• BMB Reports
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• v.34 no.5
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• pp.452-456
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• 2001

Surface plasmon resonance has been used for a biospecific interaction analysis between two macromolecules in real time. Determination of an antibody that is capable of specifically interacting with the native form of antigen is very useful for many biological and medical applications. Twenty monoclonal antibodies against the $\alpha$ subunit of E. coli DNA polymerase III were screened for specifically recognizing the native form of protein using surface plasmon resonance. Only four monoclonal antibodies among them specifically recognized the native $\alpha$ protein, although all of the antibodies were able to specifically interact with the denatured $\alpha$ subunit. These antibodies failed to interfere with the interaction between the $\tau$ and $\alpha$ subunits that were required for dimerization of the two polymerases at the DNA replication fork. This real-time analysis using surface plasmon resonance provides an easy method to screen antibodies that are capable of binding to the native form of the antigen molecule and determine the biological interaction between the two molecules.

• YAKHAK HOEJI
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• v.40 no.4
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• pp.422-427
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• 1996

The anti-Ex-A IgG was specifically labeled with stable isotopes, DL-His-2,4-$d_2$, L-Phe-$d_5$, L-Trp-$d_5$, L-Tyr-2,6-$d_2$ and L-[1-$^{13}C$]Trp, by growing hybridoma cell in serum-free medium. By use of NMR spectroscopy with selectively labeled Fab fragment, we applied a paratope mapping on antigen-antibody complex. Assignments of the observed carbonyl carbon resonances have been determined by using $^{13}C$-$^{15}N$ double labeling method in order to assign the Trp resonances. Photo CIDNP was also applied to investigate the antigen-binding site(s) on the surface residues of antibody. We found that Trp 36, which is located at the $V_H$ domain, is an important residue to bind to Ex-A, however, two Tyr on the surface of anti-Ex-A IgG plays no crucial role to bind to antigen. On the basis of these results, we demonstrate that stable isotope-aided NMR strategy can be extended to molecular structural analyses of the complex of an Fab fragment and a protein antigen.

• Parasites, Hosts and Diseases
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• v.41 no.3
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• pp.175-177
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• 2003

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a $6{\;}{\times}{\;}His$ tagged protein (Ncp43p) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1％) were found to react with Ncp43p positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43p could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T.gondii infections in other mammals.

• Korean Journal of Microbiology
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• v.40 no.1
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• pp.23-28
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• 2004

Antigenic analysis of Herpes simplex type 2 virus was performed and its major antigen was localized using an immunoelectron microscopy. Antigens of 32, 43, 59 and 69 kDa were constantly expressed during the course of infection for 48 hr in the infected Vero cell. An antigen of 51 kDa was turned out to be the major one in inducing a immune response in Western-blot analysis. The 51 kDa antigen was localized on the surface of HSV-2 by immunoelectron microscopy using colloidal golds and anti-HSV 2 polyc1onal antibody. Immunofluorescence assay indicated that viral antigens were found throughout the infected cell and, especially, on the surface of the cell.

• Journal of Convergence for Information Technology
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• v.10 no.6
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• pp.164-176
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• 2020

Efficient production of antigen specific cytotoxic T cells is critical for appropriate adoptive immune response. In vitro culture and expansion of human T lymphocyte clones are very sophisticated and subtle procedure in immune cell therapy and hard to control. Therefore, many groups devoted their efforts to manipulate artificial antigen presenting cells (aAPCs) that can induce T cell activation and clonal expansion. To mimicking of natural antigen-presenting cells, aAPCs encompass basic signal molecules required for T cell activation: MHC:antigen complexes, co-stimulatory molecules and soluble immune modulating molecules. Orchestrated organization of these molecules is important for efficient T cell activation. Here, we discuss how those molecules have been incorporated in several aAPC models, but also how physical properties od aAPC are important for interaction with T cells.

• Korean Journal of Medicinal Crop Science
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• v.17 no.2
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• pp.133-138
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• 2009

Nelumbo nucifera (lotus) is known to be a useful medicinal plant and leaf extract contains several flavonoids and alkaloids. To analyze the effect of Nelumbo nucifera leaves extract (NNL) on the HBsAg production, we treated NNL on HepG2.2.15 cells which contain the hepatitis B viral genome and secrete surface antigen into media. NNL suppressed the production of hepatitis B surface antigen as a dose-dependent manner. To analyze the effect of NNL on HBV DNA replication, PCR analysis was performed. NNL was not affected the HBV DNA replication and HBsAg mRNA expression. To understand the effect of NNL on the production of HBsAg, we carried out the analysis of lipid-metabolizing gene expression using one-step RT-PCR. NNL reduced the gene expression of FASN and SREBP2 and increased the expression of LDLR. Triglyceride content of HepG2.2.15 cells was not decreased by treatment of NNL. This result suggests a possibility that NNL may have an effect for the inhibition of hepatitis B surface antigen by modulation of lipid and cholesterol metabolism.

• IMMUNE NETWORK
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• v.6 no.2
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• pp.102-110
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• 2006

Background: Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective anti-tumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific $CD8^+$ T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. Methods: Bone marrow-derived DC (EM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of ${\beta}$-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using ${\beta}$-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. Results: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. Conclusion: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I- restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.

• Archives of Pharmacal Research
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• v.21 no.5
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• pp.521-526
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• 1998

The physicochmical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10-15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAg by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCI density gradient method was 1.19g/ml and the isoelectric point by Mono $P^{TM}$ HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and it smajr component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derved r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.

• JSTS:Journal of Semiconductor Technology and Science
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• v.7 no.3
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• pp.151-160
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• 2007

We report the label-free biomolecules detection based on nanomechanical micro cantilevers operated in dynamic mode for detection of two marker proteins (myoglobin and creatin kinase-MB (CK-MB)) of acute myocardical infarctions. When the specific binding between the antigen and its antibody occurred on the fuctionalized microcantilever surface, mechanical response (i.e. resonant frequency) of microcantilevers was changed in lower frequency range. We performed the label-free biomolecules detection of myoglobin and CK-MB antigen in the low concentration (clinical threshold concentration range) as much as 1 ng/ml from measuring the dynamic response change of micro cantilevers caused by the intermolecular force. Moreover, we estimate the surface stress on the dynamic microcantilevers generated by specific antibody-antigen binding. It is suggested that our dynamic microcantilevers may enable one to use the sensitive label-free biomolecules detection for application to the disease diagnosis system based on mechanical immuno-sensor.

• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.78-84
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• 2019

Cell therapeutic agents for treating degenerative brain diseases using neural stem cells are actively being developed. However, few systems have been developed to monitor in real time whether the transplanted neural stem cells are actually differentiated into neurons. Therefore, it is necessary to develop a technology capable of specifically monitoring neuronal differentiation in vivo. In this study, we established a system that expresses cell membrane-targeting red fluorescent protein under control of the Synapsin promoter in order to specifically monitor differentiation from neural stem cells into neurons. In order to overcome the weak expression level of the tissue-specific promoter system, the partial 5' UTR sequence of Creb was added for efficient expression of the cell surface-specific antigen. This system was able to track functional neuronal differentiation of neural stem cells transplanted in vivo, which will help improve stem cell therapies.