Determination of the epitopic peptides of fig mosaic virus and the single-chain variable fragment antibody by mass spectrometry.

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.

Comparative evaluation of resistance to potato virus Y (PVY) in three different RNAi-based transgenic potato plants.

Small interfering RNAs (siRNAs) produced from template double-stranded RNAs (dsRNAs) can activate the immune system in transgenic plants by detecting virus transcripts to degrade. In the present study, an RNA interference (RNAi) gene silencing mechanism was used for the development of transgenic potato plants resistant to potato virus Y (PVY), the most harmful viral disease. Three RNAi gene constructs were designed based on the coat protein (CP) and the untranslated region parts of the PVY genome, being highly conserved among all strains of the PVY viruses. Transgenic potato plants were generated using Agrobacterium containing pCAMRNAiCP, pCAMRNAiUR, and pCAMRNAiCP-UR constructs. The transgene insertions were confirmed by molecular analysis containing polymerase chain reaction (PCR) and southern blotting. The resistance of transgenic plants to PVY virus was determined using bioassay and evaluating the amount of viral RNA in plants by RT-PCR, dot blotting of PVY coating protein, and enzyme-linked immunosorbent assay (ELISA). Bioassay analysis revealed that more than 67% of transgenic potato plants were resistant to PVY compared with the non-transgenic plants, which showed viral disease symptoms. No phenotypic abnormalities were observed in transgenic plants. Out of six lines in southern blot analysis, four lines had one copy of the transgene and two lines had two copies of the target genes. No correlation was detected between the copy number of the genes and the resistance level of the plant to PVY. Transgenic lines obtained from all three constructs indicated more or less similar levels of resistance against viral infection; however, CP-UR lines exhibited relatively high resistance followed by CP and UR expressing lines, respectively. Meanwhile, some lines showed a delay in symptoms 35 days after infection which were classified as susceptible.

Transient expression of an scFvG8 antibody in plants and characterization of its effects on the virulence factor pthA of Xanthomonas citri subsp. citri.

Citrus bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major disease of citrus plants, causing a significant loss in the citrus industry. The pthA is a bacterial effector protein mediates protein-protein and protein-DNA interactions and modulates host transcription. Injection of pthA effector protein into the host cell induces the expression of the susceptibility gene CsLOB1 which is required for citrus canker disease development. In this study, we described in planta expression of a specific anti-pthA single-chain variable fragment (scFv) recombinant antibody, scFvG8, and assessed its function using molecular docking, immunoblotting, and indirect enzyme-linked immunosorbent assay (ELISA). Based on the results, homology-based molecular docking suggested that at least eight intermolecular hydrogen bonds are involved in pthA-scFvG8 interactions. Immunoblotting and indirect ELISA results reconfirmed specific binding of scFvG8 to pthA protein. Moreover, gene fragment encoding scFvG8 was cloned into plant expression vector and transiently expressed in leaves of Nicotiana tabacum cv. Samson by agroinfiltration method. Transient expression of scFvG8 (at the expected size of 35 kDa) in N. tabacum leaves was confirmed by western blotting. Also, immunoblotting and indirect ELISA showed that the plant-derived scFvG8 had similar activity to purified scFvG8 antibody in detecting pthA. Additionally, in scFvG8-expressing tobacco leaves challenged with Xcc, a reduction (for up to 70%) of hypersensitive response (HR) possibly via direct interaction with pthA, was observed in the necrotic leaf area compared to control plants infected with empty vector. The results obtained in this study confirm that scFvG8 can suppress the function of pthA effector protein within plant cells, thus the induction of stable expression of scFvG8 in lime trees can be considered as an appropriate approach to confer resistance to Xcc.

Conjugation of Single-Chain Variable Fragment Antibody to Magnetic Nanoparticles and Screening of Fig Mosaic Virus by MALDI TOF Mass Spectrometry.

The ability of mass spectrometry for discrimination between protein and peptide masses which are unique to specific pathogens provides an accurate and fast method for the detection of different types of pathogens, especially viruses. Capsid proteins are specific to each virus and can be used as a biomarker for detection of this pathogen. On the other hand, single-chain variable fragment (scFv) antibodies have been recently used to enhance the accuracy of immunoassay techniques. So conjugation of mass spectrometry and scFv antibody provides a very accurate and fast method for the detection of viruses. In this report, for the first time, we have immobilized scFv antibody of fig mosaic virus (FMV) on the magnetic nanoparticles (MNPs) to extract the virus capsid protein from complex biological media and subsequently identified this protein through both its intact molecular mass and peptide mass fingerprint (PMF) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Isolation of single-chain variable fragment (scFv) antibodies for detection of Chickpea chlorotic dwarf virus (CpCDV) by phage display.

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.

Simple and effective label free electrochemical immunosensor for Fig mosaic virus detection.

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.

The contribution of cell wall composition in the expansion of Camellia sinensis seedlings roots in response to aluminum.

MAIN CONCLUSION: Treatment with aluminum triggers a unique response in tea seedlings resulting in biochemical modification of the cell wall, regulation of the activity of the loosening agents, and elongation of root. Unlike most terrestrial plants, tea (Camellia sinensis L.) responds to aluminum (Al) through the promotion of its root elongation; but the real mechanism(s) behind this phenomenon is not well understood. A plausible relationship between the modifications of the cell wall and the promotion of root elongation was examined in tea seedlings treated for 8 days with 400 µM Al. The mechanical properties of the cell wall, the composition of its polysaccharides and their capacity to absorb Al, the expression of genes, and the activities of the wall-modifying proteins were studied. With 6 h of the treatment, about 40% of the absorbed Al was bound to the cell wall; however, the amount did not increase thereafter. Meanwhile, the activity of pectin methylesterase, the level of pectin demethylation, the amounts and the average molecular mass of xyloglucan in the root apices significantly decreased upon exposure to Al, resulting in the reduction of Al binding sites. On the other hand, the activity and the gene expression of peroxidase decreased, whereas the activity and gene expression of xyloglucan-degrading enzymes, the expression of expansin A and the H +-ATPase4 genes increased in the Al-treated plants. Interestingly, it was accompanied by the increase of elastic and viscous extensibility of the root apices. From the results, it can be suggested that the biochemical modification of the cell walls reduces sites of Al binding to roots and triggers the activity of the loosening agents, thereby increasing the length of tea roots.

In planta expression of specific single chain fragment antibody (scFv) against nucleocapsid protein of fig mosaic virus (FMV).

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.

Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri.

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.

Development of label-free electrochemical OMP-DNA probe biosensor as a highly sensitive system to detect of citrus huanglongbing.

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.

Development of high-throughput quantum dot biosensor against Polymyxa species.

The plasmodiophoromycete Polymyxa betae and P. graminis are eukaryotic biotrophic parasites residing in the roots of chenopodiacae and gramineae plants. They are natural transmitting agents of several important plant viruses such as are beet necrotic yellow vein virus (BNYW), beet soil borne mosaic virus (BSBMV), wheat soil-borne mosaic virus (WSBMV). Developing advanced high-throughput diagnostic methods capable of accurate detection of these pathogens could assist with the screening programs and consequently with the development of disease-resistant germplasms. In the present study, a previously developed quantum dots (QDs) FRET-based nano-biosensor was upgraded to a high-throughput version. QDs were functionalized with a specific antibody against the P. betae's specific glutathione-S-transferase (GST) protein. On the other hand, GST was conjugated to Rhodamine dye. Ninety six-well polystyrene plates were used as the detection platform. The mutual affinity of the antigen and the antibody brought the CdTe QDs 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%, and was successfully used for high-throughput screening of 96 real samples with consistent results within the course of less than 30 minutes.

A new single-chain variable fragment (scFv) antibody provides sensitive and specific detection of citrus tristeza virus.

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.

Developing of specific monoclonal recombinant antibody fused to alkaline phosphatase (AP) for one-step detection of fig mosaic virus.

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.

Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1.

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.

Generation and molecular docking analysis of specific single-chain variable fragments selected by phage display against the recombinant nucleocapsid protein of fig mosaic virus.

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. Silico 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.

Recombinant-antibody-mediated resistance against Tomato yellow leaf curl virus in Nicotiana benthamiana.

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.

Production of a polyclonal antiserum against recombinant nucleocapsid protein and its application for the detection of fig mosaic virus.

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.

Isolation and Characterization of Novel Phage Displayed scFv Fragment for Human Tumor Necrosis Factor Alpha and Molecular Docking Analysis of Their Interactions.

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.

Antibody-mediated resistance against plant pathogens.

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.