• Clinical and Experimental Vaccine Research
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• v.13 no.3
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• pp.242-252
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• 2024

Purpose: In this study, an in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated. The titer of ELISA was calculated using the reference line (RFL) method based on the standard curve drawn using the international reference anti-mouse serum NIBSC (National Institute for Biological Standards and Control) 97/642. Materials and Methods: In the development step, signal to noise was depicted to select the buffers that showed the most appropriate ratio. In the validation step, standard range, precision, dilution linearity, and specificity were confirmed, and RFL and parallel line (PLL) methods were compared in precision and dilution linearity. Results: Coating concentration for plate was achieved at 0.1 ㎍/mL for pertussis toxin (PT), 0.15 ㎍/mL for filamentous hemagglutinin antigen (FHA), and 0.25 ㎍/mL for pertactin (PRN). The signal to noise ratio was 22.02 for PT, 14.93 for FHA, and 8.02 for PRN with 0.25% goat serum in phosphate-buffered saline (PBS) as a dilution buffer, and 2% skim milk in PBS as a blocking buffer. Based on the precision results, we assessed the lower limit of quantification by 1, 0.2, and 1.5 EU/mL concentration for PT, FHA, and PRN which met the ICH (International Council for Harmonization) M10 criteria of a 25% accuracy and total error of 40%. In specificity, homologous serum was spiked into heterologous serum and the accuracy met the criteria. There was no difference in the results between RFL and PLL calculations (p-value=0.3207 for PT, 0.7394 for FHA, 0.2109 for PRN). Conclusion: ELISA validated with RFL calculation method in this study is a relatively accurate assay for mouse humoral immunogenicity test.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.33 no.4
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• pp.301-307
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• 2011

Purpose: If the tooth structure is damaged, then it is impossible to regenerate the tooth. The materials used to restore the tooth structure are not related to the composition of the tooth. The materials used to restore the structure can't replace the natural tooth because they just fill the defective structure. Calcium phosphate cement remineralizes the dentin and almost replaces the natural tooth, but there are some disadvantages. We conducted basic tests with Biomimetic CPC (Bio-CPC) to make sure of the possibility of the biomaterial to remineralize the defective tooth structure. Methods: In this study, the bioactivity and biocompatibility of Bio-CPC were evaluated for its potential value as the bio-material for regeneration of damaged tooth structure by conducting a cell toxicity assay (WST-1 assay), a cytokinesis-block micronucleus assay, a chromosomal aberration test, total RNA extraction and RT-PCR on MDPC-23 mouse odontoblast-like cells. Results: The in vitro cytotoxicity test showed that the Bio-CPC was fairly cytocompatible for the MDPC-23 mouse odontoblast-like cells. Conclusion: Bio-CPC has a possibility to be a new biomaterial and further study of Bio-CPC is needed.

• Food Science and Preservation
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• v.14 no.4
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• pp.419-424
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• 2007

Fruits and vegetable extracts are well-known as healthy foods. Such foods have been used as herbal medicines or traditional therapies for centuries. To assess biological activities in grapes, we examined the immunomodulating activities of water extracts from four kinds of grapes (cultivars Kyoho, Delaware, Campbell, and Niagara). We explored possible antioxidant and anticancer activities using antioxidant assays such as the 1,1-diphenyl-2-picrylhydrazyl (DPPH) reduction assay, the ferric iron reducing ability of plasma (FRAP) assay, a cell proliferation assay, an NO inhibition assay, a wound healing assay, and an IL-4/IL-13 elicitation assay. Methanol extracts of grapes were tested. The results showed that each grape extract had potent antioxidant activities. The grape extracts increased cell proliferation and NO production activities in tumor cell lines. IL-4 and IL-13 cytokine levels were decreased in mouse primary spleen cells by treatment with any extract. These results suggest that grape extracts can be used as biomaterials with immunomodulating activities.

• Journal of Pharmacopuncture
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• v.13 no.3
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• pp.63-71
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• 2010

Objectives: Gallic acid (GA) is the major component of tannin which could be easily founded in various natural materials such as green tea, red tea, grape juice, and Corni Fructus. The purpose of this study is to investigate the effect of Gallic acid (GA) on production of interleukin (IL) in mouse macrophage Raw 264.7 cells stimulated by lipopolysaccharide (LPS). Methods: Productions of interleukins were measured by High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on $xMAP^{(R)}$ (multi-analyte profiling beads) technology. Firstly, cell culture supernatant was obtained after treatment with LPS and GA for 24 hour. Then, it was incubated with the antibody-conjugated beads for 30 minutes. And detection antibody was added and incubated for 30 minutes. And Strepavidin-conjugated Phycoerythrin (SAPE) was added. After incubation for 30 minutes, the level of SAPE fluorescence was analyzed on Bio-plex Suspension Array System and concentration of interleukin was determined. Results: The results of the experiment are as follows. 1. GA significantly inhibited the production of IL-3, IL-10, IL-12p40, and IL-17 in LPS-induced mouse macrophage RAW 264.7 cells at the concentration of 25, 50, 100, 200 uM (p<0.05). 2. GA significantly inhibited the production of IL-6 in LPS-induced mouse macrophage RAW 264.7 cells at the concentration of 50, 100, 200 uM (p<0.05). 3. GA diminished the production of some cytokine such as IL-4, IL-5, and IL-13 in LPS-induced mouse macrophage RAW 264.7 cells. 4. GA did not show the inhibitory effect on the production of IL-$1{\alpha}$ and IL-9 in LPS-induced mouse macrophage RAW 264.7 cells. Conclusions: These results suggest that GA has anti-inflammatory activity related with its inhibitory effects on the production of interleukins such as IL-3, IL-10, IL-12p40, IL-17, and IL-6 in LPS-induced macrophages.

• Journal of Digital Convergence
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• v.16 no.7
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• pp.309-316
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• 2018

The purpose of this study was to investigate the effects of Violae Herba Water Extract (VH) on the proinflammatory factors of lipopolysaccharide (LPS)-induced on the production of inflammatory mediators in RAW 264.7 mouse macrophages cells. We examined effect of Violae Herba Water Extract on the cell viability of RAW 264.7 mouse macrophages cells. Futhermore, After 24 hours treatment we investigated anti-inflammatory effect of Violae Herba Water Extract by the production of Bio-Plex cytokine assay, concentrations of various cytokines such NO, $interleukin(IL)-1{\beta}$, tumor necrosis factor ${\alpha}(TNF-{\alpha})$ and IL-6. The water extract of Violae Herba significantly inhibited the production of NO, $IL-1{\beta}$, $TNF-{\alpha}$ and IL-6 at the concentration of 25, 50, 100 and $200{\mu}g/mL$ in the LPS-induced RAW 264.7 mouse macrophages cells with no changes in the cell viability of them. These results suggest that water extract of Violae Herba has anti-inflammatory effect related with its inhibition of proinflammatory cytokines such as $IL-1{\beta}$, $TNF-{\alpha}$ and IL-6 in the LPS-induced RAW 264.7 mouse macrophages cells. Further research is needed to develop therapeutic agents for inflammatory diseases using Violae Herba.

• Molecular & Cellular Toxicology
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• v.1 no.2
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• pp.99-105
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• 2005

Perchloroethylene (tetrachloroethylene, PCE), a dry cleaning and degreasing solvent, can enter ground-water through accidental leak or spills. PCE can be degraded to trichloroethylene (TCE), 1, 1-dichloroethylene (DCE) and vinyl chloride (VC) as potential bio-product. These compounds have been reported that they can cause clinical diseases and cytotoxicity. However, only a little genotoxic information of these compounds has been known. In this study, we investigated DNA single strand breaks of PCE, TCE, DCE and VC by single cell gel electrophoresis assay, (comet assay) which is a sensitive, reliable and rapid method for DNA single strand breaks with mouse lymphoma L5178Y cells. From these results, $37.5\;{\mu}g/ml$ of PCE, $189\;{\mu}g/ml$ of TCE and $56.4\;{\mu}g/ml$ of DCE were revealed significant DNA damages in the absence of S-9 metabolic activation system meaning direct-acting mutagen. And in the presence of S-9 metabolic activation system, $41.5\;{\mu}g/ml$ of PCE, $328.7\;{\mu}g/ml$ of TCE and $949\;{\mu}g/ml$ of DCE were induced significant DNA damage. In the case of VC, it was revealed a significant DNA damage in the presence of S-9 metabolic activation system. Therefore, we suggest that chloroethylene compounds (PCE, TCE, DCE and VC) may be induced the DNA damage in a mammalian cell.

• Reproductive and Developmental Biology
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• v.40 no.1
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• pp.7-13
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• 2016

Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat ${\beta}$-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.

• Journal of Acupuncture Research
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• v.32 no.3
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• pp.117-126
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• 2015

Objectives : This study was an analysis of the anti-inflammatory, anti-oxidative and skin whitening properties of Gamioncheong-decoctione(GMOCD) extract. Methods : GMOCD(96 g) and 2 L of distilled water were heated at $100^{\circ}C$ for four hours and then concentrated, frozen, freeze-dried, dissolved in distilled water and filtered. The following analysis was completed: cell cytotoxic effect using MTT assay, oxidative products of NO by griess assay, concentration of prostaglandin $E_2(PGE_2)$ by commercially competitive enzyme immunoassay, and cytokines($IL-1{\beta}$, IL-6 and TNF-${\alpha}$) by Bio-Plex$^{(R)}$ Suspension Array System's Bio-Plex Pro$^{TM}$ mouse cytokine, chemokine, and growth factor assay. Anti-oxidative effect was measured using the DPPH method and skin whitening effect using tyrosinase inhibition assay. Results : GMOCD water-extract did not show any toxicity at all doses and cell viability was more than 90 % at all doses. GMOCD water-extract significantly inhibited NO production at doses of 100, 200, $400{\mu}g/ml$, significantly inhibited $PGE_2$ production at doses of 200 and $400{\mu}g/ml$ and reduced the LPS-induced IL-$1{\beta}$, IL-6 and TNF-${\alpha}$ production in a dose-dependent manner. $IL-1{\beta}$ production was significantly reduced at a dose of $400{\mu}g/ml$ and IL-6 production was significantly reduced at doses of 200 and $400{\mu}g/ml$. DPPH free radical scavenging capability had a skin whitening effect rate of more than 50%. Tyrosinase inhibition activity was apparent in a dose-dependent manner. Conclusions : This study suggests that GMOCD water-extract suppressed NO and $PGE_2$ production and inhibited cytokines($IL-1{\beta}$, IL-6 and TNF-${\alpha}$). GMOCD also improved DPPH free radical scavenging capability. GMOCD water-extract increased tyrosinase inhibitory activity in a dose-dependent manner but this was not a statistically significant result.

• Journal of Microbiology and Biotechnology
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• v.23 no.9
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• pp.1327-1338
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• 2013

The humanized anti-hepatocyte growth factor (HGF) monoclonal antibody (mAb) YYB-101 is a promising therapeutic candidate for treating various cancers. In this study, we developed a bioprocess for large-scale production of YYB-101 and evaluated its therapeutic potential for tumor treatment using a xenograft mouse model. By screening diverse chemically defined basal media formulations and by assessing the effects of various feed supplements and feeding schedules on cell growth and antibody production, we established an optimal medium and feeding method to produce 757 mg/l of YYB-101 in flask cultures, representing a 7.5-fold increase in titer compared with that obtained under non-optimized conditions. The optimal dissolved oxygen concentration for antibody production was 70% $pO_2$. A pH shift from 7.2 to 7.0, rather than controlled pH of either 7.0 or 7.2, resulted in productivity improvement in 5 L and 200 L bioreactors, yielding 737 and 830 mg/ml of YYB-101, respectively. The YYB-101 mAb highly purified by affinity chromatography using a Protein A column and two-step ion exchange chromatography effectively neutralized HGF in a cell-based assay and showed potent tumor suppression activity in a mouse xenograft model established with human glioblastoma cells.

• Journal of Food Hygiene and Safety
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• v.27 no.1
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• pp.37-41
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• 2012

The current standard for testing tetrodotoxin (TTX) in foodstuffs is the mouse bioassay (MBA) in Korea as in many other countries. However, this test suffers from potential ethical concerns over the use of live animals. In addition, the mouse bioassay does not test for a specific toxin thus a sample resulting in mouse incapacitation would need further confirmatory testing to determine the exact source toxin (e.g., TTX, STX, brevotoxin, etc.). Furthermore, though the time of death is proportional to toxicity in this assay, the dynamic range for this proportional relationship is small thus many samples must be diluted and new mice be injected to yield a result that falls within the quantitative dynamic range. Therefore, in recent years, there have been many efforts in this field to develop alternative assays. High performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) has been emerged as one of the most promising options. A LC-MS-MS method involves solid-phase extraction (SPE) and followed by analysis using an electrospray in the positive ionization mode and multiple reactions monitoring (MRM). To adopt LC-MS-MS method as alternative standard for testing TTX, we performed a validation study for the quantification of TTX in puffer fish. This LC-MS-MS method showed good sensitivity as limits of detection (LOD) of $0.03{\sim}0.08{\mu}g/g$ and limits of quantification (LOQ) of $0.10{\sim}0.25{\mu}g/g$. The linearity ($r^2$) of tetrodotoxin were 0.9986~0.9997, the recovery were 80.9~103.0% and the relative standard deviations (RSD) were 4.3~13.0%. The correlation coefficient between the mouse bioassay and LC/MS/MS method was higher than 0.95.