• Bulletin of the Korean Chemical Society
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• v.25 no.6
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• pp.829-832
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• 2004

The pure product of 2,3-diaminophenazine was prepared by the enzyme-catalyzed reaction of ophenylenediamine-$H_2O_2$-horseradish peroxidase and characterized by UV/Vis spectroscopy, IR spectroscopy and NMR spectroscopy. The electrochemical behaviour of 2,3-diaminophenazine on the glassy carbon electrode was studied. The interaction between 2,3-diaminophenazine and deoxyribonucleic acid was studied by cyclic voltammetry method and UV/Vis spectroscopy, which indicated that the interaction between them is intercalation. The influence of reacting time was also studied. The binding ratio of the 2,3-diaminophenazine-DNA complex is calculated to be 1 : 2 and the binding constant is to be $5.07{\times} 10^3L{\cdot}mol^{-1}$ at room temperature.

• Bulletin of the Korean Chemical Society
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• v.18 no.1
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• pp.44-50
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• 1997

A method of dual-signal generation from two different enzymes was developed and utilized to simultaneously perform dual immunoassays in a single microwell. Two enzymes selected as tracers were horseradish peroxidase (HRP) and β-galactosidase (GAL). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) and chlorophenolred-β-galactopyranoside (CPRG) as chromogenic substrates for the respective enzyme were used. Although the two enzymes showed their maximum activities at distinct pH conditions (pH 5.1 for HRP and 7.5 for GAL), the enzyme reactions were able to be concurrently carried out at pH 5.75 in a dual-substrate solution without signal loss. This performance was achieved by increasing TMB concentration two-fold, introducing potassium salt as activator of GAL reaction, and extending total reaction time 50%. The signal generation method was then used for dual-enzyme immunoassays to detect antibodies with co-immobilized Hepatitis C virus antigens (core and NS5) and a Hepatitis B virus antigen (PreS(2)) in a microwell. Dose-response curves of the assays revealed cooperativity between different antigen-antibody complex formation, which suggested that dual immunoassays can only be used for qualitative screening tests unless the antigens immobilized were spatially separated.

• Bulletin of the Korean Chemical Society
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• v.18 no.5
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• pp.515-519
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• 1997

In a competitive enzyme immunoassay, the performance was tested with different analyte-enzyme conjugates (signal generators) in their binding constants to antibody. Analyte (progesterone)-enzyme (glucose oxidase; GO) conjugates were chemically synthesized and purified by using a gel column with an immobilized antibody to progesterone. In an elution range from the column, four peaks were detected by measuring total enzyme activities. Results from further analysis indicated that the first peak contained mainly unreacted GO while the next three peaks conjugated GO with progesterone. These three conjugate preparations were compared in dose-response curves along with the unpurified mixture. The purified conjugates showed higher detection capabilities than did the mixture. Especially, the preparation in the second peak next to the free GO peak improved the detection limit five times. This performance was comparable to that of a progesterone-horseradish peroxidase conjugate that has been identified to have one progesterone ligand.

• Animal cells and systems
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• v.4 no.4
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• pp.353-359
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• 2000

Location of the neurons in the lateral reticular nucleus projecting to dorsal horn of the cervical, thoracic, or lumbar spinal cord was investigated in the rat using the technique of retrograde transport of horseradish peroxidase. The projection was bilateral with ipsilateral predominance. Neurons projecting to the cervical spinal cord were located near the medial, dorsal, and lateral perimeter of the magnocellular division of the lateral reticular nucleus, whereas cells projecting to the thoracic and lumbar spinal cord were localized in the medial and dorsal boundaries of the magnocellular division. The labeled neurons were distinctly multipolar in shape and measured approximately 10-15 $\mu m$ in their greatest transverse diameter. A few neurons were also observed in the subtrigeminal nucleus, whereas few cells were in the parbocellular division. These observations provide an anatomical substrate for the functional implication of the lateral reticular nucleus in the regulation of spinal nociceptive transmission and vascular hemodynamics via the descending pathway into the spinal cord.

• Nuclear Engineering and Technology
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• v.53 no.1
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• pp.142-147
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• 2021

Hydrogen peroxide is a radiolysis product of water formed under gamma-irradiation; therefore, its reliable detection is crucial in the nuclear industry for spent fuel management and coolant chemistry. This study proposes an electrochemical sensor for hydrogen peroxide detection. Cysteamine (CYST), gold nanoparticles (GNPs), and horseradish peroxidase (HRP) were used in the modification of a gold electrode for fabricating Au/CYST/GNP/HRP sensor. Each modification step of the electrode was investigated through electrochemical and physical methods. The sensor exhibited strong sensitivity and stability for the detection and measurement of hydrogen peroxide with a linear range of 1-9 mM. In addition, the Michaelis-Menten kinetic equation was applied to predict the reaction curve, and a quantitative method to define the dynamic range is suggested. The sensor is highly sensitive to H₂O₂ and can be applied as an electrochemical H₂O₂-sensor in the nuclear industry.

• KSBB Journal
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• v.14 no.1
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• pp.82-90
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• 1999

In order to eventually fabricate an analytical system for infectious microorganisms, we synthesized major immunochemical components, utilized them for the construction of model system, and investigated an assay concept for bacterial whole cells. For the preparation of system components, a polyclonal antibody, against Salmonella thompson as model analyte, purified by immuno-affinity chromatography was used to chemically link to streptavidin or an enzyme, horseradish peroxidase(HRP). The antibody and streptavidin was modified with sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate and N-succinimidyl-3-[2-pyridyldithio]propionate(subsequently activated by dithiotheritol), respectively. The modified components were reacted to synthesize antibody-streptavidin conjugates which were then purified on a two-layer chromatography column of diaminobiotin gel and Sephadex G-100. For antibody-HRP conjugates, HRP molecules were activated by $NalO_4$ oxidation and then coupled to immunoglobulin. After stabilizing with ($NaCNBH_3$, the conjugates were purified by size exclusion chromatography on Biogel A5M column. To devise a model system, such produced components were combined with a dot-blotter in which a nitrocellulose membrane($12{\mu}m$ pre size) with immobilized biotin was already located. The analyte (S. thompson cells) was reacted with the both antibody conjugates in a liquid phase, and the complexes formed were captured on the membrane surfaces by applying vacuum in the bottom compartment of the blotter to invoke biotin-streptavidin reaction. Under optimal conditions, the system enabled to identify the analytical concept for bacterial whole cells, and the lower limit of detection was approximately $1{\mu}g/m{\ell}$($10^5-10^6$ cells/m$m{\ell}$). The controlling factors were the concentrations of each antibody conjugate that caused agglutination in the presence of analyte as they increased.

• Biomedical Science Letters
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• v.4 no.1
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• pp.57-63
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• 1998

The objectives of this study were to produce polyclonal antibody to adipocyte plasma membrane (APM) proteins isolated from pig, and to investigate its tissue specificity. Plasma membrane proteins from adipocyte, brain, heart, kidney, liver and spleen were isolated using a self-forming Percoll gradient. Sheep (40kg) was immunized three times at three week interval with the purified APM proteins. Blood was taken from non-immunized sheep (NS) and from immunized sheep at 10 (AS-1), 12 (AS-2), and 14 (AS-3) days after the third immunization. Antisera titers and cross-reactivity against other tissues were determined by enzyme-linked immunosorbent assay (ELISA). Antisera reacted strongly to APM proteins showing detectable amounts of antibody at 1：81,000 dilution. And antisera showed much stronger reactivity to APM proteins than any other tissue plasma membrane proteins. Furthermore, tissue specificity of antisera against APM was reconfirmed by immunoblotting using anti-sheep immunoglobulin G-horseradish peroxidase conjugate as a secondary antibody Antisera to APM proteins showed adipocyte specificity compared with other tissues. In conclusion, polyclonal antibody against APM proteins isolated from pig was developed successfully in our laboratory, and these antisera showed tissue specificity with APM.

• Korean Journal of Plant Tissue Culture
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• v.21 no.2
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• pp.91-97
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• 1994

To improve the productivity of peroxidase (POD) of cell line SP-47 derived from cell suspension cultures of sweet potato (Ipomoea batatas (L) Lam.cv White Star), we optimized culture conditions including the composition and concentration of plant growth regulators and carbon source, and the cell inoculum size. When one g (fr wt) of cells was inoculated into 50 mL TL medium supplemented with l mg/L 2,4-D and 30g/L sucrose in 300 mL Erlenmeyer flask at 25$^{\circ}C$ in the dark (100rpm), the POD activity per g cell dry wt was maximized to be about 6,800 units after 25 days of subculture, which was about 30 times higher than that of intact roots of horseradish plants grown in the greenhouse, but the cell growth was maximum after 15 days of subculture. The protein content per g cell dry wt maintained almost plateau and after 25 days of subculture decreased as culture Proceeded further whereas the POD specific activity (unit/mg protein) was about two times higher after subculture and continuously increased from 12 days to the end of cultures (40 days). The POD isozyme patterns showed almost the same regardless of cell growth stage, but some acidic isozymes were slightly increased after 25 days of subculture. These results indicate that POD activity in suspension cultures of sweet potato is closely associated with cell growth and stresses derived from cell culture renditions and medium depletion. Due to its high POD activity the SPL47cell line seems to be suitable for the mass production of POD.

• Applied Biological Chemistry
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• v.41 no.5
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• pp.384-389
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• 1998

The herbicide propanil and its metabolite, DCA were incubated with oxidative catalysts in the presence or absence of humic monomers to evaluate the incorporation of them into humic substances. Propanil and DCA underwent little or no transformation by oxidatve catalysts in the absence of humic monomers. In the presence of humic monomers, the most effective co-substrate for transformation of propanil was syringic acid by laccase and HRP, that of DCA was catechol by laccase and HRP, and protocatechuic acid by birnessite. The transformation of DCA was the highest when it was incubated with catechol at pH 8.0 during 24 hrs by laccase, and with catechol at pH 3.0 during 2 hrs by HRP, and with protocatechuic acid at pH 5.0 during 2 hrs by birnessite. The DCA transformation increased with increasing concentration of humic monomers. The transformation of DCA was increased with about 5 times when it was incubated with lactase and birnessite together than lactase alone, but that of it was not effected when it was incubated with HRP and birnessite together. When DCA was incubated with dissolved organic carbon in the presence of oxidative catalysts, the transformation of it was not increased by laccase and birnessite but increased by HRP.

• Korean Journal of Food Science and Technology
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• v.41 no.1
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• pp.111-115
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• 2009

In this study we analysed for peanuts in processed foods using an enzyme-linked fluorescent immunoassay (ELFA), and compared the results with labeled ingredients. Crude peanut protein (CPP) was immunized into rabbits to produce specific antibodies(Ab). A sandwich ELFA was established using anti-CPP Ab and Ab-horseradish peroxidase (HRP) conjugate. The cross-reactivities of the Ab toward CPP, peanuts, almonds, soybeans, and walnuts were 100, 9.8, $1.1{\times}10^{-2},\;4.4{\times}10^{-3}$, and 0%, respectively. The samples included 19 items consisting of biscuits, snacks, chocolates, and so on. The results from the sandwich ELFA showed that peanuts were contained in 7 of the processed food items, among which, 5 items were labeled as having peanuts present but 2 items were not. One of the 2 items that was peanut-detected but unlabeled was a biscuit labeled to contain almonds and assayed to contain $2.1{\times}10^{-3}%$ peanuts, which might have been due to the weak cross-reactivity of the Ab toward almonds. The other item was a snack labeled to contain soybeans and assayed to contain 0.098% peanuts, which might have been due to peanut cross-contamination during processing, since the crossreactivity of the Ab toward soybeans was very weak. These results suggest that ELFA is a good tool to detect peanuts in processed foods, and allergens in certain processed foods should be labeled correctly.