• The Journal of Korean Society of Virology
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• v.26 no.2
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• pp.251-258
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• 1996

The V3 loop, a hypervariable domain of envelope glycoprotein, has an essential role in viral infectivity and has a major epitope for type-specific neutralizing antibody. In order to investigate genetic diversity of V3 region of gp120 of human immunodeficiency virus type 1 (HIV-1) isolated from Korean patients, DNA sequences encoding the C2 to V3 region were amplified by nested polymerase chain reaction (PCR) from uncultured peripheral blood mononuclear cells obtained from 15 HIV-1 seropositive patients and nucleotide sequences were determined. All nucleotide sequences from fifteen patients were compared with 8 distinctive subtypes (A-H) and another subtype O. Phylogenetic analysis was carried out with PHYLIP ver 3.5 (Dnapars) program. Of the 15 isolates, 14 HIV-1 subjects were clustered with subtype B, while one was clustered with subtype C. Intra-subtype B distance at the nucleotide and deduced amino acid level were maximum 17.7% and 37.0%, respectively. Intra-patient distance at the nucleotide and deduced amino acid level were maximum 7.3% and 17.8%, respectively. Analysis of the nucleotide sequences revealed that Korean types have relatively well conserved sequences. These findings could be useful for assessing the source of infection and developing an AIDS vaccine.

• The Journal of Korean Society of Virology
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• v.26 no.2
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• pp.205-214
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• 1996

한탄바이러스의 내피 단백질 (N)은 이 바이러스에 대한 중요한 항원으로 작용하지만 신증후출혈열 예방과 관련된 작용은 명확히 알려져 있지 않다. 본 연구는 이러한 내피 단백질이 한탄바이러스에 대한 중화 항체를 유도할 수 있는가 하는 관점에서 수행되었다. 한탄바이러스의 내피 단백질을 대장균에서 용해된 형태로 발현하고 이를 단클론 항체를 이용한 면역친화 컬럼으로 분리 정제하였다. 정제된 내피 단백질을 기니픽에 면역하여 항혈청을 얻고 이것의 한탄바이러스에 대한 중화능력을 중화항체 플락 감소법 (plaque reduction neutralization test)을 이용하여 조사한 결과 최고 1:160의 중화능이 있음을 관찰하였다. 이는 한탄바이러스의 내피 단백질이 중화 항체를 유도할 수 있는 epitopes을 가지고 있음을 의리하며 이러한 생각은 본 연구에서 수행한 면역침강법과 N 단백질에 대한 단클론항체를 이용한 면역친화법을 통한 한탄바이러스의 정제 실험 결과에서도 뒷받침되고 있다.

• IMMUNE NETWORK
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• v.9 no.6
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• pp.265-273
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• 2009

Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.133.1-133
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• 2003

Gummy stem blight, caused by Didymella bryoniae occurs exclusively on cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. Through this study, we optimized cultural conditions for mass-production of pycnidiospore by Metal Halide Lamp irradiation. In brief, the mycelial was cultured at $26^{\circ}C$ on PDA, for 2 days under the darkness, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$, a great number of pycnidia was simultaneously formed. Thus produced pycnidiospores were used as immunogen. From fusions of myeloma cell (v-653) with splenocytes from immunifed mice were car ried out. And, two hybridoma cell lines that recognized the immunogen Didymella bryoniae were obtained. One Monoclonal Antibody, Db1, recognized the supernatant and the other monoclonal antibody, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid two MAb Db1 and Db15, were immunotyped and identified as IgG1 and IgG2b, respectively. Titer of MAb Db1 and MAb Db15 was measured absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with Didymella bryoniae but none reacted with other of fungi and CMV, CGMMV Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{3}$ well by indirect ELISA characterization of the MAb Db1, Db15 antigen by heat and protease treatments show that the epitope recognized by the MAb Bb1, Db15 were a glycoprotein.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.21-28
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• 2001

Enzyme linked-immunosorbent assay (ELISA) for a rapid detection of fungi, Aspergillus and Penicillium genera in food, were developed and their efficiencies were approved by detecting artificially contaminated agricultural commodities. Mice were immunized with partially purified Aspergillus flavus extracellualr polysaccharide (EPS) and lymph node cells of the mice were fused with the myeloma cells for production of monoclonal antibodies. Mab 1G11, one of the antibodies, was selected and purified. A sandwich ELISA was established and its detection limit toward A. flavus EPS was 1mg/ml. Among the 59 strains tested (including 18 species of Aspergillus, 16 of Penicillium, 11 of Fusarium, 1 of Absidia, 2 of Alternaria, 2 of Candida, 2 of Cladosporium, 2 of Geotrichum, 2 of Mucor, 2 of Rhizopus, 1 of Trichoderma), species of Aspergillus and penicillium had a high reactivity with Mab 1G11 even up to 10,000 times dilution of culture broths. The other genera except Cladosporium resinae showed no reactivity, thus Mab 1G11 was specific to the genera of Aspergillus and Penicillium. The epitope of A. flavus EPS against monoclonal Mab 1G11 was on the carbohydrate moiety when 1 to 100$\mu g/g$ A. flavus EPS were put into rice, potato, and mandarin orange, the average recoveries detected by sandwich ELIA were 123, 59, and 76%, respectively. Correlation was found to be linear between the EPS, and mycelium of A. flavus and Penicillium citrinum grown in a liquid medium (r=0.87 and 0.96), and also between the EPS and colony forming unit in solid media of rice of potato (r=0.91-0.99).

• Parasites, Hosts and Diseases
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• v.38 no.2
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• pp.75-84
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• 2000

Antibody responses in serum and cerebrospinal fluid (CSF) samples from patients with active and chronic paragonimiasis and in sera from patients on whom follow-up studies were done after praziquantel treatment were analyzed using antigens of Paragonimus westermani prepared from eggs, metacercariae, juveniles of 4-and 7-week old, adult worms and recombinant protein of 28 kDa cruzipain-like cysteine protease (rPw28CCP). The patient sera/CSFs of active and chronic paragonimiasis revealed strong antibody reactions against the crude extracts of 4-and 7-week old juveniles as well as against those from egg and adult. rPw28CCP also showed specific reaction to the sera with active paragonimiasis. After the treatment, levels of specific antibodies in the sera gradually decreased to negative range in most patients. In some cases with persisting high antibody levels, however, the reactions at 27 kDa egg Protein were sustained throughout the observation period of 34 months. The reactions at 35 and 32 kDa in adult extract and rPw28CCP disappeared rapidly after the treatment. Persistent antibody reactions even after successful treatment are provoked by continuous antigenic challenge from eggs which were not resolved by treatment.

• BMB Reports
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• v.29 no.2
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• pp.175-179
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• 1996

Viral surface proteins are known to play an essential role in attachment of the virus particle to the host cell membrane. In case of the hepatitis B virus (HBV) several reports have described potential receptors on the target cell side, but no definite receptor protein has been isolated yet. As for the viral side, it has been suggested that the preS region of the envelope protein, especially the preS1 region, is involved in binding of HBV to the host cell. In this study, preS1 region was recombinantly expressed in the form of a maltose binding protein (MBP) fusion protein and used to identify and visualize the expression of putative HBV receptor(s) on the host cell. Using laser scanned confocal microscopy and by FACS analysis, MBP-preS1 proteins were shown to bind to the human hepatoma cell line HepG2 in a receptor-ligand specific manner. The binding kinetic of MBP-preS1 to its cellular receptor was shown to be temperature and time dependent. In cells permeabilized with Triton X-100 and treated with the fusion protein, a specific staining of the nuclear membrane could be observed. To determine the precise location of the receptor binding site within the preS1 region, several short overlapping peptides from this region were synthesized and used in a competition assay. In this way the receptor binding epitope in preS1 was revealed to be amino acid residues 27 to 51, which is in agreement with previous reports. These results confirm the significance of the preS1 region in virus attachment in general, and suggest an internalization pathway mediated by direct attachment of the viral particle to the target cell membrane.

• BMB Reports
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• v.32 no.5
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• pp.461-467
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• 1999

Recombinant Mycobacterium smegmatis expressing ovalbumin was used to immunize C57BL/6(H-$2^b$) mice, and the humoral immunity against recombinant ovalbumin was analyzed. Antibodies were purified by denatured ovalbumin-conjugated affinity chromatography. The epitopes of the antibodies were screened with a random peptide library displayed on the tip of fUSE5 filamentous phage pIII minor coat proteins. Two peptides, IRLADR and SPGAEV, were selected predominantly by the recognition of purified antibodies using biopanning methods. The composition of the peptide sequence with the primary structure of OVA revealed that the peptide sequence analogizes to INEAGR, part of the $^{323}ISQAVHAAHAEINEAGR^{339}$ sequence previously reported as the antigenic determinant for murine Band also Th cell epitopes (I-$A^d$ binding). Also, the structures of these mimotopes obtained from restrained molecular dynamic computations resulted in the formation of a $\beta$-turn proven to be a secondary structure of the parent peptide within the ovalbumin molecule, enabling us to confirm the structural similarity. This study demonstrates that immunization with recombinant M. smegmatis can generate neutralizing antibodies identical with those induced by the administration of natural antigenic proteins and supports the potential use of mycobacteria as vaccine delivery vehicles.

• BMB Reports
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• v.30 no.6
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• pp.390-396
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• 1997

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for the quantification of human apolipoprotein A-I (apoA-I) was developed using monoclonal antibodies. For this assay, we used three monoclonal antibodies to trap and detect apo A-I. HDAI16 and HDA15 monoclonal antibodies were used for trapping apoA-I and HDAI8 monoclonal antibody was for detecting apoA-I. These three monoclonal antibodies were produced by immunizing mice with high density lipoprotein (HDL) isolated from human plasma. By immunoblot analysis, these three monoclonal antibodies were specific to apoA-I and showed no cross-reactivities with other plasma proteins. The results of competition assays for epitope cross-reactivity test also verified that these monoclonal antibodies identified separate and distinct epitopes on HDL and apoA-I. Affinity constants of monoclonal antibodies were measured by ELISA. Their association constants ranged from $10^7$ to $10^8$ $M^{-1}$. For this assay, pure apoA-I was isolated by affinity chromatography using monoclonal antibodies. In this sandwich assay, the amount of HRP-labeled HDAI8 bound to apoA-I trapped by HDAI16 and HDAI5 was proportional to apoA-I concentration in the range of 0 to 500ng/ml. ApoA-I concentration in plasma was calculated from the linear regression equation of standard curve. The precision and reliability of the assays are reflected in the low intra-and interassay coefficients of variation that averaged 3.25% and 4.30%, respectively. This assay is sensitive, simple, reproducible, convenient in incubation interval, and does not use radioisotope: thus it can be widely applied in clinical laboratories.

• The Plant Pathology Journal
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• v.20 no.4
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• pp.252-257
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• 2004

We developed a polyclonal antibody based-ELISA system to monitor inocula accurately and rapidly before onset of anthracnose on soybean sprouts. Titer of mouse antisera against conidia of Colletotrichum gloeosporioides, determined by indirect ELISA, was high enough to be detectable up to ${\times}$25,600 dilutions. Both PAb1 and PAb2 had the highest level of reactivity to Colletotrichum gloeosporioides. Absorbance readings exceeded 0.15. Sensitivity of PAb to C. gloeosporioides was precise enough to detect spore concentration as low as 500 conidia/well by indirect ELISA. Both antibodies are very sensitive and highly specific to the target pathogen Colletotrichum gloeosporioides, apparently discriminating other unrelated pathogen, or epiphytes. This kit fulfills the requirements far detecting inocula before infection and onset of anthracnose. Our ELISA system should also be feasible to detect C. acutatum (Mungbean sprouts rot) and G. cingulata (C. gleosporioides), (apple, pepper). It was remarkable that absorbance value was not reduced even after 4 consecutive washings (Fig.4), suggesting that antigenic determinants are on the surface of conidia. Antigenic determinant was characterized by heating and enzyme treatment: Both PAb1 and PAb2 bind to protein epitope that does not contain residue of amino acid, arginine, and Iysine, even though more work needs to be done.