroplasma Viroplasm 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 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 |
| 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_short | 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 rio cuarto virus major viroplasm protein p9 1 for diagnostic sandwich elisa and immunodetection |
| topic | Nanobiotechnology ELISA Immunodiagnosis Nanobiotecnología Inmunodiagnóstico Mal de Río Cuarto virus Virus del Mal de Río Cuarto Viroplasma Viroplasm |
| 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 |
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