• Toxicological Research
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• v.27 no.2
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• pp.125-131
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• 2011

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1-carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with ${\lambda}$-type light chains. The $IC_{50}s$ of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml ($R^2$ > 0.99) and from 1 to 100 ng/ml ($R^2$ > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

• Journal of Life Science
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• v.14 no.1
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• pp.26-31
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• 2004

Redox factor-1 (Ref-1), known as a redox regulator, controls the DNA binding of AP-1 and is activated in HT29 colon cancer cells by hypoxia in vitro. REF-1 also increases tile DNA binding affinity of Hypoxia-inducible Factor-lalpha$ (HIF-lalpha$), HIF-like Factor (HLF) and early growth response-1 (Egr-1) which induce expression of the genes involved in angiogenesis, so that we speculate that REF-1 may play a role in hypoxia-induced angiogenesis. In this research we tried to detect novel proteins interacting with REF-1 using Yeast two-hybrid system using full-length REF-1 cDNA as bait. As result of such screening we detected 3 positive clones. DNA sequencing and GeneBank search revealed that one of the clones contained the same sequences as M.musculus cDNA for tioredoxin.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.4
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• pp.298-304
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• 2006

In this study, we establish a thin-layer chromatography (TLC) immunostaining method for detecting starfish gangliosides. A new monoclonal antibody (MAb) against AG-2, the major gangliosides molecular species of Acanthaster planci, was produced by fusing hybridoma with splenocytes immunized to liposomal AG-2. BALB/c male mice were injected with liposomal AG-2 antigen, and immunized. Their splenocytos were isolated and fused with hypoxanthine-aminopterine-thimidine (HAT)-sensitive mouse myeloma cells. Hybridomas producing MAb reactive to AG-2 were cloned using the limited dilution method. Established hybridomas were cultured in eRDF medium. Crude MAb produced from clone 8D4 was purified with a magnesium pyrophosphate column. Enzyme immunoassay and TLC immunostaining of AG-2 were performed using the purified MAb. Structurally related gangliosides did not cross-react with anti-AG-2 antibodies. The detection limit of TLC immunostaining was 50 ng of AG-2. The newly established immunostaining method was further developed for detecting AG-2 distribution and qualitative analysis in tissues and/or organs. Our results show that the majority of AG-2 is present in the stomach of male A. planci, while AG-2 is distributed not only in the stomach but also in the the pyloric caeca of female A. planci.

• Plant Breeding and Biotechnology
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• v.6 no.4
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• pp.313-320
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• 2018

Rice is the staple food of more than 50% of the world population. Cultivated rice has the AA genome (diploid, 2n = 24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized transposable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos have been used to generate gene-tagged populations. To date T-DNA and maize transposable element systems have been utilized as main insertional mutagens in rice. The Ac/Ds system offers the advantage of generating new mutants by secondary transposition from a single tagged gene. To enhance the efficiency of gene detection, advanced gene-tagging systems (i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of insertional mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been conducted by collaborative works in Korea.

• The Plant Pathology Journal
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• v.15 no.5
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• pp.270-279
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• 1999

cDNAs complementary to the 3'-terminal regions of two potyvirus genomes were cloned and sequenced. The clone G7 contains one open reading frame (ORF) of 1,338 nucleotides and a 3' untranslated region (3'-UTR) of 403 nucleotides at the 3'-end excluding the 3'end poly(A) tail. The putative viral coat protein (CP) shows 55%-92% amino acid sequence homology to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.0 kb by Northern blot analysis. Five cDNA clones were screened out using GPV2 oligonucleotide as a probe. One of these clones, DEA72, which has a longest cDNA insert, contains one ORF of 1,459 nucleotides and a 3'-UTR of 590 nucleotides at the 3'-end excluding the 3'-end poly(A) tail. The putative viral CP shows 57%-88% amino acid sequence homologies to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.6 kb by Northern blot analysis. The results of immunoblot and Northern blot analyses suggest that almost all of the tested garlic plants showing mosaic or streak symptoms are infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus in variable degrees but rarely infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus. Immunoelectron microscopy using anti-DEA72 CP antibody shows that this potyvirus is about 750 nm long and flexuous rod shaped.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1377-1383
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• 2006

Among 46 Acinetobacter baumannii isolates collected in 2004, two imipenem-resistant isolates were obtained from clinical specimens taken from patients hospitalized in Busan, Republic of Korea. Two carbapenemase-producing isolates were further investigated to determine the mechanism of resistance. These isolates were analyzed by antibiotic susceptibility testing, microbiological tests of carbapenemase activity, determination of pI, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. Two cases of infection by A. baumannii producing the IMP-1 ${\beta}$-lactamase were detected. The isolates were characterized by a modified cloverleaf synergy test and EDTA-disk synergy test. Isoelectric focusing of crude bacterial extracts revealed nitrocefin-positive bands with a pI value of 9.0. PCR amplification and characterization of the amplicons by direct sequencing indicated that the isolates carried a $bla_{IMP-l}$ determinant. The isolates were characterized by a multidrug resistance phenotype, including penicillins, extended-spectrum cephalosporins, carbapenems, and aminoglycosides. These results indicate that the observed imipenem resistance of two Korean A. baumannii isolates was due to the spread of an IMP-1-producing clone. Our microbiological test of carbapenemase activity is simple to screen class B metallo-${\beta}$-lactamase-producing clinical isolates to determine their clinical impact and to prevent further spread. This study shows that the $bla_{IMP-l}$ resistance determinant, which is emerging in Korea, may become an emerging therapeutic problem, since clinicians are advised not to use extended-spectrum cephalosporins, imipenem, and aminoglycosides. This observation emphasizes the importance of having effective control measures in Asian hospitals, such as early detection of colonized patients, isolation procedures, and a judicious use of antibiotics.

• Korean Journal of Clinical Laboratory Science
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• v.49 no.4
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• pp.446-454
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• 2017

Human papillomaviruses (HPVs) are major causes of cervical cancer. Sixteen high risk HPVs, including HPV 16, HPV 18, HPV31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 53, HPV 56, HPV 58, HPV 59, HPV 66, HPV 68, and HPV 69 are found in cervical cancer. HPVs 16 and 18 are mainly presented in 70% of cervical cancer. Therefore, identifying the presence of these high-risk HPVs is crucial. The objective of this study is to establish the HPV ViroCheck for detecting 16 HR-HPVs and genotypes of HPVs 16 and 18, as well as to analyze the analytical performance of HPV ViroCheck. We performed the analytical sensitivity of HPV E6 / E7 genes of 16 high risk HPVs to confirm the limit of detection. Then, a cross reactivity of HPV ViroCheck with microorganisms and viruses related to the cervix were analyzed for analytical specificity. Analytical sensitivity of high risk HPV genotypes ranged from 1 to 100 copies when using cloned DNAs. The limit of detection was 10 cells for both SiHa and HeLa cells. Cervical-related microorganisms and viruses did not show cross-reactivity to HPV DNA. Moreover, the intra- and inter-assay coefficient variations (CVs) were below 5%. In conclusion, HPV Virocheck will be useful for the detection of 16 HR HPVs, as well as HPV 16 and HPV 18 genotypes rapidly.

• Journal of fish pathology
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• v.32 no.2
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• pp.59-67
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• 2019

We developed and subsequently characterized mouse antibodies (MAbs) against viral hemorrhagic septicemia virus (VHSV, genotype IVa), the causative agent of VHS. Five hybridoma clones secreting MAbs against VHSV were established. The MAbs recognized the glycoprotein (MAbs 2C10, 18H4, 23H6, and 30B7) and nucleocapsid protein (15E10) of VHSV by western blot analysis. All five MAbs reacted with VHSV-infected cells and tissue homogenates of VHSV-infected olive flounder (Paralichthys olivaceus) by western blot analysis. Whereas, no reactivity was observed in normal cells and tissue homogenates of normal olive flounder. Moreover, these MAbs reacted with VHSV, but did not react with other fish viruses (infectious hematopoietic necrosis virus, hirame rhabdovirus, spring viraemia of carp virus, infectious pancreatic necrosis virus, marine birnavirus, and nervous necrosis virus) by enzyme linked immunosorbent assay (ELISA). These results indicate that the MAbs are specific to VHSV and can be of value in VHSV detection.

• Korean Journal of Microbiology
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• v.33 no.1
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• pp.1-6
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• 1997

5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase is the sixth enzyme of the shikimate pathway that synthesizes aromatic amino acids. The enzyme is a primary target for the glyphos'lte which is a broad-spectrum and environmetally safe herbicide. As a first step toward development of glyphpsate-resistant EPSP synthase, the EPSP synthase gene(aroA) was amplified by polymerase chain reaction and cluned into pET-25b vector. In this construct. designated pET-aro, the aroA gene is expressed under control of strong T7 promoter. and the EPSP synthase is produced as a fusion protein with pelB leader at N-terminus and HSV-tag and His-tag at C-terminus. When the pET-aro clone was induced to produce the enzyme, it was found that the EPSP synthase was successfully exported to peri plasmic space. The periplasmic transport was greatly dependent on the induction temperatures. Among the induction temperatures examined($25^{\circ}C$, $30^{\circ}C$, $34^{\circ}C$ and $37^{\circ}C$). induction at $34^{\circ}C$ gave rise to maximal periplasmic transport. The recomhinant EPSP synthase could have been purified hy $Ni^{2+}$ -affinity chromatography using the His-tag. and detected hy anti-HSV -tag antibody. The recombinant EPSP synthase also hound to phosphocellulose resin and was eluted hy shikimate 3-phosphate and phosphoenolpyruvate. as expected. The recombinant EPSP synthase purified from phosphocellulose resin showed typical EPSP synthase activity.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2001.10a
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• pp.61-86
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• 2001

All cancers are caused by abnormalities in DNA sequence. Throughout life, the DNA in human cells is exposed to mutagens and suffers mistakes in replication, resulting in progressive, subtle changes in the DNA sequence in each cell. Since the development of conventional and molecular cytogenetic methods to the analysis of chromosomal aberrations in cancers, more than 1,800 recurring chromosomal breakpoints have been identified. These breakpoints and regions of nonrandom copy number changes typically point to the location of genes involved in cancer initiation and progression. With the introduction of molecular cytogenetic methodologies based on fluorescence in situ hybridization (FISH), namely, comparative genomic hybridization (CGH) and multicolor FISH (m-FISH) in carcinomas become susceptible to analysis. Conventional CGH has been widely applied for the detection of genomic imbalances in tumor cells, and used normal metaphase chromosomes as targets for the mapping of copy number changes. However, this limits the mapping of such imbalances to the resolution limit of metaphase chromosomes (usually 10 to 20 Mb). Efforts to increase this resolution have led to the "new"concept of genomic DNA chip (1 to 2 Mb), whereby the chromosomal target is replaced with cloned DNA immobilized on such as glass slides. The resulting resolution then depends on the size of the immobilized DNA fragments. We have completed the first draft of its Korean Genome Project. The project proceeded by end sequencing inserts from a library of 96,768 bacterial artificial chromosomes (BACs) containing genomic DNA fragments from Korean ethnicity. The sequenced BAC ends were then compared to the Human Genome Project′s publicly available sequence database and aligned according to known cancer gene sequences. These BAC clones were biotinylated by nick translation, hybridized to cytogenetic preparations of metaphase cells, and detected with fluorescein-conjugated avidin. Only locations of unique or low-copy Portions of the clone are identified, because high-copy interspersed repetitive sequences in the probe were suppressed by the addition of unlabelled Cotl DNA. Banding patterns were produced using DAPI. By this means, every BAC fragment has been matched to its appropriate chromosomal location. We have placed 86 (156 BAC clones) cytogenetically defined landmarks to help with the characterization of known cancer genes. Microarray techniques would be applied in CGH by replacement of metaphase chromosome to arrayed BAC confirming in oncogene and tumor suppressor gene: and an array BAC clones from the collection is used to perform a genome-wide scan for segmental aneuploidy by array-CGH. Therefore, the genomic DNA chip (arrayed BAC) will be undoubtedly provide accurate diagnosis of deletions, duplication, insertions and rearrangements of genomic material related to various human phenotypes, including neoplasias. And our tumor markers based on genetic abnormalities of cancer would be identified and contribute to the screening of the stage of cancers and/or hereditary diseases