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. 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.

vector in a 3:1 molar ratio. Expressions and purifications were performed using the same protocols as for MRCV P9-1.

Plant material
Cultivated maize (Zea mays) samples were collected in Río Cuarto, Province of Córdoba, Argentina, where MRCV is endemic. Three fully developed leaves of symptomatic and asymptomatic maize plants were cut and kept in dry ice. Next, each sample was grinded with liquid nitrogen using a mortar and stored at -80 °C until use.

Direct ELISA for detection of recombinant MRCV P9-1
Plates were coated at 4 °C ON with 100 µl of P9-1 or P9-1 ∆C-arm in two-fold dilutions in triplicates.
After blocking residual protein binding sites with 5% skimmed milk, 100 µl of 1.25 ng/µl of Nb:AP dissolved in extraction buffer were added to each well and incubated at 37 °C for 1h. Finally, ELISAs were developed by adding 100 µl of 2 mg/ml of pNPP and absorbance was read at 405 nm.

Production of polyclonal antisera against MRCV P9-1
To raise polyclonal antibodies against P9-1, three guinea pigs received two subcutaneous injections with 53 µg of purified recombinant P9-1, one at day 0 with complete Freund's adjuvant, and the second at day 28 with incomplete Freund's adjuvant. The immune sera titres were monitored by direct ELISA at days 0, 16 and 34, while the antiserum was collected at day 40 after the first immunisation. The ELISAs were performed as indicated in the llama immunisation section. Sera from the three guinea pigs were pooled, used at two-fold serial dilutions from 1/800 to 1/51200, and detection was performed with 1/3000 dilution of goat anti-guinea pig-HRP (KPL, Sera Care, USA). For sera titration at day 40, four-fold serial dilutions from 1/3200 to 1/13107200 of each serum were employed.
Guinea pigs handling, inoculation, and sample collection were conducted by supervision of veterinarians under animal welfare protocols and guidelines approved by INTA Institutional Animal Care and use Committee IACUC 7 and in compliance with the ARRIVE guidelines 8 . All methods were carried out in accordance with relevant international guidelines and regulations.

Confocal imaging
Samples were analysed in a Nikon Eclipse CS1i confocal microscope using a Plan Apo VC 20.0x objective (AN 0.75). eGPF, in Nb1:eGFP, was excited with the 488-nm Argon laser line running at 10% and the emission was collected through DM 480 and BA 515/30 filters. For the observation of the Nb13:RFP, RFP was excited with a Helium-Neon (He-Ne) laser (543 nm) and the emission was collected through DM 545 and BA 605/75 filters. The laser scan for the acquisition of the definitive images was performed with a residence time of 5 µs and a pinhole opening of 30 µm. Bleeding between channels was avoided by acquiring the images in the "Frame lambda" mode of the EZ-C1 acquisition software (Silver Version 3.91). In this mode, each channel is acquired sequentially, starting with the lowest energy laser (He-Ne, in this case). This laser was also used to acquire images of the transmitted light channel.
In addition, "sham" samples were processed without Nb1:eGFP nor Nb13:RFP in order to subtract autofluorescence background. Therefore, the gain of each channel was set to a minimum with these samples and this configuration was used for the acquisition of all the images, with a resolution of