• 대한수의학회지
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• 제57권1호
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• pp.31-36
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• 2017

Japanese encephalitis (JE) is an important zoonosis caused by the mosquito-transmitted JE virus (JEV), which is a causative agent of reproductive failure in pregnant sows. Detection of JEV antibodies in swine is performed by hemagglutination inhibition (HI), virus neutralization (VN), and the plaque reduction neutralization test (PRNT). The most stringent PRNT is the 90% endpoint PRNT ($PRNT_{90}$). These conventional assays are difficult to carry out in diagnostic laboratories with insufficient instruments or cell culture systems. An alternative assay that is easily conducted and time efficient is required. In this study, we improved the indirect enzyme-linked immunosorbent assay (I-ELISA) with clarified antigen for the detection of JEV antibodies. The I-ELISA results obtained from 175 swine serum samples were compared with HI, VN, and $PRNT_{90}$ results. The sensitivity of I-ELISA was 91.8%, 95.0%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. The specificity of I-ELISA was 92.2%, 94.7%, and 94.7% compared with HI, VN, and $PRNT_{90}$ results, respectively. Moreover, the I-ELISA results were significantly correlated with the HI (r = 0.93), VN (r = 0.95), and $PRNT_{90}$ (r = 0.92) results. These results suggest that the improved I-ELISA is useful for serosurveillance of JEV in swine.

• 대한한의학회지
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• 제31권3호
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• pp.55-65
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• 2010

Background: Nitric oxide (NO) is a reactive free radical gas and a messenger molecule. NO has many physiological functions, but excessive NO production induces neurotoxicity. Objective: The present study investigated whether the aqueous extract of Polygala tenuifolia Willdenow possesses a protective effect on NO-induced apoptosis in human neuroblastoma cell line SK-N-MC. Method: For this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, DNA fragmentation assay, reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and caspase-3 enzyme assay were performed. Result: Sodium nitroprusside (SNP) exposure significantly decreased the viability of cells. The cells treated with SNP exhibited several apoptotic features such as increasing of Bax expression, caspase-3 enzyme activity and inhibiting of Bcl-2 expression. On the other hand, the viability of cells pre-treated with the aqueous extract of Polygala tenuifolia Willdenow was increased dose-dependently. The cells pre-treated for 1 h with the aqueous extract of Polygala tenuifolia Willdenow followed by treatment with SNP showed a decreased occurrence of apoptotic features like decreasing Bax expressions, caspase-3 enzyme activity and increasing Bcl-2 expressions. The aqueous extract of Polygala tenuifolia Willdenow reduced apoptotic cell death in neuroblastoma cell line SK-N-MC through the inhibition of Bax-dependent caspase-3 activation and the increasing of Bcl-2 expression. Conclusion: Based on the present results, it is possible that Polygala tenuifolia Willdenow has therapeutic value for the treatment of a variety of NO-induced brain diseases.

• 한국광학회:학술대회논문집
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• 한국광학회 2002년도 제13회 정기총회 및 2002년도 동계학술발표회
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• pp.234-235
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• 2002

• 한국어병학회지
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• 제21권3호
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• pp.247-257
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• 2008

To understand the activity of non-specific defence factors in cultured Sebastes, the antibacterial effect of the serum, skin mucus and homogenate of various organs from cultured oblong rockfish (Sebastes oblongus) and rockfish(Sebastes schlegeli) against pathogenic bacteria, Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum, and Streptococcus sp. was compared with that of flounder(Paralichthys olivaceus) and seabass(Leteolabrax japonicus). And the activities of proteolytic enzyme, chitinolytic enzyme and haemolycin as non-specific defence factor were investigated on the oblong rockfish and rockfish. Samples from oblong rockfish showed the highest antibacterial activity by lysoplate assay on agar plate mixed with pathogens, followed in descending order by rockfish, seabass, and flounder. Turbidimetric assay was carried to evaluate the lysozyme activity of fish samples against lyophilized cells of Micrococcus lysodeiktikus. The serum, kidney, liver, stomach, intestine and eyeball of oblong rockfish and the mucus and gill of rockfish appeared to have the highest lysozyme activity among the fish strains investigated. All samples except skin mucus, liver, and eyeball of oblong rockfish and rockfish showed proteolytic enzyme activity. Chitinolytic enzyme activity was showed in random sampling and haemolytic activity was remarkable in oblong rockfish. Therefore, Sebastes strain was proved to have effective defense mechanisms based on the antibacterial activities, and lysozyme, proteolytic enzyme, chitinolytic enzyme, and haemolycin were considered to act as the non-specific defence factor of Sebastes.

• KSBB Journal
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• 제21권2호
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• pp.85-89
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• 2006

F. trogii ATCC 200800으로부터 원형질체 분리를 통해 단일균주들을 선별하였으며, 선별된 균주들의 고체배양 및 agar plug assay를 통해 효소생산을 위한 균주를 대량 선별하였다. Agar plug assay를 통해 4일 동안 100여종 이상의 균주를 동시에 배양, 분석이 가능하였으며, 염료분해환을 형성하지 않은 균주는 액체배양 확인 결과 MnP의 생산이 거의 일어나지 않는 것으로 나타났다. 이러한 방법으로 선별된 균주를 이용하여 UV 돌연변이를 통해 모균주로부터 유전적 변이를 유도해 새로운 균주선별을 시도하였으며, 이로부터 모균주와 비교하여 효소생산성이 향상되고 안정성이 증대된 균주를 선별할 수 있었다. 모균주와 선별된 균주의 명확한 유전적 차이를 규명하기보다는 배양시 형태학적 특성이 상이함을 확인하였다. 또한, 선별된 균주를 이용하여 배양액내에 다양한 종류의 inducer를 첨가에 따라 효소생산에 미치는 영향을 확인하였다. 과량의 inducer 물질이 첨가될 경우, 균체성장은 물론 효소생산성도 크게 떨어졌으며, 균체성장이 어느 정도 이루어진 상태 즉, 이차대사가 이루어지는 시점에서 inducer를 첨가할 경우 그 효율이 최대였으며, 동시에 inducer를 첨가할 경우 보다 미량씩 일정량을 나누어 주입할 경우 효소생산에 더 유리한 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.2110-2115
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• 2015

A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

• 대한의생명과학회지
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• 제3권2호
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• pp.115-123
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• 1997

인간 $\alpha$-fetoprotein (AFP)은 간암, 위암, 생식기 종양 및 신경관 이상인 환자를 검사하고 진단하는데 유용한 지표로 알려져 있다. 본 연구에서는 사람의 AFP를 분리정제 하여 폴리클로날 항체를 생산하고 인간 혈장과 양수내의 AFP를 측정하기 위한 경쟁적 효소면역 분석법을 개발하고자 하였다. 친화크로마토그래피법과 SDS-polyacrylamide 전기영동법을 이 용하여 양수로부터 AFP를 분리하였다. 정제된 AFP를 토끼에 주사하여 폴리클로날 항체를 생산하였으며, 이중면역확산법과 Western blot 분석법을 사용하여 본 연구실에서 제조된 항체의 항원 특이성이 대단히 높음을 확인하였다. AFP와 항혈청을 이용하여 표준곡선을 얻었으며, 민감도는 5ng/ml이었고, 작용범위는 5~l,000ng/ml이었다. 분석내 CV는 4.5%이었고, 분석간 CV는 8.5%이었다. 따라서 이러한 결과로 보아 본 연구에서 개발된 경쟁적 효소면역분석법이 AFP를 측정하기에 적절하며, 간암 등의 기초연구에도 많은 기여를 할 것으로 생각된다.

• 한국가축번식학회지
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• 제16권2호
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• pp.87-102
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• 1992

본 연구는 우 casein을 신속, 정확하게 분석할 수 있는 특수 면역효소분석법을 개발하였다. Biotin이 연결된 casein과 peroxidase-conjugated avidin을 사용하였으며 면역화시킨 닭의 난황으로부터 추출한 항체를 이용하여 분석하였다. Sulfo-N-hydroxy succinimido biotin을 사용하여 casein에 biotin을 연결시키고 microplate에 고정한 뒤 peroxidase-conjugated avidin을 결합시켰다. 생산된 항체는 $\alpha$-와 $\beta$-casein에 특이적이었으며, 유청단백질, IgG, 우혈청 알부민과 교차반응은 면역효소분석법과 Western blot에서 나타나지 않았다. 본 분석법의 민감도는 2ng에서 20$\mu\textrm{g}$이었으며 Standard와 시료의 분석시 뚜렷한 평행곡선이 형성되었다. Intra-assay와 Inter-assay의 변이계수는 각각 5.5와 5.7%이었다. 그리고 비유 초기의 casein량을 조사한 결과 분만전 3일경부터 급격히 상승하는 것을 알 수 있었다.

• Osong Public Health and Research Perspectives
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• 제5권4호
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• pp.187-192
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• 2014

Objectives: Measurement of the incidence of the human immunodeficiency virus (HIV) is very important for epidemiological studies. Here, we determined the recency period with the AxSYM avidity assay and the BED-capture enzyme immunoassay (BED-CEIA) in Korean seroconverters. Methods: Two hundred longitudinal specimens from 81 seroconverters with incident HIV infections that had been collected at the Korea National Institute of Health were subjected to the AxSYM avidity assay (cutoff = 0.8) and BED-CEIA (cutoff = 0.8). The statistical method used to estimate the recency period in recent HIV infections was nonparametric survival analyses. Sensitivity and specificity were calculated for 10-day increments from 120 days to 230 days to determine the recency period. Results: The mean recency period of the avidity assay and BED-CEIA using a survival method was 158 days [95% confidence interval (CI), 135-181 days] and 189 days (95% CI, 170-208 days), respectively. Based on the use of sensitivity and specificity, the mean recency period for the avidity assay and BED-CEIA was 150 days and 200 days, respectively. Conclusion: We determined the recency period to estimate HIV incidence in Korea. These data showed that the nonparametric survival analysis often led to shorter recency periods than analysis of sensitivity and specificity as a new method. These findings suggest that more data from seroconverters and other methodologies are needed to determine the recency period for estimating HIV incidence.

• Korean Chemical Engineering Research
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• 제54권3호
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• pp.380-386
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• 2016

본 연구에서는 인플루엔자 바이러스 표면에 존재하는 neuraminidase 효소의 활성을 평가 할 수있는 종이칩 기반의 분석 시스템을 구축하였다. 종이칩의 장점을 살려 분석 전문가와 장비 없이 현장 진단(Point-of-care)이 가능하도록 X-Neu5Ac 기질을 이용한 비색분석법을 통해 시료 내 neuraminidase 효소의 존재를 정량적으로 확인 할 수 있도록 설계 및 제작하였다. Neuraminidase 효소의 활성을 확인할 수 있는 종이칩 센서(Paper-based neuraminidase assay sensor; PNAS) 성능 실험 결과 neuraminidase를 0.004 U/mL 농도부터 검출 가능하였으며, 인간 혈청에 각기 다른 농도로 존재하는 neuraminidase 효소의 양을 활성 평가를 통해 정량적으로 검출할 수 있음을 입증하였다($R^2$ > 0.99). 또한, 보관기간에 따른 종이칩의 안정성 평가 결과 빛이 차단 된 $4^{\circ}C$ 환경에서 보관 시 70일까지 초기 성능이 안정하게 유지됨을 확인하였다. 마지막으로, PNAS 상에서 효소 반응의 신뢰성 평가를 위해 미카엘리스-멘텐 동역학 (Michaelis-Menten kinetics)을 적용하여 X-Neu5Ac 기질에 대한 neuraminidase의 동역학 분석 결과 $K_m$ 값은 $8.327{\times}10^{-3}M$으로 확인되었으며, 이 값은 용액상에서의 효소 반응 속도 계산으로부터 산출된 값과($K_m=8.327{\times}10^{-3}M$) 근사한 수치임을 확인하였다. 본 연구로부터 개발된 종이칩 기반의 neuraminidase 효소 활성 평가 시스템은 인플루엔자 바이러스의 신속하고 안전한 검출에 다양하게 응용 될 수 있을 것으로 생각된다.