• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.75.2-75
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• 2003

To develop an effective protection strategy against tomato bushy stunt virus (TBSV), tobacco plants expressing single-chain Fv antibodies (scFv), were established. A previous had shown that the replication activity of viral replicase was inhibited by the selected scFvs. Moreover, no systemic symptom was found after virus inoculation on leaves of wt N. benthamiana infiltrated with an Agrobacterium suspension resulting i3l expression of the scFvs. However, control plants showed systemic symptoms. In this study the localization of the scFvs within two transgenic plant lines, (CP28H3, CP-P55) was demonstrated using immunogold labelling. The gold particles, indicating the presence of scFv, were mostly found In the cytoplasm of the plant cells including chloroplasts and in the cell walls. However, they were hardly found in the vacuole, nucleoplasm and intercellular spaces. Gold particles often accumulated in either the cytosol or chloroplasts showing a specific labeling, There was no difference in type of gold labeling between both transgenic lines. The localization of the scFv in the cytoplasm further conforms the inhibition of the RNA-dependent RNA polymerase (RdRp) by the selected scFv because it is known that the RdRp is localized to membraneous cytosolic structures.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.10-17
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• 2016

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

• BMB Reports
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• v.42 no.11
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• pp.731-736
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• 2009

The generation of functional recombinant antibodies from hybridomas is necessary for antibody engineering. However, this is not easily accomplished due to high levels of aberrant heavy and light chain mRNAs, which require a highly selective technology that has proven complicated and difficult to operate. Herein, we attempt to use an alkaline phosphate (AP)-fused form of single-chain variable fragment (scFv) for the simple identification of a hybridoma-derived, functional recombinant antibody. As a representative example, we cloned the scFv gene from a hybridoma-producing mouse IgG against branched-chain keto acid dehydrogenase complex-E2 (BCKD-E2) into an expression vector containing an in-frame phoA gene. Functional recombinant antibodies were easily identified by conventional enzyme-linked immunosorbent assay (ELISA) by employing scFv-AP fusion protein, which also readily serves as a valuable immuno-detective reagent.

• IMMUNE NETWORK
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• v.3 no.1
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• pp.23-28
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• 2003

Background: Intracellular antibody specific to hepatitis B virus X protein (HBx) might be useful for studying the role of HBx in hepatocellular carcinogenesis and HBV replication. Methods: With variable region genes for H7 monoclonal anti-HBx Ab, we constructed a vector for bacterial expression of single chain Ab (scFv) and a vector for eukaryotic cell expression of it. The expression of H7 scFv and its binding activity against HBx was examined by immunoblotting and immunofluorescence microscopy. Results: H7 scFv expressed in bacterial cells retained reactivity to HBx. We demonstrated its intracytoplasmic expression in CosM6 eukaryotic cells. Conclusion: This is the first study showing the expression of intracellular anti-HBx Ab in eukaryotic cells. H7 scFv may be a good tool to study the function of HBx in HBV infection.

• Journal of Plant Biotechnology
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• v.8 no.1
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• pp.9-14
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• 2006

As an alternative method to produce low cost reagents for immunodiagnosis and protect the plants from viral disease, a gene encoding a single chain variable fragment(scFv) recombinant antibody targeted to the coat protein of cucumber mosaic virus (CMV) was expressed in Nacotiana tabacum. The source of the scFv recombinant antibody gene was from spleen tissue of an immunized mouse. The gene was initially cloned into the pCANTAB5E phagemid and expressed in E. coli. In the following study, the antibody gene was subcloned into the plant expression vector, pCAMBIA-1301 and introduced into tobacco leaf tissue via Agrobacterium tumefacients mediated transformation. After transformation, 56 out of 58 plants were shown to carry the desired anti-CMV scFv gene by PCR analysis. Overall, only 12.5% of the 56 putative transgenic plants were found to express the antibody to a detectable level.

• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.18 no.1
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• pp.295-301
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• 2017

Botulinum neurotoxin (BoNT/A) is a neurotoxin that selectively attacks the peripheral cholinergic nerve endings. It is produced by Gram -positive, endospore-forming strict anaerobic bacteria, Clostridium botulinum. Since BoNT/A could be a biothreat agent, as well as a contaminator of food and water supplies, the development of sensitive assays for toxin detection and potent antitoxin for the treatment of intoxication is necessary. In this study, for the purpose of producing monoclonal antibodies (mAbs) that are capable of neutralizing Botulinum neurotoxin type A (BoNT/A), scFv (single-chain variable domain fragment) libraries from the rabbit antisera against BoNT/A was fused to a human IgG. The resulting recombinant scFvIgG antibody protein was expressed in stable cell lines and was purified using a protein A affinity chromatography. The efficacy of scFvIgG mAb was confirmed by ELISA and was evaluated for the neutralization of BoNT/A in vivo. Such an in vivo toxin neutralization assay was performed using mice. Although scFvIgG antibody proteins (10 ug) failed to fully protect the mice challenged with BoNT/A (100,000 $LD_{50}$), it significantly prolonged the survival time. These results suggest that scFvIgG mAb may be capable of neutralizing BoNT/A single-chain variable domain fragment.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1186-1190
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• 2008

Single-chain Fv (scFv) antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells owing to the important biological roles of c-Met in the proliferation of malignancies. Here, we show that the productivity of scFv against c-Met in Escherichia coli is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders (i.e., $V_L$-linker-$V_H$ and $V_H$-linker-$V_L$), expressed them in the cytoplasm of E. coli trx/gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with $V_H/V_L$ orientation was more than five times higher than that with $V_L/V_H$ format. Coexpression of DsbC enhanced the yield of soluble amounts of anti-c-Met scFv protein for both constructs. The purified scFv antibodies of the two different formats exhibited almost the same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with $V_H/V_L$ or $V_L/V_H$ formats and obtained similar results to the case of scFv antibodies.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.148-156
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• 2021

Single-chain antibodies against epidermal growth factor receptor variant III (EGFRvIII) are potentially promising agents for developing antibody-based cancer treatment strategies. We described in our previous study the successful expression of an anti-EGFRvIII scFv antibody in Escherichia coli. However, we could also observe the formation of insoluble aggregates in the periplasmic space, limiting the production yield of the active product. In the present study, we investigated the mechanisms by which growth conditions could affect the expression of the soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures, attempting to maximize production. The secreted scFv molecules were purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and western blot analyses. We used the ImageJ software for protein quantification and determined the antigen-binding activity of the scFv antibody against the EGFRvIII protein. Our results showed that the highest percentage of soluble scFv expression could be achieved under culture conditions that combined low IPTG concentration (0.1 mM), low growth temperature (18℃), and large culture dish surface area. We found moderate-yield soluble scFv production in the culture medium after lactose-mediated induction, which was also beneficial for downstream protein processing. These findings were confirmed by conducting western blot analysis, indicating that the soluble, approximately 30-kDa scFv molecule was localized in the periplasm and the extracellular space. Moreover, the antigen-binding assay confirmed the scFv affinity against the EGFRvIII antigen. In conclusion, our study reveals that low-speed protein expression is preferable to obtain more soluble anti-EGFRvIII scFv protein in an E. coli expression system.

• BMB Reports
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• v.40 no.6
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• pp.875-880
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• 2007

Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody(scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.