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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Published inScientific reports Vol. 11; no. 1; pp. 20013 - 13
Main Authors Llauger, Gabriela, Monti, Demián, Adúriz, Matías, Romão, Ema, Dumón, Analía Delina, Mattio, María Fernanda, Wigdorovitz, Andrés, Muyldermans, Serge, Vincke, Cécile, Parreño, Viviana, del Vas, Mariana
Format Journal Article
LanguageEnglish
Published London Nature Publishing Group UK 08.10.2021
Nature Publishing Group
Nature Portfolio
Subjects
631/1647/664/1257
631/1647/664/1467
631/1647/664/1881
631/1647/664/2089
631/1647/664/2228
631/1647/664/2229
631/326/2521
Alkaline phosphatase
Animals
Camelids, New World - immunology
Chromatography
Cloning
Corn
Disease
Disease transmission
E coli
Enzyme-linked immunosorbent assay
Enzyme-Linked Immunosorbent Assay - methods
Epidemiology
Escherichia coli - genetics
Humanities and Social Sciences
Immunologic Tests - methods
Immunology
Infections
multidisciplinary
Nanobodies
Phosphatase
Plant breeding
Plant Diseases - virology
Plants
Proteins
Recombinant Proteins - analysis
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Reoviridae - immunology
Reoviridae - isolation & purification
Reoviridae - metabolism
Science
Science (multidisciplinary)
Surface plasmon resonance
Vectors
Viral Proteins - analysis
Viral Proteins - biosynthesis
Viral Proteins - genetics
Viroplasm
Viruses
Zea mays - virology
Online AccessGet full text
ISSN2045-2322
2045-2322
DOI10.1038/s41598-021-99275-z

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Abstract 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.
AbstractList 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.
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.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.
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.
Abstract 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.
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.
ArticleNumber 20013
Author Mattio, María Fernanda
Llauger, Gabriela
Adúriz, Matías
Wigdorovitz, Andrés
Vincke, Cécile
del Vas, Mariana
Parreño, Viviana
Romão, Ema
Dumón, Analía Delina
Monti, Demián
Muyldermans, Serge
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BackLink https://www.ncbi.nlm.nih.gov/pubmed/34625580$$D View this record in MEDLINE/PubMed
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Snippet 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...
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...
Abstract 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...
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Alkaline phosphatase
Animals
Camelids, New World - immunology
Chromatography
Cloning
Corn
Disease
Disease transmission
E coli
Enzyme-linked immunosorbent assay
Enzyme-Linked Immunosorbent Assay - methods
Epidemiology
Escherichia coli - genetics
Humanities and Social Sciences
Immunologic Tests - methods
Immunology
Infections
multidisciplinary
Nanobodies
Phosphatase
Plant breeding
Plant Diseases - virology
Plants
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Recombinant Proteins - analysis
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Reoviridae - immunology
Reoviridae - isolation & purification
Reoviridae - metabolism
Science
Science (multidisciplinary)
Surface plasmon resonance
Vectors
Viral Proteins - analysis
Viral Proteins - biosynthesis
Viral Proteins - genetics
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Title Development of Nanobodies against Mal de Río Cuarto virus major viroplasm protein P9-1 for diagnostic sandwich ELISA and immunodetection
URI https://link.springer.com/article/10.1038/s41598-021-99275-z
https://www.ncbi.nlm.nih.gov/pubmed/34625580
https://www.proquest.com/docview/2580182511
https://www.proquest.com/docview/2580701217
https://pubmed.ncbi.nlm.nih.gov/PMC8501053
https://doaj.org/article/c0685c6872914794bdfc53f26ee2fc9c
Volume 11
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