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

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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.

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The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research

Nathalie Percie Du Sert, Viki Hurst, Amrita Ahluwalia … (2021)

Reproducible science requires transparent reporting. The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were originally developed in 2010 to improve the reporting of animal research. They consist of a checklist of information to include in publications describing in vivo experiments to enable others to scrutinise the work adequately, evaluate its methodological rigour, and reproduce the methods and results. Despite considerable levels of endorsement by funders and journals over the years, adherence to the guidelines has been inconsistent, and the anticipated improvements in the quality of reporting in animal research publications have not been achieved. Here, we introduce ARRIVE 2.0. The guidelines have been updated and information reorganised to facilitate their use in practice. We used a Delphi exercise to prioritise and divide the items of the guidelines into 2 sets, the “ARRIVE Essential 10,” which constitutes the minimum requirement, and the “Recommended Set,” which describes the research context. This division facilitates improved reporting of animal research by supporting a stepwise approach to implementation. This helps journal editors and reviewers verify that the most important items are being reported in manuscripts. We have also developed the accompanying Explanation and Elaboration (E&E) document, which serves (1) to explain the rationale behind each item in the guidelines, (2) to clarify key concepts, and (3) to provide illustrative examples. We aim, through these changes, to help ensure that researchers, reviewers, and journal editors are better equipped to improve the rigour and transparency of the scientific process and thus reproducibility.

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Nanobodies: natural single-domain antibodies.

Serge Muyldermans (2013)

Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine.

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Naturally occurring antibodies devoid of light chains.

C. Hamers-Casterman, T Atarhouch, S Muyldermans … (1993)

Random association of VL and VH repertoires contributes considerably to antibody diversity. The diversity and the affinity are then increased by hypermutation in B cells located in germinal centres. Except in the case of 'heavy chain' disease, naturally occurring heavy-chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains or cloned VH domains. Here we investigate the presence of considerable amounts of IgG-like material of M(r) 100K in the serum of the camel (Camelus dromedarius). These molecules are composed of heavy-chain dimers and are devoid of light chains, but nevertheless have an extensive antigen-binding repertoire, a finding that calls into question the role of light chains in the camel. Camel heavy-chain IgGs lack CH1, which in one IgG class might be structurally replaced by an extended hinge. Heavy-chain IgGs are a feature of all camelids. These findings open new perspectives in the engineering of antibodies.

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Author and article information

Contributors

Mariana del Vas: delvas.mariana@inta.gob.ar

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 8 October 2021

Publication date PMC-release: 8 October 2021

Publication date Collection: 2021

Volume: 11

Electronic Location Identifier: 20013

Affiliations

[1 ]Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CICVyA INTA, UEDD INTA/CONICET, Buenos Aires, Argentina

[2 ]INCUINTA, Instituto de Virología e Innovaciones Tecnológicas (IVIT), CICVyA INTA, UEDD INTA/CONICET, Buenos Aires, Argentina

[3 ]GRID grid.8767.e, ISNI 0000 0001 2290 8069, Lab of Cellular and Molecular Immunology, , Vrije Universiteit Brussel, ; Brussels, Belgium

[4 ]GRID grid.423606.5, ISNI 0000 0001 1945 2152, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), , Unidad de Fitopatología y Modelización Agrícola (UFYMA), ; Córdoba, Argentina

[5 ]GRID grid.419231.c, ISNI 0000 0001 2167 7174, Instituto Nacional de Tecnología Agropecuaria (INTA), Centro de Investigaciones Agropecuarias (CIAP), , Instituto de Patología Vegetal (IPAVE), ; Córdoba, Argentina

[6 ]GRID grid.30055.33, ISNI 0000 0000 9247 7930, Liaoning Key Laboratory of Molecular Recognition and Imaging, School of Bioengineering, , Dalian University of Technology, ; Dalian, People’s Republic of China

[7 ]GRID grid.510970.a, Myeloid Cell Immunology Lab, , VIB Center for Inflammation Research, ; Brussels, Belgium

Article

Publisher ID: 99275

DOI: 10.1038/s41598-021-99275-z

PMC ID: 8501053

PubMed ID: 34625580

SO-VID: 58f2e90e-0576-46ee-b0a5-71c76d5f3970

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 14 April 2021

Date accepted : 13 September 2021

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100003074, Agencia Nacional de Promoción Científica y Tecnológica;

Award ID: PICT 2017-2537

Award ID: PICT start up 2014-3754

Award Recipient : Gabriela Llauger Mariana del Vas

Funded by: FundRef http://dx.doi.org/10.13039/501100010677, Instituto Nacional de Tecnología Agropecuaria;

Award ID: I081

Award Recipient : Demián Monti

Custom metadata

ScienceOpen disciplines: Uncategorized

Keywords: antibody generation,antibody isolation and purification,elisa,immunoblotting,immunohistochemistry,immunoprecipitation,infectious-disease diagnostics

Data availability:

ScienceOpen disciplines: Uncategorized

Keywords: antibody generation, antibody isolation and purification, elisa, immunoblotting, immunohistochemistry, immunoprecipitation, infectious-disease diagnostics

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

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UCL: UN SDG 01 No Poverty

Most cited references 70

The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research

Nanobodies: natural single-domain antibodies.

Naturally occurring antibodies devoid of light chains.

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