• Title/Summary/Keyword: microplate

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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.

Monitoring of Environmental Arsenic by Cultures of the Photosynthetic Bacterial Sensor Illuminated with a Near-Infrared Light Emitting Diode Array

  • Maeda, Isamu;Sakurai, Hirokazu;Yoshida, Kazuyuki;Siddiki, Mohammad Shohel Rana;Shimizu, Tokuo;Fukami, Motohiro;Ueda, Shunsaku
    • Journal of Microbiology and Biotechnology
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    • v.21 no.12
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    • pp.1306-1311
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    • 2011
  • Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 ${\mu}g$/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.

Characterization of Vibrio ponticus KJS1 Isolated from Larvae of Maroon clownfish, Premnas biaculeatus (Maroon clownfish, Premnas biaculeatus 자어로부터 분리한 Vibrio ponticus KJS1의 특성)

  • Kim, Jong-Su;Rho, Sum;Kang, Bong-Jo
    • Journal of fish pathology
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    • v.20 no.1
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    • pp.25-31
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    • 2007
  • Vibriosis is one of the most important diseases affecting marin fish. In this study, a bacterium belonging to the Vibrio were isolated from maroon clownfish (Premnas biaculeatus) larvae (3~7 days after hatching). The bacterium was identified as Vibrio ponticus KJS1 by 16S rRNA gene sequence analysis. The result of biochemical test using the GN2 microplate (BioLog Inc, USA) showed that it has the ability to use 35 substrates including dextrin and sucrose was detected, could not degrade 52 substrates including α-cyclodextrin, and showed the weak ability to use 7 substrates including glycogen. It was sensitive to oxolinic acid, flumequine, ciprofloxacine, ofloxacin, norfloxacin, nalidixic acid, pefloxacin, and doxycycline hydrochloride.

Detection of Antibody to Infectious Laryngotracheitis Virus by Enzyme Linked Immunosorbent Assay (효소면역법에 의한 닭 전염성 후두기관염 바이러스 항체 측정에 관한 연구)

  • 임숙경;위성하;최정옥;고홍남
    • Korean Journal of Veterinary Service
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    • v.15 no.1
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    • pp.32-45
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    • 1992
  • In order to establish and enzyme-linked immunosorbent assay to ILTV, field virus strain of ILTV was propagated in chorioallantoic membrane of the embryonated eggs. purified and used as antigen. The antisera selected from the field samples and immunized chickens based on serum neutralization test were used as the standard positive and negative sera in all tests. It was found that optimal antigen concentration was $2{\mu}g$ of protein per well and a 1 : 100 dilution of standard serum showed low background optical density with negative serum and high P/N values of positive sera. A 1 : 500 dilution of the rabbit anti-chicken IgG peroxidase conjugate produced a high P/N values and thirty minutes was chosen as suitable time to read the optical density of the enzyme substrate reaction and optical density was consistent during the 16 hours after stopper was treated. When coated antigen was kept on microplate for varying time up to 16 hours at $4^{\circ}C$ or $37^{\circ}C,$ no significant difference was observed between the treatment. The coated antigen could be kept without change of antigenicity for at least one month at $-70^{\circ}C,\; -20^{\circ}C,\; 4^{\circ}C$ and room temperature. When blocking buffer contanining bovine serum albumin was mixed directly with conjugate and serum at 10% level induced higher P/N values compared to blocking antigen coated microplate with the same blocking buffer. The coefficience of correlation between ELISA and SN test was 0.577. When antibody response of chickens, vaccinated with ILTV, was examined by ELISA and SN test, antibody rising and decay pattern between the two test was similar until 11 weeks of age. However 12 weeks onward antibody titer checked on by SN test was slightly lower than that tested by ELISA.

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Microplate Fixation without Maxillomandibular Fixation in Double Mandibular Fractures

  • Song, Seung Wook;Burm, Jin Sik;Yang, Won Yong;Kang, Sang Yoon
    • Archives of Craniofacial Surgery
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    • v.15 no.2
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    • pp.53-58
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    • 2014
  • Background: Maxillomandibular fixation (MMF) is usually used to treat double mandibular fractures. However, advancements in reduction and fixation techniques may allow recovery of the premorbid dental arch and occlusion without the use of MMF. We investigated whether anatomical reduction and microplate fixation without MMF could provide secure immobilization and correct occlusion in double mandibular fractures. Methods: Thirty-four patients with double mandibular fractures were treated with open reduction and internal fixation without MMF. Both fracture sites were surgically treated. For bony fixations, we used microplates with or without wire. After reduction, each fracture site was fixed at two or three points to maintain anatomical alignment of the mandible. Interdental wiring was used to reduce the fracture at the superior border and to enhance stability for 6 weeks. Mouth opening was permitted immediately. Results: No major complications were observed, including infection, plate exposure, non-union, or significant malocclusion. Five patients experienced minor complications, among whom the only one patient experienced a persistant but mild malocclusion with no need for additional management. Conclusion: This study showed that double mandibular fractures correction with two-or three-point fixation without MMF simplified the surgical procedure, increased patient comfort, and reduced complications, due to good stability and excellent adaptation.

Enzyme-Linked Immuno-Sorbent Assay for Bovine Caseins (우 Casein의 면역효소분석법)

  • 염행철
    • Korean Journal of Animal Reproduction
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    • v.16 no.2
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    • pp.87-102
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    • 1992
  • A rapid, sensitive, and specific enzyme-linked immuno-sorbent assay (ELISA) for bovine casein was developed. Biotinylated casein and peroxidase-conjugated avidin were used in the assay with antibody separated from yolks of immunized hens. Caseins were biotinylated with sulfo-N-hydroxy succinimido biotin and peroxi-dase-conjugated avidin bound the biotinylated casein which became bound to immobilized anti-body on a microplate. The antibodies were specific for bovine $\alpha$- and $\beta$-caseins, and their cross-reactivities with whey proteins, IgG, and serum albumin from bovine were not detectable by ELISA and Western blot. Various sensitivities ranging from 2ng/ml to 20${\mu}\textrm{g}$/ml of casein were achieved, and were controlled by adding vanous concentrations of the biotinylated casein. Parallelism was observed between standard and sample curves. The coefficients of variation of intra-assays and inter-assays from the most sensitive assay were 5.5 and 5.7%, respectively, at the 50% displacement. Casein contents of peripaturient milk samples showed that casein secretion rapidly increased 3d prepartum.

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Microplate-Based Oxygen Radical Absorbance Capacity (ORAC) Assay of Hydrophilic and Lipophilic Compartments in Plasma

  • Kwak Ho Kyung;Blumberg Jeffrey B.;Chen Chung Yen;Milbury Paul E.
    • Nutritional Sciences
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    • v.9 no.1
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    • pp.48-54
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    • 2006
  • Methods have been developed to evaluate the total antioxidant capacity of foods and plasma but limitations are associated with their ability to determine precisely the contribution of lipophilic antioxidants in a lipid milieu as well as interactions among them Thus, we modified the Oxygen Radical Absorbance Capacity (ORAC) assay to determine the peroxyradical scavenging ability of both hydrophilic and lipophilic compartments in plasma The hydrophilic ORAC assay was performed in a phosphate buffer system utilizing 2,2'-azobis (2-amidinopropane) dihydrochloride as a peroxyradical generator and fluorescein as the target The lipophilic ORAC assay was carried out in a dimethylsulfoxide :butyronitrile (DMSO/BN, 9:1 v/v) system using 2,2'-azobis (2,4-dimethyl valeronitrile) as a peroxyradical generator and BODIPY C11 581/591 as the target Analyses were conducted in bovine serum supplemented with water - and lipid - soluble antioxidants and in human plasma. Albumin (0.5$\sim$5 g/dL) and uric acid (0.1$\sim$0.5 $\mu$mol/L) increased hydrophilic ORAC values in a dose-dependent fashion ($R^{2}$=0.97 and 0.98, respectively) but had no impact on lipophilic ORAC values. $\alpha$-Tocopherol (15$\sim$200 $\mu$mol/L) increased lipophilic ORAC values in a dose-dependent fashion ($R^{2}$=0.94); neither $\alpha$-tocopherol nor $\beta$-carotene had an impact on hydrophilic ORAC values. However, addition of $\beta$-carotene at physiological concentration (0.23$\sim$1.86 $\mu$mol/L), either alone or in combination with other carotenoids, had no significant impact on lipophilic ORAC values. Thus, while assays of 'total antioxidant capacity' in biological matrices would be a useful research and clinical tool, existing methods are limited by the lack of complete responsiveness to the full range of dietary antioxidants.

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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The Isolation of Acetylcholinesterase Inhibitory Constituents from Lycoris radiata using On-line HPLC-biochemical Detection System

  • Yang, Hee-Jung;Yoon, Kee-Dong;Chin, Young-Won;Kim, Young-Choong;Kim, Jin-Woong
    • Natural Product Sciences
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    • v.16 no.4
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    • pp.228-232
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    • 2010
  • Bioactivity-guided fractionation using on-line HPLC biochemical detection system on $CHCl_3$-soluble fraction of Lycoris radiata led to the isolation of deoxylycorenine (1), O-demethylhomolycorine (2), galanthamine (3), lycoramine (4), mixture of $6{\alpha}$-and $6{\beta}$-haemanthidine (5), and lycorine (6), identified by spectroscopic data and physicochemical property. Among the isolated compounds, 1, 3 and 6 showed acetylcholinesterase inhibitiory activities with $IC_{50}$ values of 18.0, 12.0 and $16.6\;{\mu}M$, respectively, in in vitro colorimetric microplate assay.

Sandwich ELISA 방법을 이용한 숙주유래 단백질의 검출

  • 성혜윤;김창민;민홍기;용군호
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 1994.04a
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    • pp.254-254
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    • 1994
  • 현재 국내에서 시판되고 있는 생물공학 의약품매 혼입될수 있는 숙주유래 단백질을 검출하기 위하여 숙주계로 사용되고 있는 Saccaromyces cerevisiae KCTC 1720과 Escherichis coli k12의 total protein을 분리 정제하여 토끼와 guinea pig으로부터 total protein 항체를 얻었다. 이때 토끼항체의 단백질 농도는 yeast의 경우에 4.05mg/m1, E. coli의 경우에 7.14mg/m1이었고, guinea pig의 단백질농도는 yeasat의 경우에 1.90mg/m1이었고 E. coli의 경우에 7.17mg/m1이었다. S. cerevisiae와 E. coli를 숙주로 하여 생산된 생물공학 의약품의 숙주유래 단백질을 검출하기 위하여 guinea pig항체를 96 well microptate에 흡착시키고 검체와 토끼항체의 순으로 microplate에 첨가하는 방법인 sandwich ELISA방법올 사용하였다. 이 방법을 생물공학 의약품의 숙주유래 단백질 검출에 적용한 결과 사람 성장 호르몬의 경우에는 5ng/vial 이하로 검출되었다. 또한 생물학적 제제 생물공학 제품의 경우에는, B형 간염백신제재와 인터페론 감마는 1ng/vial 이하로 검출되었고 인터페론 알파의 경우에는 25ng/vial이하로 검출되었다. 또한 이 방법은 현재 개발되어 시판되고 있는 생물공학 의약품 내에 혼입된 숙주유래 단백질을 검출하는데 쓰일 것이다.

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