Development of monoclonal antibodies against melon necrotic spot virus and their use for virus detection

Journal Pre-proof Development of monoclonal antibodies against melon necrotic spot virus and their use for virus detection ´ Manuel Miras, Covadonga Torre, Cristina Gomez-Aix, Yolanda Hernando, Miguel A. Aranda

PII:

S0166-0934(19)30209-5

DOI:

https://doi.org/10.1016/j.jviromet.2020.113837

Reference:

VIRMET 113837

To appear in:

Journal of Virological Methods

Received Date:

10 May 2019

Revised Date:

11 February 2020

Accepted Date:

11 February 2020

´ Please cite this article as: Miras M, Torre C, Gomez-Aix C, Hernando Y, Aranda MA, Development of monoclonal antibodies against melon necrotic spot virus and their use for virus detection, Journal of Virological Methods (2020), doi: https://doi.org/10.1016/j.jviromet.2020.113837

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Development of monoclonal antibodies against melon necrotic spot virus and their use for virus detection Manuel Miras1, Covadonga Torre2, Cristina Gómez-Aix2, Yolanda Hernando2 and Miguel A. Aranda1,* [email protected] 1

Center of Edaphology and Applied Biology of the Segura (CEBAS)-CSIC, Department of

Stress Biology and Plant Pathology, PO Box 164, 30100 Espinardo, Murcia, Spain

Abiopep S.L., R&D Department, Parque Científico de Murcia, Ctra. de Madrid, Km 388,

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2

Complejo de Espinardo, Edf. R, 2º, 30100 Espinardo, Murcia, Spain

*

Seven hybridoma cell lines secreting specific mAbs against the melon necrotic spot

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

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Correspondence: (M.A.A.); tel.: +34 968396200 Ext 6355

virus (MNSV) coat protein were obtained.

mAb 2D4H4 was purified and showed high specificity and reactivity to three different

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

MNSV isolates in Western blots. A sensitive TAS-ELISA protocol for the routine detection of MNSV was developed.

mAb 2D4H4 was used to localize MNSV CP in infected cells by TEM

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immunocytochemistry, illustrating the usefulness of mAb 2D4H4 for advanced cellular

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biology studies.

ABSTRACT Melon necrotic spot virus (MNSV) is endemic in cucurbit crops worldwide, causing epidemic outbreaks from time to time. MNSV is transmitted in nature by a soil-inhabiting fungus and also through seeds, making its detection in seed certification programs a necessity. Polyclonal

antisera and RT-PCR-based detection assays have been developed for MNSV, but up to now no monoclonal antibodies (mAbs) have been described for this virus. In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10-3 - 10-4 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as IgG2a and kappa light-chain. Western-blot

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analyses showed that mAb 2D4H4 provided sensitive and specific detection of MNSV. A TASELISA protocol was developed for mAb 2D4H4. Using this protocol, limits of detection of 1:20,480 and 1:10,240 (g/mL, w/v) were attained for the homologous isolate and a

heterologous MNSV isolate, respectively. Moreover, mAb 2D4H4 was used successfully to localize the MNSV CP in infected cells by immunocytochemistry/transmission electron

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microscopy, illustrating the usefulness of this mAb for advanced cellular studies.

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Keywords: cucurbit; detection; diagnosis; immunocytochemistry; melon; TAS-ELISA.

1. Introduction

Melon necrotic spot virus (MNSV) is a carmovirus within the family Tombusviridae (Hibi and

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Furuki, 1985; Riviere and Rochon, 1990) that is endemic in cucurbit crops worldwide (Lecoq and Desbiez, 2012). MNSV has a small isometric particle (30 nm in diameter) which encapsidates a single-stranded (+)-sense, uncapped RNA genome of 4.3 kb that lacks a poly(A)

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tail and encodes at least five open reading frames (ORFs) (Riviere and Rochon, 1990). The 5’end ORF encodes a protein of 29 kDa (p29) terminating with an amber codon, with its read-

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through resulting in a larger gene product of 89 kDa (p89), with both being essential for MNSV replication. ORF 3 and 4 code for two small polypeptides, each about 7 kDa (p7A and p7B), that are involved in cell-to-cell movement. Lastly, the 3’-proximal ORF encodes the 42 kDa coat protein (CP) (Genoves, Navarro, and Pallas, 2006; Riviere et al., 1989; Riviere and Rochon, 1990). MNSV can be transmitted mechanically, by the zoospores of the fungus Olpidium bornovanus and through seeds (Campbell and Sim, 1994; Herrera-Vásquez et al., 2010; Lange and Insunza, 1977). Characteristic symptoms include local necrotic spots on

leaves, and necrosis on stems and petioles and fruit size reduction (Matsuo et al., 1991). The MNSV host range is rather narrow, affecting only cucurbits, although isolates able to infect plants outside the family Cucurbitaceae have been reported (Díaz et al., 2004). Recently, MNSV outbreaks have been reported in several countries, including Spain, United States, China, Canada and Brazil (Li et al., 2015; Marine et al., 2017; Miras et al., 2014; Moura et al., 2018; Wu et al., 2016). Several diagnostic methods have been developed for MNSV, including polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), nucleic acid hybridization and InmunoStrip® (Lecoq and Desbiez, 2012). Plant pathogen diagnostic

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companies have developed polyclonal antibodies (pAb) with high sensitivity and specificity against the MNSV virions, but to our knowledge, no monoclonal antibodies (mAbs) against MNSV have been produced and/or commercialized yet, in spite of their theoretical higher

sensitivity and specificity as compared to pAbs. In this study, a mAb against the CP of MNSVMα5 was produced in BALB/c mice and its performance was evaluated with various detection

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techniques such as enzyme-linked immunosorbent assay (ELISA), Western blot and

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immunocytochemistry/transmission electron microscopy (TEM).

2. Materials and methods

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2.1. Virus sources

MNSV-Mα5, MNSV-264, MNSV-N and Moroccan watermelon mosaic virus (MWMV) were collected and characterized previously (Díaz et al., 2004; Miras, Juárez, and Aranda, 2019)

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2.2. Expression and purification of the MNSV-Mα5 recombinant coat protein The strategy used to produce recombinant MNSV CP was essentially similar to that used by Liu et al. (2017). The CP coding sequence was PCR-amplified from plasmid L2-T7 (Nieto et

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al., 2006) and cloned into the NdeI/HindIII sites of the pET30a vector (Novagen, UK). The E. coli strain BL21 Star (D3) was transformed with the recombinant plasmid. A single colony was

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used to inoculate LB medium containing kanamycin and cultured at 37 ºC. When the OD600 reached 1.2, the cell culture was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 15 ºC for 16 h. Cells were harvested by centrifugation and the pellet was resuspended in Nickel buffer A (25 mM HEPES pH 7.5, 400 mM NaCl, 20 mM imidazole, 10 % glycerol) supplemented with DNase I (Roche) and protease inhibitor cocktail EDTA-free (Roche) followed by sonication. The precipitate after centrifugation was dissolved using urea. The denatured protein in the supernatant after centrifugation was purified by affinity chromatography using HisTrap 5 mL (GE, Healthcare) according to the manufacturer´s

instructions. The target protein was buffer exchanged and refolded by gel filtration, sterilized through a 0.22 µm filter and then stored at -80 ºC. The concentration of the purified protein was determined by the BCA protein assayTM (ThermoFisher) with bovine serum albumin (Sigma) as the standard. The protein purity and molecular weight were determined by SDS-PAGE along with Western blot for confirmation. 2.3. Preparation of mAbs against the recombinant CP mAbs were produced using standard methods (Dunbar and Skinner, 1990; Li et al., 2015; Wu et al., 2014). Briefly, the purified MNSV recombinant CP was used as an immunogen and injected into five 6 weeks old BALB/c mice. Production of hybridoma secreting mAbs against

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the MNSV-CP was performed according to Kohler and Milstein (1975) with minor modifications. Hybridoma supernatants were screened for the presence of anti-CP antibodies by indirect ELISA in 96-well plates. Positive hybridoma clones were cultured in Dulbecco´s

Modified Eagle´s Medium (DMEM) high glucose (WISENT Bioproducts), supplemented with 10 % fetal bovine serum (WISENT Bioproducts) and 1 % Penicillin-Streptomycin (Sigma).

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Cells were allowed to grow at 37 ºC and supplemented with 5.5 % of CO2.

The hybridoma were then injected intraperitoneally into pristine-primed syngeneic

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BALB/c mice to produce ascites. After 7–10 days, the ascites samples were collected and their titres were determined by indirect ELISA. mAbs isotypes were determined using an isotyping

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kit as per the manufacturer’s instructions (Sigma-Aldrich). Anti-CP IgG was purified from ascites with an immobilized protein-G affinity column (GE Healthcare, Bucks, UK) according to the manufacturer’s manual. Purified antibodies were stored at −80 °C.

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2.4. Indirect and TAS-ELISA

Indirect ELISA experiments were conducted as described elsewhere (Crowther, 2000). Briefly, plates were coated with recombinant MNSV-CP protein in phosphate-buffered saline (PBS) (1

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μg/mL and 100 μL/well) for 12 h at 4 °C. After three washes with PBS with 0.1 % Tween 20 (PBST), 100 μL/well of the hybridoma cell line culture described above were added and

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incubated for 2 h at 37 °C. Lastly, peroxidase-AffiniPure Goat Anti-Mouse IgG was added and incubated for 30 min at 37 °C. The enzymatic activity was measured by adding 100 μL of substrate and incubating the plate for 15 min, after which the absorbance was read at 405 nm. The His-tag protein was included as a negative control. Sap extracts from healthy and MNSV-infected melon plants were prepared by grinding leaf tissues in extraction buffer (PBST, 2 % PVP). Ninety-six-well plates were coated with diluted (1:200) commercial polyclonal anti-CP of MNSV (DSMZ GmbH, Braunschweig, Germany). After washing three times with PBST, crude extracts were added (100 μL/well) for

12 h at 4 °C. After three washes with PBST, the CP-mAb was added and incubated at 37 °C for 4 h (100 μL/well). After the plates were washed with PBST, 100 μL/well of an anti-mouse polyclonal antibody conjugated with alkaline phosphatase (anti-mouse IgG/AP) (SigmaAldrich, St. Louis, Missouri, USA) was added and incubated for 4 h at 37 °C. Finally, after three washes with PBST, 100 μL of substrate was added to each well and incubated for 90 min at 37 °C, and then the absorbance at 405 nm was measured. The threshold absorbance value (cut-off) for considering a sample as positive was established at 2.5 times the absorbance of the negative control. The optimal working concentrations of the CP-mAb and the anti-mouse IgG/AP were determined by a checkerboard titration test as described before (Crowther, 2000).

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2.5. Western blot Expressed recombinant CP and total protein extracts (extraction buffer: 0.1 M Tris HCl pH 9.0, 0.1 M NaCl, 5 M urea, 10 mM EDTA, 0.1 M β-mercaptoethanol) from mock and MNSVinfected leaves were separated with a 12 % SDS-PAGE and blotted onto nitrocellulose

membranes (GE Amersham) for 1 h at 100 mA. After blocking, membranes were incubated in

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hybridoma cultures or in purified CP-mAb at a dilution of 1:1 and 1:3000, respectively. Blots were incubated in Agrisera® matching secondary antibody (anti-mouse IgG conjugated)

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diluted to 1:25000 in tris-PBST for 1 h at room temperature with agitation. Membranes were developed for 2 min with SuperSignalTM West Pico PLUS (ThermoFisher).

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2.6. Immunocytochemistry electron microscopy

Immunocytochemistry was performed as describe previously (Gomez-Aix et al., 2015). Briefly, tissue from healthy melon plants and MNSV lesions from infected melon plants were sampled

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and fixed in 0.5 % of glutaraldehyde in 0.1 M (pH 7.2) sodium phosphate buffer. Ultrathin sections on formvar-coated nickel grids were incubated with 1:50 diluted MNSV mAb. Sections were then incubated with a commercial secondary antibody IgG conjugated with 10

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nm colloidal gold (1:50).

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3. Results and discussion

3.1. Preparation of mAbs against the MNSV coat protein

To produce mAbs against MNSV, the MNSV-Mα5 full-length CP coding sequence was cloned into an expression vector and the CP was overexpressed and purified from E. coli as a histidine-tag fusion. PAGE analysis confirmed the expected size (42 kDa) of the recombinant CP (Fig. 1A). Expression of the fusion protein was also checked by Western blot using an antiHis antibody (Fig. 1B); the faint bands observed below the CP could be the result of partial proteolysis of the purified CP.

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Fig. 1: PAGE analysis of the expressed of MNSV-Mα5 recombinant coat protein (CP). A)

SDS-PAGE followed by Coomassie blue staining of the purified recombinant MNSV CP. B) Western blot analysis of the recombinant MNSV CP using mouse anti-His monoclonal

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antibody. M, protein molecular weight markers.

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The recombinant CP was then used to immunize BALB/c mice to obtain ten hybridoma cell lines secreting mAbs against MNSV. Three hybridomas (2D4H4, 3H12A11 and 4A12C4)

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secreted IgG2a-type antibodies, five (1A12E1, 4A11E4, 4B11A6, 10E4E1 and 10H10G5) secreted IgG2b antibodies, and two (3C6D5 and 4H1E3) IgG1 antibodies. All of these cell lines were tested as positive by indirect ELISA using the fusion CP protein as the coating antigen

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(Table 1). Titers of the antibodies against the recombinant MNSV CP ranged from 1:810 to >1:2430 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL (Table 1). Supernatants from the ten hybridoma cell lines were then evaluated through Western

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blot analysis using MNSV-infected leaf extract; seven of them recognized the MNSV CP, five (4B11A6, 2D4H4, 3H12A11, 10H10G5 and 4A11E4) giving rise to a clean, single-band

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pattern, and two (4A12C4, 4H1E3) producing more complex patterns in Western-blots (Fig. 2).

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Fig. 2: Reactivity of the hybridoma cell lines to MNSV-Mα5-infected melon leaf extracts in Western blot. Lane 1 of each blot was loaded with healthy plant extracts and lane 2 with extracts from melon plants infected with MNSV-Mα5 (0.1 g in 1 mL buffer). M, protein molecular weight markers.

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After three rounds of cloning, the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of its immunoglobulin class was identified as IgG2a and kappa light-chain. Serological reactivity of mAb 2D4H4, as determined by Western

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blot, showed that it strongly reacted with up to 50 ng of the MNSV CP recombinant protein and did not recognize a histidine-tagged control (Fig. 3A). The mAb was then assessed for its ability to detect other MNSV isolates and for its specificity. Western blot results revealed that

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mAb 2D4H4 recognized the CP from purified virus particles and infected leaf extracts of isolate MNSV-Mα5 as well as from resistant-breaking isolates MNSV-264 and MNSV-N.

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Previous phylogenetic analysis showed that the CP of MNSV-Mα5 was the most distant to other European isolates and clustered between these and the Asian isolates (Nieto et al., 2011; Herrera-Vásquez et al., 2010). Since mAb 2D4H4 also reacted to isolates MNSV-264 and MNSV-N, this confirmed its ability to detect phylogenetically rather different isolates by Western blot. To date, no commercial polyclonal antibodies have been described to detect the MNSV CP in Western blots and the polyclonal anti-CP developed by Mochizuki et al. (2009) showed low specificity in infected leaf extracts but also reacted to mock-inoculated plants. In

contrast, 2D4H4 mAb did not react at all with extracts from mock-inoculated melon and squash

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plants nor with the potyvirus MWMV (Fig. 3B).

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Fig. 3: Specificity of mAb 2D4H4 as observed in Western blots. A) The 2D4H4 mAb detected

inputs of 100 ng (lane 1) and 50 ng (lane 2) of the recombinant CP-MNSV protein. The His-tag

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protein (lane 3) was used as negative control. B) Reactivity of mAb 2D4H4 against plant leaf extracts. Lanes 1 and 2 were extracts from melon and squash mock-inoculated plants, respectively; lane 3 corresponded to 10 ng of MNSV-Mα5 from a virus preparation; lanes 4, 6

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and 7 were extracts from melon plants inoculated with isolates MNSV-Mα5, MNSV-264 and MNSV-N, respectively. Lane 5 corresponded to a MWMV-infected melon plant sample. Lane

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8 corresponded to a Nicotiana benthamiana leaf sample infected with MNSV-264 and lane 9 to a squash plant sample inoculated with MNSV-Mα5. 3.2. Use of the MNSV mAb in TAS-ELISA and TEM immuno-labelling

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To study whether mAb 2D4H4 could be used for routine diagnostic purposes, a TAS-ELISA method was developed. First, checkerboard titration tests were performed to determine the

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optimal working concentration for mAb 2D4H4 and the anti-mouse IgG/AP antibodies; results indicated that the optimal concentrations were 1 μg/mL and 0.33 μg/mL, respectively. For mAb 2D4H4, concentrations of 0.5 μg/mL could be sufficient in the case that the availability of this antibody becomes a limiting factor. TAS-ELISA sensitivity was determined by evaluating its limit of detection (LD). For this, serial dilutions were made from extracts of melon cotyledons infected with isolates MNSV-Mα5 or MNSV-264 (Fig. 4). The LD was evaluated for the optimal concentration of mAb 2D4H4 obtained in the checkerboard titration test (1 μg/mL). For this concentration, MNSV-264 was detected up to a dilution of 1:10,240 (g/mL, w/v) and

MNSV-Mα5 up to 1:20,480 (g/mL, w/v) (cut-off = 0.174) (Fig. 4). Note that MNSV-264 is phylogenetically distant to MNSV-Mα5 and its RNA accumulates to lower concentrations than MNSV-Mα5 RNA in susceptible melon plants (Miras et al., 2014; Nieto et al., 2011), possibly explaining the difference in sensitivity for these two isolates. In comparison with commercial polyclonal antibodies, a previous report showed that the sensitivity limit for the detection of MNSV in infected leaves by DAS-ELISA was 160 μg/mL, corresponding to a dilution of 1:6,250 (g/mL, w/v) (Gosalvez et al., 2003). The sensitivity of the mAb 2D4H4 seemed to be much higher, although it must be considered that TAS-ELISA could be up to 5 times more

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sensitive than DAS-ELISA (Alioto et al., 1999).

Fig. 4: Sensitivity test of the mAb 2D4H4 by the triple antibody sandwich (TAS)-enzyme-

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linked immunosorbent assay (ELISA), using a Log2 serial dilution of the MNSV-infected melon tissues extracted in PBS buffer. The dotted line corresponded to the negative control cutoff absorbance value of the experiment (0.174). The arrows indicated the limits of detection

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(LD), with the orange arrow marking the LD for the dilution of the MNSV-Mα5 extract (1:20,482 [g/mL, w/v]) and the green arrow the LD for the dilution of MNSV-264 extract

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(1:10,240 [g/mL, w/v]). C-: serial dilutions of healthy melon leaf extract in PBS.

In order to determine whether mAb 2D4H4 could be used in more refined cellular

biology analyses, TEM immuno-cytochemistry was carried out. Previously, it was shown that modified mitochondria are the MNSV factories, and that a significant proportion of the CPspecific immunogold-labelling signal remained associated to these structures in MNSVinfected cells (Gomez-Aix et al., 2015). Thus, mAb 2D4H4 was used in an TEM immunogoldlabelling experiment to detect MNSV CP in virus-infected cells. The TEM results showed that

the gold particles were primarily detected in the cytoplasm and the altered mitochondria (Fig. 5), in agreement with previous results (Gomez-Aix et al., 2015). No immunogold labeling was detected in sections from mock-inoculated plants, showing the specificity of the 2D4H4 antibody for MNSV CP immunolocalization. In contrast, previous attempts to detect MNSV CP using polyclonal antibodies failed and it was only accomplished when the antibody was subjected to an absorption process, with the resulting loss of sensitivity (Gomez-Aix et al., 2015). The highly specific and sensitive detection of MNSV CP with mAb 2D4H4 would allow the study of the cellular roles of the CP and its localization in the zoospores of O. bornovanus

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to study fungal transmission (Mochizuki et al., 2008).

Fig. 5: Immunogold-labeling of MNSV CP in melon plant tissues in transmission electron microscopy (TEM). Sections from a healthy leaf (A) and a MNSV-Mα5-infected leaf (B-C).

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Specific localization of gold labeling in the modified mitochondria (B) and the cytoplasm (C).

4. Conclusion

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Chl, chloroplast; CW, cell wall; Mit, mitochondria. Bars: 1 μm.

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Seven hybridoma cell lines secreting specific mAbs against the MNSV CP were obtained. Of these, mAb 2D4H4 was purified and showed high specificity and reactivity to three different MNSV isolates in Western blots. Moreover, we developed a sensitive TAS-ELISA protocol for the routine detection of MNSV. Lastly, we successfully used mAb 2D4H4 to localize MNSV CP in infected cells by TEM immunocytochemistry, illustrating the usefulness of mAb 2D4H4 for advanced cellular biology studies.

AUTHOR STATEMENT

MM, CT and CG-A performed the experiments. YH and MAA conceived the work. MM and MAA wrote the paper.

Acknowledgements

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C.T. and C. G.-A. were recipients of fellowships DI-14-06825 and PTQ-15-07646 from the Industrial Doctoral and Torres-Quevedo programs, respectively, from the Ministry of

Economy, Industry and Competitiveness (Spain). M.A.A. acknowledges funding from the

Ministry of Economy, Industry and Competitiveness (Spain) through research grant AGL2015-

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72804EXP. We thank Mari Carmen Montesinos (CEBAS-CSIC, Murcia, Spain) for technical

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assistance and Mario Fon ([email protected]) for editorial assistance.

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Negative

1:30

1:90

1:270

1:810

1:2430

2.846

2.592

2.065

1.34

0.669

0.332

0.172

0.14

0.109

0.108

0.107

0.106

2.436

2.17

1.65

1.048

0.523

0.157

0.145

0.144

0.13

0.118

2.552

2.41

2.02

1.445

0.123

0.123

0.084

0.08

2.3

7.947

1.319

0.716

0.113

0.09

0.086

2.721

2.514

0.24

0.108

2.64

2.316

1.846

0.111

0.107

2.92

2.82

0.102

0.102

10H10G5

1:10

B

0.257

0.102

1:2430

A

0.109

0.078

<1:10

B

1:2430

A

0.069

0.068

0.078

<1:10

B

0.367

0.189

0.102

1:810

A

0.085

0.078

0.069

0.078

<1:10

B

2.161

1.645

0.96

0.453

0.102

1:2430

A

0.084

0.078

0.075

0.069

0.078

1:10

B

1.139

0.591

0.334

0.102

1:2430

A

0.095

0.092

0.081

0.079

0.078

<1:10

B

2.582

1.911

1.181

0.595

0.102

>1:2430

A

0.098

0.098

0.097

0.096

0.078

<1:10

B

Pr

na l

Jo ur

10E4E1

0.078

0.102

4A11E4

4H1E3

A

0.377

3H12A11

4B11A6

1:2430

0.798

3C6D5

4A12C4

0.102

Control

e-

2D4H4

Coating*

pr

1A12E1

Titer

oo

1:10

f

Supernatant Dilution Cell lines

2.623

2.469

2.153

1.435

0.771

0.347

0.102

1:2430

A

0.091

0.085

0.083

0.08

0.073

0.063

0.078

<1:10

B

2.908

2.803

2.611

1.997

1.357

0.735

0.102

>1:2430

A

0.103

0.102

0.093

0.091

0.082

0.076

0.078

<1:10

B

2.512

2.206

1.723

1.022

0.535

0.246

0.102

1:2430

A

0.093

0.083

0.083

0.075

0.073

0.071

0.078

<1:10

B

Isotype

Supernatant concentration

IgG2b,K

6.009 µg/mL

IgG2a,K

3.588 µg/mL

IgG1,K

3.917 µg/mL

IgG2a,K

2.363 µg/mL

IgG2b,K

4.290 µg/mL

IgG2a,K

1.512 µg/mL

IgG2b,K

1.741 µg/mL

IgG1,K

4.420 µg/mL

IgG2b,K

2.096 µg/mL

IgG2b,K

4.081 µg/mL

Table 1: Properties and characterization of hybridoma cell lines. Hybridoma supernatants were screened for the presence of anti-CP antibodies by indirect ELISA in 96-well plates. *Coating antigen: A, MNSV CP protein; B, His-tag protein.