• Journal of mucopolysaccharidosis and rare diseases
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• 제2권1호
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• pp.31-31
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• 2016

Purpose: To describle clinical features and enzyme activity of Vietnamese patients with Mucolipidosis type II. Methods: Clinical features, laboratory and plasma lysosom enzyme activity by 4 MU-Fluorometric assay was studied from 2014-2015 at the Northern referral center of Pediatrics - National Children's Hospital. Results: 16 cases (7 girls and 9 boys) were diagnosed with I-cell bases on clinical symptoms and enzyme activities studies. Diagnosis age was $5.93{\pm}4.28$ years, onset age was recognised from birth to 4 years (median 1.25) with the feature of joint stiffness and bone deformation. All cases presented with the feature of joint stiffness, chest deformation and kyphoscoliosis; Fifteen cases (93.7%) had coarse facial features. No patients had hepatosplenomegaly on abdominal ultrasound, 5/15 patients had heart valves disease. Enzyme assay showed ${\alpha}$-Hexosaminidase of $1,885.9{\pm}338.7$ (nmol/mg plasma/17 hrs), ${\alpha}$-Iduronate sulfatase of $4,534.8{\pm}1,062.9nmol/mg$ plasma/4 hrs). Conclusion: Mucolipidosis II seriously affected the life of the patients with skeletal deformities, contractures develop in all joints and cardiac involvement.

• 대한한의학회지
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• 제26권4호
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• pp.12-21
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• 2005

Objectives: In the present study, we investigated whether an aqueous extract of Armeniacae semen induces apoptotic neuronal cell death upon mouse N2a neuroblastoma cells. Methods: 1. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTI) assay. 2. For in situ detection of apoptotic cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAPI) staining. 3. The fraction of cells was revealed by flow cytometric analysis used that. 4. For detection of apoptotic DNA cleavage, DNA fragmentation assay was performed. 5. For detection of bax and bcl-2, Western blot analysis was performed. 6. Caspase enzyme activity was measured using caspase-3 assay. Results: From the present results, N2a neuroblastoma cells treated with Armeniacae semen extract exhibited several characteristics of apoptosis. A treatment of Armeniacae semen extract was shown to increase the expression of Bax, a proapoptotic protein, and the treatment decreased the expression of Blc2, an anti-apoptotic protein. In addition, Armeniacae semen extract increased the caspase-3 enzyme activity. Conclusions: The present results show that Armeniacae semen extract induces apoptotic cell death in mouse N2a neuroblastoma cells.

• 대한본초학회지
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• 제24권3호
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• pp.97-102
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• 2009

Objectives : We determined the anti-inflammatory activity of KCNS-001 that is a herbal formula including 6 medicinal plants and that are used to mitigate atopic dermatitis in oriental medicine. Methods : To evaluate anti-inflammatory effect of KCNS-001, we measured the production of reactive oxygen species (ROS), nitric oxide (NO) and cyclooxygenase-2 (COX-2) in LPS-activated Raw 264.7 cells. Cell viability was determined by MTT assay. The concentrations of ROS and relative level of NO were measured with DPPH assay and Griess reagent, respectively. COX-2 and TNF-$\alpha$ were detected by enzyme immuno assay (EIA) and enzyme-linked immunosorbent assay (ELISA). Results : ROS and NO production were reduced by KCNS-001 in a dose-dependent manner. KCNS-001 significantly inhibited activity of COX-2 and suppressed the release of tumor necrosis factor-alpha (TNF-$\alpha$). Conclusions : These results indicate that the KCNS-001 may have an anti-inflammatory agent for the treatment of various inflammatory disease.

• 대한의생명과학회지
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• 제12권3호
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• pp.225-231
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• 2006

Fibrinolytic enzyme was purified from the fruiting bodies of Lepista nuda, using DEAE-Cellulose chromatography, Phenyl Sepharose chromatography, and Mono-S column chromatography. The substance has a molecular weight of 30006.62 Da as measured by MALD-TOF mass spectrometry. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. The activity of the enzyme was inhibited by PMSF, indicating that the enzyme is a serine protease. No inhibition was found with E-64, pepstatin, and EDTA. It has broad substrate specificity for synthetic peptides. The enzyme was stable up to $30^{\circ}C$. The enzyme hydrolyzes both Aa and y chains of human fibrinogen but did not show any reactivity for $B{\beta}$ chain of human fibrinogen.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.727-727
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• 2005

• 한국식품위생안전성학회지
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• 제9권3호
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• pp.123-131
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• 1994

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of residual gentamicin(GM) in edible animal products. The immunogen(GM-KLH conjugate) and coating antigen(GM-BSA conjugate) were prepared by coupling GM sulfate to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, respectively. Polyclonal antibody to GM was produced in rabbits(New Zealand White, female) by using the immunogen and the antibody titer was measured by indirect ELISA. A competitive ELISA was developed using GM-bovine serum albumin conjugate as a coating antigen, GM(as standards or sample), polyclonal antibody to GM, secondary antibody conjugated with horseradish peroxidase as an enzyme, and H2O2 and o-phenylenediamine dihydrochloride as a substrate and a chromophore, respectively. The detection limit of GM was 10 ng/ml and the standard curve of GM(n=26) was linear up to 10 $\mu\textrm{g}$/ml in this competitive ELISA system. There were no cross-reactivities of the partially purified antibody between GM and the various antibiotice such as amikacin, benzyl-penicillin, chloramphenicol, erythromycin, furazlidone, kanamycin, neomycin, oleandomycin, streptomycin, sulfathiazole and thiamphenicol(CR50<0.05%)

• 대한수의학회지
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• 제29권4호
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• pp.503-515
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• 1989

Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to avian infections bronchitis virus(IBV) were standardized. By adopting the optimized conditions an equation calculating ELISA antibody titers from the observations at single serum dilution was formulated. The purified antigen of IBV Mass-41 strain was dispensed into polystyrene microplate wells at a concentration of 300ng per well($100{\mu}l$) and the plates were coated by completey drying at $37^{\circ}C$. Diluted chicken serum and horseradish peroxidase conjugated goat anti-chicken IgG were added in order in $100{\mu}l$ volumes per well and allowed to react for 30 minutes each at room temperature. Just before use and after each reaction the plates were washed three times with distilled water. Finally o-phenylenediamine solution was added as an enzyme substrate. After incubation for another 15 minutes at room temperature absorbances were read at 492nm. Hyperimmune serum against Mass-41 strain was used as internal reference positive(IRP) serum. After repeated titration of IRP and negative serum, a constant titer of IRP was determined. Serum titrations were carried out for various sample sera together with IRP and negative sera and the observed titers of sample sera were corrected by reflecting the ratio between observed and constant titers of IRP serum. These corrected titers of the sample sera were plotted against sample/positive(S/P) OD ratios. All the OD's measured in the serum titrations were also corrected by substracting negative serum OD. The following equation was formulated from the above data; $Log_{10}$ ELISA titer=$5.568({\log}_{10}S/P)+4.161$ Thus it was possible to calculate ELISA titer by measuring absorbance at 1/400 single serum dilution. Titer measured by cross ELISA tests employing Mass-41 strain and three local IBV isolates were similar. These results suggest that the ELISA tests standardized in this study can be used for evaluating not only vaccinal immunity but also for infection status against fields IBV's.

• 한국동물위생학회지
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• 제15권1호
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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.

• BMB Reports
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• 제29권3호
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• pp.192-199
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• 1996

A rapid and sensitive method has been developed to detect a-fetoprotein (AFP) in human serum by a two-site sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) for human AFP within 1 h. To obtain the most sensitive and reliable MAbs. 12 kinds of MAbs (HPJ1 to HPJ12) as a capture antibody and 4 kinds of horseradish peroxidase (HRP) conjugated MAbs as a tracer antibody were investigated. Among these, only HPJ 10-HRP conjugated HPJ 1 (HPJ 10-HPJ $1^*$) and HPJ 11-HRP conjugated HPJ 10 (HPJ 11-HPJ $10^*$) were chosen as candidates based on the linearity of the standard curve and the sensitivity of the assay. To further characterize these two pairs. MAbs against human AFP were purified from hybridoma cells. conjugated with HRP. and then characterized to optimize the two-site sandwich ELISA The HPJ 10-HPJ $1^*$ pair showed a sensitivity of 1 ng/ml and a better reproducibility than the HPJ 11-HPJ $10^*$ pair when the human sera were incubated at $37^{\circ}C$ for 30 min. The results obtained for 480 randomly selected human sera showed 0~20 ng/ml of AFP values for the normal human sera. To test the utility of our kit, AFP concentrations were determined for 951 human sera (including 85 normal sera, 480 random blood sera, 213 HBsAg-positives. 50 anti-HCV antibody positives. and 47 malignant diseases) and compared with other commercially available AFP detecting kits. These results show that the present two-site sandwich ELISA method is a rapid, sensitive, and reliable procedure for detecting AFP in human serum.

• 대한의생명과학회지
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• 제5권1호
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• pp.11-15
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• 1999

갑상선 질환의 진단에 필요한 갑상선 호르몬의 측정시 thyroxine (T$_4$)농도 측정방법이 가장 흔히 이용되고 있다. 과거에 많이 활용되어온 방사면역측정법 (RH)이 폐기물 처리 및 관리인원 등의 인적, 물적인 문제가 크게 대두되고 있는 점을 감안하면 효소결합면역측정법의 확립은 큰 의미가 있다고 하겠다. 본 연구에서는 thyroxine (T$_4$)에 대한 단클론 항체 (monoclonal antibody)를 이용하여 T$_4$에 horseradish-peroxidase를 화학적으로 결합시킨 microtiter plate에 HRP-conjugate T$_4$와 혈청내의 T$_4$간의 서로 경쟁적인 반응을 이용한 표준곡선을 얻었다. 그리고 이렇게 확립한 기법을 임상적인 갑상선질환 환자의 혈청을 기존의 chemiluminescence 방법을 이용하여 얻은 결과와 비교하여 본 실험에서 확립한 ELISA검사 수치의 유용성을 검토하였으며 이 방법의 임상이용 가능성을 검토하였다.