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

Raeisi, Hamideh; Safarnejad, Mohammad Reza; Moeini, Pedram; Safarpour, Hossein; Sokhansanj, Y.

doi:10.1007/s00705-020-04813-1

Cited by 7 publications

(13 citation statements)

References 42 publications

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“…Additionally, the Western blotting result showed a single band at the expected size to detect rHrpE, indicating high specificity of scFvH6. Previous studies have demonstrated that scFv antibodies can be reliable tools with high specificity and sensitivity to detect different antigens 17 , 22 , 23 , 31 , 32 , which is in accordance with our results. Additionally, our previous work revealed that the use of specific scFvs against components of T3SS of Xcc can show high specificity for detecting Xcc infection in plants 18 , 33 .…”

Section: Discussionsupporting

confidence: 93%

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

Raeisi,

Safarnejad,

Alavi

et al. 2024

Sci Rep

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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.

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Section: Discussionsupporting

confidence: 93%

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

Raeisi,

Safarnejad,

Alavi

et al. 2024

Sci Rep

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“…Additionally, rAbs can be used as an alternative attractive strategy to mAbs and pAbs. Successful application of scFv antibodies in various ELISA platforms is reported in many studies; for instance, the development of ELISA based on phage-displayed scFv antibodies was reported for detecting B. melitensis ( Hayhurst et al, 2003 ), IFN-γ antigen ( Yang et al, 2018 ), cowpea chlorotic dwarf virus ( Raeisi et al, 2020 ), citrus tristeza virus ( Raeisi et al, 2022b ; Chen et al, 2015b ), the protein components of type three secretion system of Xanthomons citri ( Raeisi et al, 2019 ), B. thuringiensis ( Dong et al, 2018 ) and so on. Moreover, several publications have reported the development of diagnostic VHH-based ELISA assays for rapid diagnosis of specific targets.…”

Section: Development Of Rapid Detection Methods Based On Recombinant ...mentioning

confidence: 99%

“…In this regard, antibodies are exploited due to their high affinity and specific binding activity toward target molecules, which highlights their effectiveness as theranostics tools. In recent decades, recombinant antibody (rAb) technologies have widely received much attention as diagnostic tools ( Xu et al, 2017 ; Shali et al, 2018 ; Fouladi et al, 2019 ; Raeisi et al, 2019 ; Alibeiki et al, 2020 ; Raeisi et al, 2020 , 2022b ; Berry et al, 2022 ). The rAb technologies that involve the construction of large libraries of different antibody fragments, such as fragment antigen-binding (Fab), single-chain fragment variable (scFv), minibodies, and nanobodies, can screen antibodies in vitro based on their binding properties, thus help develop more cost-effective antibodies with high sensitivity and specificity ( Bockstaele et al, 2000 ; Angela Chiew Wen et al, 2016 ; Raeisi et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Rapid-format recombinant antibody-based methods for the diagnosis of Clostridioides difficile infection: Recent advances and perspectives

et al. 2022

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Clostridioides difficile, the most common cause of nosocomial diarrhea, has been continuously reported as a worldwide problem in healthcare settings. Additionally, the emergence of hypervirulent strains of C. difficile has always been a critical concern and led to continuous efforts to develop more accurate diagnostic methods for detection of this recalcitrant pathogen. Currently, the diagnosis of C. difficile infection (CDI) is based on clinical manifestations and laboratory tests for detecting the bacterium and/or its toxins, which exhibit varied sensitivity and specificity. In this regard, development of rapid diagnostic techniques based on antibodies has demonstrated promising results in both research and clinical environments. Recently, application of recombinant antibody (rAb) technologies like phage display has provided a faster and more cost-effective approach for antibody production. The application of rAbs for developing ultrasensitive diagnostic tools ranging from immunoassays to immunosensors, has allowed the researchers to introduce new platforms with high sensitivity and specificity. Additionally, DNA encoding antibodies are directly accessible in these approaches, which enables the application of antibody engineering to increase their sensitivity and specificity. Here, we review the latest studies about the antibody-based ultrasensitive diagnostic platforms for detection of C. difficile bacteria, with an emphasis on rAb technologies.

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“…According to the principle of color development, it is divided into enzyme-linked immunosorbent assay (ELISA) (see Figure 1 ) and immunocolloidal gold assay [ 57 , 58 ]. ELISA can determine the affinity of phage to CpCDV-CP [ 14 ], and ELISA or tissue blot immunoassay (TBIA) is used to detect chickpea virus in infected tissues, to accurately distinguish FBNYV from Luteoviridae (or Geminiviridae) [ 15 ]. However, the ELISA is cumbersome and requires special equipment.…”

Section: Detection Technology Of Chickpea Virusmentioning

confidence: 99%

Identification of New Chickpea Virus and Control of Chickpea Virus Disease

Cun

2022

Evidence-Based Complementary and Alternative Medicine

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Objective. The objective of the study was to discuss the classification, virus characteristics, detection methods, and control measures of chickpea virus, with an aim to provide a theoretical basis for identification of new chickpea virus and control of chickpea virus disease. Methods. The domestic and foreign studies were reviewed, and the virus coat protein or nucleic acid sequence was identified by immunological and molecular diagnostic techniques. Results. There were 14 main types of chickpea viruses attacking, and seven Luteoviridae viruses were reported, namely, chickpea chlorotic stunt virus (CpCSV), bean leafroll virus (BLRV), beet western yellows virus (BWYV), soybean dwarf virus (SbDV), cotton leafroll dwarf virus (CLRDV), cucurbit aphid-borne yellows virus (CABYV), and phasey bean mild yellows virus (PhBMYV). The family Geminiviridae includes chickpea chlorotic dwarf virus (CpCDV), chickpea chlorosis virus (CpCV), chickpea redleaf virus (CpRLV), chickpea yellows virus (CpYV), and mastrevirus. The family Nanoviridae is dominated by the faba bean necrotic yellows virus (FBNYV). The family Bromoviridae includes cucumber mosaic virus (CMV) and alfalfa mosaic virus (AMV). Conclusion. At present, there are mainly 12 types of viruses infecting chickpeas, which are transmitted by leafhoppers or aphids and are associated with symptoms such as yellowing, chlorosis, and stunted pod development, resulting in serious yield loss. Correct use of various molecular diagnostic tools to detect and identify chickpea virus can accurately assess chickpea virus infection and provide a basis for the prevention and treatment of chickpea virus disease.

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

Cited by 7 publications

References 42 publications

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

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

Rapid-format recombinant antibody-based methods for the diagnosis of Clostridioides difficile infection: Recent advances and perspectives

Identification of New Chickpea Virus and Control of Chickpea Virus Disease

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