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Development of Nanobodies against Mal de Río Cuarto virus major viroplasm protein P9-1 for diagnostic sandwich ELISA
Gabriela Llauger1, Demián Monti1, Matías Adúriz2
1Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CICVyA INTA, UEDD INTA/CONICET, Buenos Aires, Argentina.
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.
Insights
Researchers developed llama-derived Nanobodies to detect Mal de Río Cuarto virus (MRCV) in maize. These Nanobodies offer a highly sensitive and specific diagnostic tool for MRCV, aiding disease management and research.
Area of Science:
- Plant Virology
- Immunotechnology
- Molecular Biology
Background:
- Mal de Río Cuarto virus (MRCV) causes significant damage to maize crops.
- MRCV replication and assembly occur in specialized structures called viroplasms.
- Effective diagnostic tools are needed for MRCV detection and management.
Purpose of the Study:
- To isolate and characterize llama-derived Nanobodies (Nbs) targeting the P9-1 protein, a key component of MRCV viroplasms.
- To develop a sensitive and specific diagnostic assay for MRCV detection using these Nanobodies.
- To explore the utility of these Nanobodies in studying MRCV structure and epidemiology.
Main Methods:
- Selection and affinity measurement of Nanobodies against recombinant P9-1 protein using surface plasmon resonance.
- Development of a sandwich ELISA assay utilizing Nanobody-alkaline phosphatase and Nanobody-eGFP fusions.
- Application of Nanobodies in pull-down assays and immunofluorescence microscopy of infected maize tissues.
Main Results:
- High-affinity Nanobodies (nanomolar range) were generated against MRCV P9-1.
- The developed sandwich ELISA demonstrated high diagnostic sensitivity (99.12%) and specificity (100%), with a low detection limit (0.236 ng/ml).
- Nanobodies successfully detected P9-1 in infected maize extracts and localized the virus within phloem cells.
Conclusions:
- Llama-derived Nanobodies provide a robust platform for MRCV diagnostics.
- These Nanobodies are valuable tools for epidemiological studies, maize breeding programs, and fundamental research on viroplasm biology.
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