• Title/Summary/Keyword: microplate method

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Comparison of Methods for Measuring Histamine by ELISA and HPLC-MS Assay In Vitro (In Vitro에서 히스타민 측정 시 ELISA법과 HPLC-MS 분석법의 비교)

  • Lee, In Hee;Kim, Yoo Hyun
    • Korean Journal of Clinical Laboratory Science
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    • v.47 no.4
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    • pp.306-312
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    • 2015
  • The measurement of histamine is to determine the degree of allergy because the allergic reaction can lead to the release of histamine. In general, the antigen-antibody reaction was quantified by measuring absorbance using a microplate reader. In this study, we compare the method using a general antigen-antibody reaction and the method using a high performance liquid chromatography mass spectrometer (HPLC-MS) of chemical analysis in the measurement of histamine secretion. The cell line used was RBL-2H3, an allergic reaction was induced by stimulation with C48/80 (compound 48/80). Allergy-induced cells degranulation rate was confirmed by measurement of ${\beta}$-hexosaminidase and cytotoxicity was performed for the validity of the experiment. The quantitative determination of histamine showed a significant difference, since the quantitative limit of the measurement by the antigen-antibody reaction was 10.257 ppm while the quantitative limit of the measurement by HPLC-MS was 0.020 ppm. Measurement of histamine in allergic activity and anti-allergy tests showed that the HPLC-MS analysis rather than the analysis of the antigen-antibody reaction is a more precise and accurate test.

A detection method for vibrio vulnificus using monoclonal antibodies

  • Chung, Mi-Sun;Rim, Bung-Moo;Boong, Uhm-Tae;Park, Moon-Kook
    • Journal of Microbiology
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    • v.35 no.2
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    • pp.87-91
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    • 1997
  • Monoclonal antibodies were prepared in order to an assay method for Vibrio vulnificus. Sixteen mouse ybridoma cell lines were established by immunization of whole cell antigen to BALB/c mice, fusion with SP2/O myeloma cells, and cloning. Most of them secreted IgM.lambda. antibodies. A sandwich enzyme-linked immunosorbent assay was developed with rabbit anti-V. vulnificus polyclonal antibodies as capture antibody, an IgM monoclonal antibody as detector antibody, and goat anti-mouse IgM-alkaline phosphatase conjugate as developer antibody. The range of detection was 10$\^$4/ to 10 V. vulnificus cells per microplate well. When four related Vibrio species were tested for cross-reactions, V. parahaemolyticus showed 3.5% reactively and V. carchariae, V. fluvialis, and V. furnisii showed negligibal (<1%) cross-reactivity.

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TREATMENTS OF COMMINUTED MANDIBULAR FRACTURES (하악골 분쇄골절의 치료)

  • Jeon, Woo-Jin;Kim, Su-Gwan;Kim, Hyeon-Ho;Kim, Hak-Kyun
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.27 no.1
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    • pp.71-75
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    • 2005
  • This study evaluated retrospectively the treatment method and postoperative complications of communited mandibular fractures. We analyzed the clinical and radiologic data of 14 patients with the comminuted mandibular fractures who were admitted to Chosun University Dental Hospital from January 1998 to December 2003. We reviewed the cause of trauma, fracture sites, treatment methods, and postoperative complications. Thirteen patients (93%) had a successful treatment outcome without complications. Only one patient developed postoperative osteomyelitis requiring early plate removal and sequestrectomy. For the comminuted fractures of mandible, internal fixation using micro- or mini-plate was an effective treatment method with a low incidence of major complications.

Development of an Agar Diffusion Method to Measure Elastase Inhibition Activity Using Elastin-Congo Red

  • Jung Kyung-Hwan;Kim Hyun-Joo
    • Journal of Microbiology and Biotechnology
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    • v.16 no.8
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    • pp.1320-1324
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    • 2006
  • The pancreatic and neutrophil elastases are associated with several illnesses including lung and vascular diseases, various cancers, and pancreatitis. The development of a potent and specific inhibitor to the elastases could lead to new therapies. In this study, an agar diffusion method was modified to include a substrate-dye conjugate (Elastin-Congo red) as a substrate of elastase and an indicator of elastase inhibitory activity. The Elastin-Congo red agar plates consisted of 0.1 % Elastin-Congo red and 2.5% agar. The elastase and elastase inhibitors were simultaneously loaded into wells, ultimately resulting in halo formations in which the halo diameter decreased as the concentration of elastase inhibitor increased. The concentration of elastase inhibitor in the samples, therefore, was inversely proportional to the halo diameters. This simplified method provided an excellent correlation with the standard microplate technique, which uses a chromogenic substrate. The concentration of elastase inhibitor obtained from the culture supernatant of a recombinant elastase inhibitor produced by the yeast Pichia pastoris was easily determined. This study has established a simple modified and inexpensive agar diffusion method that is potentially useful for the identification, quantification, and screening of new elastase inhibitors.

Determination of Remained Host Derived Proteins in the Commercially Available Biotechnological Products Using a Sandwich Enzyme Linked Immunosorbent Assay Method (Sandwich ELISA 방법을 이용한 생물공학의약품에 잔류하는 숙주유래단백질의 검출법개발)

  • 성혜윤;최규실;김창민;민홍기;용군호
    • Biomolecules & Therapeutics
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    • v.2 no.2
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    • pp.120-125
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    • 1994
  • We obtained the total protein antibodies of Saccharomyces cerevisiae KCTC 1720 and Escherichia coli K-12 from the rabbit and the guinea pig to determine the host-derived proteins which may be remained in biotechnological products. The protein concentration of rabbit antibodies was 4.05 mg/mι in the case of yeast, 7.14 mg/mι in the case of E. coli and that of guinea pig antibodies was 1.90 mg/mι in the case of yeast, 7.17 mg/mι in the case of E. coli, respectively. To determine remained host-derived proteins in biotechnological products which produced by the hosts, S. cerevisiae or E. coli, we used a sandwich enzyme linked immunosorbent assay method in 96 well microplate. When the method applied to determine the remained host-derived proteins in commercial biotechnological products, it detected less than 3.5 ng/vial in human growth hormone, less than 1 ng/vial in hepatitis B vaccine and interferon-${\gamma}$ and 2~23 ng/vial in interferon-$\alpha$. The method can be used to determine the remained host-derived protein in biotechnological products.

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Isolation and Microbiological Characteristics of Bacillus megaterium SMY-212 for Prepartion of Black Bean Chungkugjang (검정콩 청국장 조제를 위한 Bacillus megaterium SMY-212의 분리 및 균학적 특성)

  • 손미예;권선화;성찬기;이상원;박석규
    • Journal of Life Science
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    • v.11 no.4
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    • pp.304-310
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    • 2001
  • In order to produce a high quality and functional black bean Chungkugjang, a bacterium which has potent enzyme activities(protease:124.8 U/$m\ell$, $\alpha$-amylase: 78.2U/$m\ell$, glucoamylase; 13.9U/$m\ell$, ), was isolated by using the halo zone method and identified by morphological, cultural and biochemical properties, and then finally confirmed by GP-microplate identification system as Bacillus megatrium SMY-212. The isolated SMY-212 system was also high in the formation of mucoid material and fibrinolytic activity.

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Study on the seroprevalence of Salmonella Pullorum in boiler farms in Daejeon area (대전지역 양계 농가의 추백리 항체 조사)

  • Han, So-Young;Chung, Nyun-Ki;Kim, Jong-Ho;Park, Jong-Min;Han, Su-Jin
    • Korean Journal of Veterinary Service
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    • v.31 no.1
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    • pp.31-35
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    • 2008
  • In order to investigate the seroprevalence of Salmonella Pullorum in boilers reared in Daejeon area, 509 samples were collected from 10 boiler farms randomly selected from 38. The survey was carried out during 6 months from April to October in 2007. Out of 509 chickens, 35 (6.9%) were seropositive and average titer was $2^{4.8}$. The seroprevalence by district was 5.5% (5/90) in Dong-gu, 4.1 % (4/96) in Seo-gu, 7.9% (24/303) in Yuseong-gu, 10.0% (2/20) in Daedeok-gu.

Conditions for Preparing Glycyrrhiza uralensis Extract for Inhibiting Biofilm Formation of Streptococcus mutans

  • Ham, Youngseok;Kim, Tae-Jong
    • Journal of the Korean Wood Science and Technology
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    • v.47 no.2
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    • pp.178-188
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    • 2019
  • Licorice, which has an extensive history of use as an herbal medicine, has been suggested to have oral health benefits. However, to date, no systematic study has been conducted on the preparation method of licorice extracts for oral health. In this study, licorice extracts prepared using water and ethanol were investigated for its ability to inhibit the biofilm formation of Streptococcus mutans. The licorice extract prepared with around 60% ethanol effectively inhibited the biofilm formation of S. mutans. Licorice extracted with 50% ethanol almost completely inhibited the biofilm formation at 1.5 g/L of licorice extract. This inhibitory activity was confirmed in a microplate assay and a flow cell system. Glycyrrhetic acid was extracted from licorice effectively with 60% ethanol concentration. The strong inhibitory activity of glycyrrhetic acid and the synergistic inhibition with glycyrrhizin on biofilm formation were suggested as major reasons for a concentration-specific extraction. These results suggest that licorice extract prepared using around 60% ethanol effectively inhibits the biofilm formation of S. mutans.

Micro-screening Method for the Anticomplement Substances from Natural Resources (천연유래의 항보체 활성물질 선발을 위한 미량탐색법)

  • Oh, Sei-Ryang;Jung, Keun-Young;Lee, Hyeong-Kyu
    • Applied Biological Chemistry
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    • v.39 no.2
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    • pp.147-152
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    • 1996
  • To screen inhibitors on complement system from natural resources, micro-screening method was established by using hemolytic complement assay. Complement fixation reaction was carried out in the microplate system. For standard hemolysis (50% hemolysis) of the classical pathway (CP), hemolysin and complement serum were diluted to $1/75{\sim}1/100\;and\;1/80{\sim}1/120$, respectively, when sheep erythrocytes were $5.0{\times}10^8\;cells/ml$. In case of the alternative pathway (AP), complement serum was diluted to 1/5 and EGTA and $Mg^{2+}$ were added 4 mM, $4{\sim}8\;mM$, respectively, when rabbit erythrocytes were $4.0{\times}10^8\;cells/ml$. Dimethyl sulfoxide was used for the assay of non-aquous soluble compounds or extracts and its final concentration was not more than 1%. Three phenylpropanoids showed anticomplementary activities in proportion to the concentration for both pathways and rosmarinic acid exihibited the highest inhibitory activities: $5.4{\pm}3.6%(0.063\;mM){\sim}95.8{\pm}0.2%(0.5\;mM)\;and\;35.1{\pm}0.9%(0.063\;mM){\sim}95.6{\pm}1.1%(1\;mM)$ on the CP and the AP, respectively.

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Determination of the Antioxidant Capacity of Korean Ginseng Using an ORAC Assay (ORAC Assay 에 의한 인삼의 항산화 활성 연구)

  • Kim, Sung-Hwan;Kim, Young-Mok
    • Journal of the East Asian Society of Dietary Life
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    • v.17 no.3
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    • pp.393-401
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    • 2007
  • This study was performed to investigate the antioxidant activity of Korean ginseng using an ORAC(Oxygen Radical Absorbance Capacity) assay. Four fractions each (80% ethanol, ethyl acetate, water saturated 1-butanol, and water) were obtained from different ginseng samples (White Ginseng: ; 6 yrs-., 5 yrs-., ; Cork Ginseng: ; 5 yrs-., 4 yrs-.). The saponin content of each fraction was quantified by LC/MS, and the antioxidant capacity of the ginseng was measured by the ORAC assay. The ORAC method, which was recently validated using automatic liquid handling systems, has been adapted for manual handling with the use of a conventional fluorescence microplate reader. Furthermore, the ORAC assay provides a direct measure of hydrophilic chain-breaking antioxidant capacity against peroxy radical, which is the exiting and emission of 2,2'-Azobis (2-methylpropionamidine)-dihychloride (AAPH). As a result of our experiments, ginsenosides Rg1 and Rb1 were the two major saponins found in the ginseng samples, and Rc, Rb2, Re, Rd, Rg3, and Rh1 were detected in a small quantities. For the antioxidant capacities of the fractions (80% ethanol, ethyl acetate, butanol, and water), we found that the organic solvent fraction had similar antioxidant capacities, and were higher than the capacity of the water fraction. When determining the similarities in each fraction, only the ethyl acetate fraction showed similarity compared to other fractions (p>0.05). The antioxidant capacity of ginseng may come from phenolic compounds and some nonpolar saponins. However, based on the results of this study, we hypothesize that some acidic polysaccharides and other biological components may contribute to its antioxidant capacity. Additional research is required to determine other possible biological response modifiers that contribute to the antioxidant capacity of ginseng.

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