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The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3−/4− on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease. [ABSTRACT FROM AUTHOR]

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease. [ABSTRACT FROM AUTHOR]

Beet necrotic yellow vein virus (BNYVV) is one of the main contributors to economic losses in sugar beet production. The present study generated a polyclonal antibody that detects BNYVV. The conserved genomic region of all BNYVV isolates gene encoding the viral major coat protein (BNYVV-CP) was amplified, cloned, sequenced, and expressed in E. coli strain BL21 (DE3). The expressed protein was purified under native conditions by affinity chromatography. The purified BNYVV CP was used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-CP-IgG detected the BNYVV-CP recombinant protein and BNYVV in infected sugar beet by indirect-ELISA, dot immunobinding assay, and western blot analysis blotting. The serological assays strongly proved the sensitivity and specificity of anti-BNYVV-CP-IgG against BNYVV. These results suggest that the generated anti-BNYVV-CP-IgG polyclonal antibodies can be used as reliable and quick means for the BNYVV virus detection under field conditions. [ABSTRACT FROM AUTHOR]

Tobacco mosaic virus (TMV) is a significant viral pathogen that infects various economically important crops, including tobacco, tomatoes, and cucumbers. The current study aimed to develop and optimize essential oil-loaded nanoemulsions using Response Surface Methodology (RSM) to enhance antiviral activity against TMV in Nicotiana tabacum plants. Essential oils from tea tree (Melaleuca alternifolia) and lime peel (Citrus latifolia) were chemically analyzed using gas chromatography-mass spectrometry (GC-MS). Terpinene-4-ol (70.92 %) and trans-isolimonene (33.82 %) were identified as the major components in tea tree and lime peel oils, respectively. RSM was used to optimize the nanoemulsion formulations based on oil concentration, hydrophilic-lipophilic balance (HLB), and stirring speed. Optimal tea tree oil (TTEO) and lime peel oil (LPEO) nanoemulsions were developed and characterized using Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and particle size analysis. The antiviral efficacy of the optimized formulations was tested against TMV using RT-qPCR analysis and physiological measurements. TTEO nanoemulsions significantly inhibited TMV expression by 89.78 %, while LPEO nanoemulsions achieved a 42.22 % reduction after six days. Additionally, these essential oils (EOs) enhanced plant defense mechanisms by modulating the activities of phenylalanine ammonia-lyase (PAL) and peroxidase (POD), along with maintaining chlorophyll levels. This study demonstrates that TTEO and LPEO nanoemulsions offer promising eco-friendly antiviral agents for TMV management in tobacco plants. The research underscores the potential of essential oil-based formulations in mitigating viral infections in crops, providing a sustainable alternative to synthetic chemicals. [Display omitted] • The successful development and optimization of tea tree and lime peel essential oil nanoemulsions. • A significant inhibition of TMV expression by 89.78 % with tea tree oil nanoemulsions. • The enhancement of plant defense mechanisms and maintenance of chlorophyll levels, indicating a broader impact on plant health. [ABSTRACT FROM AUTHOR]

Present study was performed to develop a fusion recombinant monoclonal antibody for one-step and accurate detection of FMV with a specific single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) named as scFv(FMV-NP)-AP. The gene encoding-specific scFv recombinant antibody binding to nucleocapsid protein of Fig Mosaic Virus (FMV-NP) was fused to upstream of AP gene and integrated in pET26b bacterial expression vector. As vector contain pelB signal peptide, the expressed protein is secreted into periplasmic compartment. Recombinant fusion protein was produced in transformed E. coli following induction by IPTG. Extraction and purification of fusion protein was performed under denatured condition. The results of SDS-PAGE and western blot analysis indicated high integrity and purity with a single band protein with expected size of 72 kDa. The total yield of purified scFv(FMV-NP)-AP fusion protein estimated around 0.5–1 mg/l cultured medium. Subsequent colorimetric analysis confirmed presence of alkaline phosphatase activity in prepared scFv-AP fusion protein. Specificity of generated recombinant fusion antibody against cognate antigen and the native virus presented in infected plant extracts was assessed by ELISA, western blot and dot blot assays. Results revealed that scFv(FMV-NP)-AP is able to detect the presence of FMV in infected fig plants. The novel approach, implementing specific recombinant fusion antibody developed in this research, leads to one-step detection of FMV in plants by avoiding the use of chemical enzyme-labeled secondary antibodies. [ABSTRACT FROM AUTHOR]

Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus), has a wide host range and geographic distribution in many parts of the world, and it is one of the most important legume-infecting viruses. Detection of CpCDV-infected plants in the field and evaluation of viral resistance of plant cultivars are possible by conducting serological assays. Here, development and characterization of a specific recombinant monoclonal antibody for CpCDV as a diagnostic tool are described. For this purpose, the coat protein of CpCDV was expressed in Escherichia coli strain Rosetta (DE3) and used to screen a Tomlinson phage display antibody library to select a specific single-chain variable fragment (scFv). In each round of biopanning, the affinity of the phage for CpCDV-CP was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the specificity of the eluted phages increased after each round of panning. Testing of individual clones by ELISA showed that five clones of the monoclonal phage were more strongly reactive against CpCDV than the other clones. All selected positive clones contained the same sequence. The phage-displayed scFv antibody, which was named CpCDV-scFvB9, did not bind to other tested plant pathogens and showed high sensitivity in the detection of CpCDV. A Western blot assay demonstrated that CpCDV-scFvB9 reacted with the recombinant coat protein of CpCDV. Finally, the interaction CpCDV-scFvB9 and CpCDV-CP was analyzed in a molecular docking experiment. This is the first report on production of an scFv antibody against CpCDV, which could be useful for immunological detection of the virus. [ABSTRACT FROM AUTHOR]

Fig mosaic disease (FMD) poses a significant threat to fig (Ficus carica) trees globally. Fig mosaic virus (Emaravirus fici, Family Fimoviridae) is one of the main causes involved in FMD. E. fici has been reported in many countries, including Iran. This research investigated the genetic diversity and distribution of this virus in Kermanshah Province, the third fig‐producing region in Iran. In 2021 and 2022, 128 samples, mainly displaying FMD symptoms, were collected from fig trees across nine counties. Using DAS‐ELISA with polyclonal antibodies, 86 samples tested positive for fig mosaic virus. The 2‐year‐old healthy fig saplings grafted with scions from infected trees exhibited mosaic symptoms and tested positive for the virus in DAS‐ELISA. Reverse transcription‐polymerase chain reaction (RT‐PCR) by RdRp‐specific primers (E5‐s and E5‐a) also amplified a DNA fragment with the expected size of about 302 bp from the ELISA‐positive samples. Five of the 12 symptomless samples showed positive results in both DAS‐ELISA and RT‐PCR. This indicates that the absence of symptoms in propagation sources does not imply that they are virus‐free. Nucleotide sequences of RT‐PCR products confirmed the fig mosaic virus infection. BLAST analysis of 20 samples (GenBank accession numbers OQ552535 to OQ552554) revealed that 19 sequences show high nucleotide identity (93.4%–96.8%) with an isolate from Lorestan Province, Iran (GenBank accession number KT732024). Notably, isolate FM203 showed distinct genetic divergence (86.6%–90.2% nucleotide identity) and shared similarities with some other Iranian isolates, including those from Kermanshah and Mazandaran provinces. Phylogenetic analysis grouped most isolates with the Lorestan isolate, while FM203 was placed in a separate cluster. This study highlighted the widespread presence of fig mosaic virus in Kermanshah Province, emphasizing varying infection rates across different counties, with Dalahoo County (Rijab) showing the highest prevalence. [ABSTRACT FROM AUTHOR]

Highlights • Fig mosaic emaravirus (FMV) induces "mosaic disease" (MD) on fig. • The detection of this virus in infected plant material is possible through RT-PCR and RT-LAMP; whereas no serological tools are available. • A polyclonal antiserum against the nucleocapsid protein of FMV was developed and evaluated in different serological techniques for FMV detection. Abstract Mosaic disease (MD), caused by Fig mosaic emaravirus (FMV), is the most important and devastating virus disease of fig trees worldwide. The detection of FMV in infected plants is possible only through the use of molecular techniques, i.e. RT-PCR and LAMP, which both offer high sensitivity of detection, but are also considered laborious when dealing with a large number of samples. To cope with this restriction, a polyclonal antiserum through the immunization of a rabbit by injecting the recombinant nucleocapsid protein (NP) of FMV was raised and evaluated for its efficacy in Western Blot, Dot immuno-binding and DAS-ELISA. The results obtained showed that the raised antiserum was able to identify the nucleocapsid protein of FMV (p3) which was found to have an estimated molecular weight of ca. 35 KDa. In addition, the antiserum, when used in the three serological assays, was able to detect the p3 of FMV in protein extracts of infected plants with different levels of efficacy. Dot immuno-binding, using denatured plant protein extract, proved to be the most efficient serological assay for detecting FMV in samples collected from different fig orchards. This is the first report on an antiserum raised against FMV that could be used for immunological detection of the virus. [ABSTRACT FROM AUTHOR]

Abstract Here, the construction and characterization of the first immunosensor for highly sensitive and label free detection of Fig mosaic virus (FMV) is reported. The specific antibody against nucleocapsid of the virus was raised and immobilized at the surface of 11-mercaptoundecanoic acid (MUA) and 3-mercapto propionic acid (MPA) modified gold electrode, via carbodiimide coupling reaction. The immunosensor fabrication steps were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical detection of FMV was conducted using differential pulse voltammetry in ferri/ferrocyanide solution as a redox probe. The proposed immunosensor exhibited high selectivity, good reproducibility and high sensitivity for FMV detection in a range from 0.1 nM to 1 μM with a detection limit of 0.03 nM. Moreover, good results were obtained for determination of FMV in real samples, indicating the feasibility of the developed immunosensor for detection of fig mosaic disease, without the need for molecular (e.g. PCR) amplification. Graphical abstract Image 1 Highlights • The first reported immunosensor for fig mosaic diagnosis. • Excellent specificity against possible interferences from the real samples was achieved. • The immunosensor can be considered as a promising alternative method to RT-PCR for FMV detection. • The method could be modified and extended for production of portable devices. [ABSTRACT FROM AUTHOR]

The study of antibody-antigen interactions, through epitope mapping, enhances our understanding of antibody neutralization and antigenic determinant recognition. Epitope mapping, employing monoclonal antibodies and mass spectrometry, has emerged as a rapid and precise method to investigate viral antigenic determinants. In this report, we propose an approach to improve the accuracy of epitopic peptide interaction rate recognition. To achieve this, we investigated the interaction between the nucleocapsid protein of fig mosaic virus (FMV-NP) and single-chain variable fragment antibodies (scFv-Ab). These scFv-Ab maintain high specificity similar to whole monoclonal antibodies, but they are smaller in size. We coupled this with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The experimental design involved using two different enzymes to digest FMV-NP separately. The resulting peptides were then incubated separately with the desired scFv-Ab at different incubation times and antibody concentrations. This allowed us to monitor the relative rate of epitopic peptide interaction with the antibody. The results demonstrated that, at a 1:1 ratio and after 2 h of interaction, the residues 122–136, 148–157, and 265–276 exhibited high-rate epitopic peptide binding, with reductions in peak intensity of 78%, 21%, and 22%, respectively. Conversely, the residues 250–264 showed low-rate binding, with a 15% reduction in peak intensity. This epitope mapping approach, utilizing scFv-Ab, two different enzymes, and various incubation times, offers a precise and dependable analysis for monitoring and recognizing the binding kinetics of antigenic determinants. Furthermore, this method can be applied to study any kind of antigens. [Display omitted] • Recombinant antibodies, with their small size and high specificity, can be considered for epitope mapping. • MALDI-TOF MS provides accurate and reliable results for linear epitope mapping. • The kinetic of epitopic residues can be followed in different incubation times and concentrations. [ABSTRACT FROM AUTHOR]

Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was analyzed by indirect enzyme-linked immunosorbent assay (ELISA) followed by blotting assays. Threedimensional (3D) structure of the selected scFv antibody fragment and VP3 protein were predicted through in silico analysis. Structural characterization of the antibody-antigen complexes was carried out by computational docking analysis. Results: The serological results obtained from the ELISA and blotting analysis showed that the selected clones produced specific scFv antibody fragments that were capable of effectively detecting infectious bursal disease (IBD) in the infected animal tissue. Biodiversity analysis by BstNI finger printing and nucleotide sequencing revealed that there was no major difference in nucleotide sequences of the selected clones. Further analysis demonstrated that this recombinant fragment of the antibody was able to bind to VP3 structural protein of IBDV with a molecular weight of ~30 kDa. Molecular docking results revealed that the binding energy of scFv to IBDV-VP3 was 545 kj/mol. Conclusion: The developed scFv antibody fragments possess great potentials for the diagnosis of IBD. The findings of the present study confirm the feasibility of using phage display technology for rapid production of antibodies against IBD diseases by applying naïve scFv libraries. [ABSTRACT FROM AUTHOR]

This work presents a new electrochemical immunosensor for sensitive and rapid detection of infectious bursal disease virus (IBDV) based on the combination of a modified glassy carbon (GC) electrode and fast Fourier transforms square wave voltammetry. The immunosensorwasa GC modified electrodeposited gold NPs, self-assembled monolayer and immobilised anti-IBDV antibodies. The interaction between the antibody and antigen was followed by measuring the change in the redox currents of the probe, using fast Fourier transform square wave voltammetry technique. The obtained linear range was 40–2500 particle mL−1 with the detection limit of 14 particle mL−1. This immunosensor was successfully applied to determine IBDV in real samples. The prepared immunosensor is selective and displayed excellent stable sensitivity over a long period of the observed time. [ABSTRACT FROM AUTHOR]

Rhizomania is the most destructive disease in sugar beet throughout the world. The disease is caused by Beet necrotic yellow vein virus (BNYVV). Polymyxa betae (Keskin) is the only known vector of BNYVV, for transmission of the virus between the plants. Developing molecular diagnostic methods has a major impact on forecasting and epidemiological studies as well as screening sugar beet plants used in resistance breeding programmes. In the present study, quantum dots (QDs) were biofunctionalized with a specific antibody against P. betae. The glutathione-S-transferase protein's (GST) corresponding antibody (anti-GST) was effectively conjugated to Tioglicolicacid-modified Cadmium-Telluride QDs (CdTe-QDs) synthesized in an aqueous solution via electrostatic interaction. The dye (Rhodamine) molecules were attached to the GST. Donor–acceptor complexes (QDs-Ab-GST-Rhodamine) were then formed based on the antigen–antibody interaction. The mutual affinity of the antigen and the antibody brought the CdTeQDs and rhodamine together close enough to allow the resonance dipole–dipole coupling required for fluorescence resonance energy transfer (FRET) to occur. The immunosensor constructed showed a high sensitivity and specificity of 100%, acceptable stability and could be used for real sample detection with consistent results. To the best of our knowledge, this is the first report on designing a nano-based biosensing tool for the detection of plant pathogenic fungi. [ABSTRACT FROM PUBLISHER]

Abstract: Plant diseases have a significant impact on the yield and quality of crops. Many strategies have been developed to combat plant diseases, including the transfer of resistance genes to crops by conventional breeding. However, resistance genes can only be introgressed from sexually-compatible species, so breeders need alternative measures to introduce resistance traits from more distant sources. In this context, genetic engineering provides an opportunity to exploit diverse and novel forms of resistance, e.g. the use of recombinant antibodies targeting plant pathogens. Native antibodies, as a part of the vertebrate adaptive immune system, can bind to foreign antigens and eliminate them from the body. The ectopic expression of antibodies in plants can also interfere with pathogen activity to confer disease resistance. With sufficient knowledge of the pathogen life cycle, it is possible to counter any disease by designing expression constructs so that pathogen-specific antibodies accumulate at high levels in appropriate sub-cellular compartments. Although first developed to tackle plant viruses and still used predominantly for this purpose, antibodies have been targeted against a diverse range of pathogens as well as proteins involved in plant–pathogen interactions. Here we comprehensively review the development and implementation of antibody-mediated disease resistance in plants. [Copyright &y& Elsevier]

Tomato yellow leaf curl virus (TYLCV) is a geminivirus species whose members cause severe crop losses in the tropics and subtropics. We report the expression of a single-chain variable fragment (scFv) antibody that protected Nicotiana benthamiana plants from a prevalent Iranian isolate of the virus (TYLCV-Ir). Two recombinant antibodies (scFv-ScRep1 and scFv-ScRep2) interacting with the multifunctional replication initiator protein (Rep) were obtained from phage display libraries and expressed in plants, both as stand-alone proteins and as N-terminal GFP fusions. Initial results indicated that both scFvs and both fusions accumulated to a detectable level in the cytosol and nucleus of plant cells. Transgenic plants challenged with TYLCV-Ir showed that the scFv-ScRep1, but more so the fusion proteins, were able to suppress TYLCV-Ir replication. These results show that expression of a scFv-ScRep1-GFP fusion protein can attenuate viral DNA replication and prevent the development of disease symptoms. The present article describes the first successful application of a recombinant antibody-mediated resistance approach against a plant DNA virus. [ABSTRACT FROM AUTHOR]

• A specific single-chain variable fragment antibody (scFvF10) was raised against the recombinant coat protein of CTV. • Homology-based molecular modeling and docking analysis confirmed the interaction between CTV-CP and scFvF10. • scFvF10 can be applied as an efficient tool for CTV-infected plant diagnosis in multiple diagnostic assays. Citrus tristeza virus (CTV) is the most economically important virus disease of citrus worldwide. To develop a specific serological assay for CTV, a Tomlinson phage display antibody library of single chain variable fragments (scFv) was screened with a recombinant CTV coat protein (CTV-CP) heterologously expressed in Escherichia coli. The phage clones were checked by ELISA to identify clones with high specificity for CTV-CP. Eight clones were strongly reactive with CTV-CP. Nucleotide sequencing of these clones revealed that all of them contained the same sequence. Thus, the phage-displayed scFv antibody was termed scFvF10. Evaluation of scFvF10 binding to CTV-CP by plate-trapped antigen ELISA (PTA-ELISA) and immunoblotting, showed that it was specific and allowed sensitive detection of CTV-CP. Homology-based molecular modeling and docking analysis confirmed that the interaction between CTV-CP and scFvF10, with a binding energy of -738 kj mol-1, occurred mainly by 12 intermolecular hydrogen bonds. Moreover, triple-antibody sandwich (TAS)-ELISA using scFvF10 as second antibody showed high sensitivity in the detection of CTV infected samples. The CTV detection limit of scFvF10 by PTA-ELISA and TAS-ELISA were 0.05 and 0.01 μg CP/mL, respectively. Our results with different diagnostic assays demonstrated that scFvF10 has the potential to be used as an efficient tool for CTV-infected plant diagnosis. [ABSTRACT FROM AUTHOR]

• Specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFvs) were raised against FMV. • Molecular docking analysis confirmed the scFv and FMV-Np interactions with the anti-FMV-Np scFv. • Specific recombinant FMV-Np scFvs can be applied in development of antibody-mediated protection strategy against FMV. The mosaic disease caused by fig mosaic virus (FMV) is considered the plague of fig worldwide. A naïve phage display library, raised against the recombinant nucleocapsid protein of FMV (FMV-Np) was screened to obtain specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFvs). After three rounds of biopanning, the bacterially expressed FMV-Np was used as an antigen for selecting specific phages for the production of specific soluble scFvs to be used in immunological assays. The binding specificity of scFvs against FMV-infected fig samples was evaluated by immunoblotting and Plate trapped antigen-ELISA (PTA-ELISA), which revealed efficient of the resultant scFvs to the target antigen. S ilico homology-modelling and molecular docking analysis confirmed the scFv and FMV-Np interactions with the anti-FMV-Np scFv through an estimated binding energy of -650 kj mol−1; considered to be generated from the interactions between 13 amino acids residues predicted as putative epitopes in the interface pocket of FMV-Np and scFv antibody. This high affinity was further confirmed in the specificity of ELISA and immunoblotting assays. This is the first report on the application of phage display technology to generate specific recombinant scFvs against FMV that can be applied in development of antibody-mediated protection strategy to control the fig mosaic disease. [ABSTRACT FROM AUTHOR]

Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus -specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus -specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops. • Aspergillus is a major fungal pathogen. • Early A. flavus detection is crucial for fungal control. • Efficient fungal detection relays on availability of highly specific antibodies. • mAbs specific to A. flavus a ntigenic cell wall mannoprotein 1 (Aflmp1). [ABSTRACT FROM AUTHOR]

Fig mosaic virus (FMV) is recognized as the main viral agent associated with the mosaic disease (MD) of fig trees (Ficus carica). Due to its worldwide occurrence, FMV represents the most significant global threat to the production of fig fruit. A disease management strategy against the MD in fig orchards has never been effective; and therefore, expression of recombinant antibody in plant cells could provide an alternative approach to suppress FMV infections. In this study we focused on expressing a specific recombinant antibody, a single-chain variable fragment (scFv), targeting the nucleocapsid protein (NP) of FMV in planta. To accomplish this objective, we inserted the scFv gene into a plant expression vector and conducted transient expression in leaves of Nicotiana tabacum cv. Samson plants. The construct was transiently expressed in tobacco plants by agroinfiltration, and antibody of the anticipated size was detected by immunoblotting. The produced plantibody was then assessed for specificity using ELISA and confirmed by Western blot analysis. In this study, the plantibody developed against FMV could be considered as a potential countermeasure to the infection by conferring resistance to MD. • Specific single chain variable fragments (scFvs) expressed against Nucleoprotein of FMV in tobacco plant cells. • Transient expression of scFv fragment against the nucleoprotein of FMV in cytosolof tobacco leaf cells. • Specific recombinant FMV-Np scFvs expressed in plant can be applied in antibody-mediated protection strategy against FMV. • High binding capacity of the plantibody to react with the crude FMV-infected material or the recombinant FMV-NP. [ABSTRACT FROM AUTHOR]

Highlights • The first electrochemical immunosensor for Citrus canker diagnosis is introduced. • The immunosensor architecture incorporates CNTs-IL composite, PB and GNPs. • The reported immunosensor exhibited low detection limit and excellent specificity. • The method could be modified and extended for the detection of other targets. Abstract A novel label free electrochemical immunosensor was developed for rapid and direct detection of the effector protein PthA, for citrus canker diagnosis. The immunosensor fabrication was based on the sequential electrodeposition of Prussian blue (PB) and gold nanoparticles (GNPs) on the surface of bamboo-like multiwall carbon nanotubes-ionic liquid nanocomposite (BCNT-IL) modified glassy carbon electrode. The morphology of the modified electrode surface was studied by means of field emission scanning electron microscope (FE-SEM). Moreover, the role of each component in the modified electrode was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results indicated that the used modification strategy effectively enhanced the stability and electroactivity of the PB as electron mediator. After immobilization of the anti-PthA antibody and blocking with BSA, the immunosensor was used for determination of different concentrations of PthA antigen under the optimal conditions. The linear dynamic range of fabricated immunosensor was from 0.1 to 50 nM with a detection limit of 0.028 nM. The proposed immunosensor exhibited high selectivity, long-term stability and good reproducibility. Moreover, good results were obtained for the determination of PthA in real samples, indicating the great potential of the developed immunosensor for detection of citrus canker disease. [ABSTRACT FROM AUTHOR]

This paper presents the first label-free and sensitive electrochemical immunosensor for efficient and rapid detection of Citrus tristeza virus (CTV). The specific antibody against coat protein (CP) of CTV was successfully immobilized on 11-mercaptoundecanoic acid (MUA) and 3-mercapto propionic acid (MPA) modified gold electrode via carbodiimide coupling reaction using N -(3-dimethylaminopropyl)- N ′-ethyl carbodiimide hydrochloride (EDC) and N -HydroxySuccinamide (NHS). Electrochemical characterizations of the immunosensor (anti-CTV/MUA-MPA/Au electrode) were carried out by cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical detection was performed by differential pulse voltammetry in [Fe(CN) 6 ] 3−/4− solution. The decrease in the [Fe(CN) 6 ] 3−/4− current upon increasing antigen concentrations on the immunosensor surface was determined in the range of 1 nM to 5 μM. The developed immunosensor provided a much lower limit of detection (LOD = 0.27 nM) than ELISA and other reported immunoassay techniques for CTV detection. The specificity of the immunosensor was also examined and no significant interference from other analytes in the real sample was observed. The proposed immunosensor is simple, relatively inexpensive, and shows good sensitivity for this application. [ABSTRACT FROM AUTHOR]

A novel label-free electrochemical immunosensor was fabricated for selective and sensitive detection of Candidatus Phytoplasma Aurantifolia (CPA), the main factor of witches broom diseases, in tree sap samples. CPA antibody was immobilized on a thiol terminated surface of a gold electrode modified by gold nanoparticles; the surface was then activated by mixture of N -(3-dimethylaminopropyl)- N -ethylcarbodiimide hydrochloride (EDC) and N -HydroxySuccinamide (NHS). Scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed for surface characterization. All the electrochemical measurements of CPA were accomplished based on the [Fe(CN) 6 ]3−/4− solution response as the standard redox probe. CPA detection was carried out using square wave voltammetry (SWV) technique, at frequency of 20 Hz and amplitude of 0.1 V. In order to reach the maximum efficiency in performance of the immunosensor, Anti-CPA 1 mg mL−1, pH 7.4 and the incubation time of 45 min were selected as optimized experimental conditions. According to the calibration curve data, the obtained linear range was 0.005 to 100 μg mL−1, with limit of detection (LOD) of 1.5 ng mL−1, which was lower than the value reported for ELISA method applied for CPA detection. The immunosensor demonstrated adequate selectivity and stability besides; there was no significant interference observed from other probable protein based components in the real sample matrix. Unlabelled Image • The first label-free electrochemical immunosensor for Candidatus Phytoplasma Aurantifolia (CPA) detection. • Adequate specificity of fabricated immunosensor against interferences from sap samples. • Noticeable LOD that is considerably lower than reported records for CPA detection. [ABSTRACT FROM AUTHOR]

A novel label-free electrochemical immunosensor was developed for sensitive and reliable detection of PthA effector protein, for citrus canker diagnosis. In this approach, Prussian blue (PB), as an electron transfer mediator, was electrodeposited onto the carbon nanotube-ionic liquid (BCNT-IL) modified glassy carbon electrode. In the next step, gold nanoparticles (GNP) were electrodeposited onto the as-prepared film for biomolecules anchoring. The morphology of modified electrode surface was studied by means of field emission scanning electron microscope (FE-SEM). Moreover, the role of each component in the modified electrode was further investigated by cyclic voltammetry (CV). The results indicated that the proposed modification strategy effectively enhanced the stability and electroactivity of PB. After the immobilization of anti-PthA antibody, the immunosensor was used for determination of different concentrations of PthA antigen under the optimal conditions, using Fast Fourier transform square wave voltammetry (FFT-SWV) technique. The dynamic linear range of this electrochemical immunosensor for detecting PthA protein was from 0.03 to 100 nM, with a detection limit of 0.01 nM. The prepared immunosensor displayed high selectivity, long term stability, good repeatability and great potential for application in real sample analysis. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor alpha (TNF-α) expression amplifies to excess amounts in several disorders such as rheumatoid arthritis and psoriasis. Although, Anti-TNF biologics have revolutionized the treatment of these autoimmune diseases, formation of anti-drug antibodies (ADA) has dramatically affected their use. The next generation antibodies (e.g. Fab, scFv) have not only reduced resulted immunogenicity, but also proved several benefits including better tumor penetration and more rapid blood clearance. Using affinity selection procedures in this study, a scFv antibody clone was isolated from naïve Tomlinson I phage display library that specifically recognizes and binds to TNF-α. The TNF-α recombinant protein was expressed in genetically engineered Escherichia coli SHuffle® T7 Express, for the first time, which is able to express disulfide-bonded recombinant proteins into their correctly folded states. ELISA-based affinity characterization results indicated that the isolated novel 29.2 kDa scFv binds TNF-α with suitable affinity. In-silico homology modeling study using 'ModWeb' as well as molecular docking study using Hex program confirmed the scFv and TNF-α interactions with a scFv-TNF- α binding energy of around -593 kj/mol which is well in agreement with our ELSIA results. The cloned scFv antibody may be potentially useful for research and therapeutic applications in the future. [ABSTRACT FROM AUTHOR]

Chickpea chlorotic dwarf virus (CpCDV) is one of the most important pathogenic viruses of chickpea that can have devastating effects on the economy and food security of people. Herein, we aim to develop a new electrochemical label-free immunosensor to detect Chickpea chlorotic dwarf virus (CpCDV) with high sensitivity and rapidity. To achieve this, a combination of room temperature ionic liquid (IL), reduced graphene oxide (rGO), and Prussian blue (PB) was first used to modify the glassy carbon electrode (GCE). In addition, Au nanoparticles (AuNPs) were electrodeposited on the electrode surface. The morphology of the surface was investigated using a field emission scanning electron microscope (FE-SEM). The techniques of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to discover the properties of each component in the function of the fabricated electrode. The results showed that the AuNPs/PB-rGO-IL/GCE electrode possessed excellent electrochemical activity, which led to a significantly improved signal response of PB. Subsequently, the anti-CpCDV was immobilized and the non-responsive sites were blocked by BSA. Then, the different parameters affecting the immunosensor response, such as pH, incubation time, and electrodeposition time of AuNPs, were investigated. Interestingly, the fast Fourier transform square wave voltammetry (FFT-SWV) technique was used to detect the CpCDV antigen. The presented immunosensor demonstrated a linear behavior in a wide range from 0.05 to 50 μg/mL and a detection limit (S/N= 3) of 0.01 μg/mL. In addition, the proposed immunosensor can be considered an acceptable candidate for the detection of CpCDV due to its satisfactory selectivity in the real matrix and its reproducibility, durability and stability. [Display omitted] • Novel label free electrochemical immunosensor for CpCDV detection • Highly sensitive surface reached by RTIL-rGO and Prussian blue • Direct detection ability of the immunosesnor for CpCDV in the real sample [ABSTRACT FROM AUTHOR]