• The Journal of the Korean Society for Microbiology
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• v.21 no.1
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• pp.151-161
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• 1986

In order to develop sensitive and sepcific assay methods for E. coli heat labile enterotoxin(LT) hybridoma cell lines secreting LT specific monoclonal antibody were obtained. LT was purified from cell lysate of E. coli O15H11. The steps included disruption of bacteria by French pressure, DEAE Sephacel ion exchange chromatography, Sephadex G200 gel filtration, and second DEAE Sephacel ion exchange chromatography, successively. Spleen cells from Balb/c mice immunized with the purified LT and $HGPRT^{(-)}$ plasmacytomas, $P3{\times}63Ag8.V653$ were mixed and fused by 50% (w/v) PEG. Hybrid cells were grown in 308 wells out of 360 wells, and 13 wells out of them secreted antibodies reacting to LT. Among these hybridoma cell 1G8-1D1 cell line was selected since it had produced high-titered monoclonal antibody continuously. By using culture supernatant and ascites from 1G8-1D1 cells the monoclonal antibody was characterized, and an assay system for detecting enterotoxigenic E. coli was established by double sandwich enzyme-linked immunosorbent assay (ELISA). The following results were obtained. 1. Antibody titers of culture supernatant and ascites from 1G8-1D1 hybridoma cells were 512, and 102, 400, respectively by GM1-ELISA and its immunoglobulin class was IgM. 2. The maximum absorption ratio of 1G8-1D1 cell culture supernatant to LT was 90% at $300\;{\mu}g/ml$ of LT concentration. LT concentration shown at 50% absorption ratio was $103.45{\mu}g$ and the absorption ratio was decreased with tile reduction of LT concentration. This result suggests that monoclonal antibody from 1G8-1D1 hybridoma cell bound with LT specifically. 3. The reactivities of 1G8-1D1 cell culture supernatant to LT and V. cholerae enterotoxin(CT) were 0.886 and 0.142(O.D. at 492nm) measured by the GM1-ELISA, indicating 1G8-1D1 monoclonal antibody reacted specifically with LT but not with CT. 4. The addition of 0.1ml of ascites to 0.6mg and 0.12mg of LT decreased the vascular permeability factor to 41% and 44% respectively, but it did not completely neutralize LT. 5. By double sandwich ELISA using monoclonal antibody, as little as 75ng of the purified LT per ml could be detected. 6. The results by assay of detecting LT in culture supernatants of 14 wild strains E. coli isolated from diarrhea patients by the double sandwich ELISA were almost the same level as those by reverse passive latex agglutination.

• The Korean Journal of Food And Nutrition
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• v.26 no.4
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• pp.797-805
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• 2013

The varying characteristics between Korean's commercial and traditional soy sauces may be initiated by raw materials and fermentation techniques such as the koji and mezu process. We have examined properties of polysaccharides isolated from two different soy sauces which were made by the commercial process (CSP-0) and the traditional Korean process (KTSP-0) as well as their macrophage activities. Two polysaccharides have not effected the RAW 264.7 cells viability. The effects of CSP-0 and KTSP-0 on RAW 264.7 cells were demonstrated by the production of nitric oxide (NO), and reactive oxygen species (ROS). The CSP-0 and KTSP-0 significantly augmented NO and ROS productions by RAW 264.7 cells under a dose dependent manner. However, the activity of KTSP-0 was more potent than that of the CSP-0 at $1,000{\mu}g/m{\ell}$. The productions of IL-6 and TNF-${\alpha}$ were determined by real-time PCR and ELISA. mRNA expression levels of IL-6 and TNF-${\alpha}$ by KTSP-0 at $1,000{\mu}g/m{\ell}$ indicated 63 and 71 times higher than negative controls, respectively. Also, the production of IL-6 and TNF-${\alpha}$ by KTSP-0 at $1,000{\mu}g/m{\ell}$ showed 32.1 and 4.5 times higher than those by the CSP-0. To assess phagocytosis activities, the effects of CSP-0 and KTSP-0 on mRNA expression of Fc receptor I and II (FcR I, II) are being determined by RT-PCR products. Only the KTSP-0 showed enhanced expressions of mRNA expression for FcR I in a dose dependent manner, whereas the CSP-0 did not affect either the FcR I or II expressions. The above data lead us to conclude that the macrophage activations of Korean traditional soy sauce polysaccharide are higher than that of the commercial soy sauce polysaccharide.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.22 no.2
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• pp.1-18
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• 2009

Objective : Moutan Cortex (the root bark of Paeonia suffruticosa Andr.) is widely used in oriental medicine as a remedy for inflammation. However, as yet there is no clear explanation of how MC(Moutan Cortex) affects the production of inflammatory cytokine. This study was to determine the effects of Essence extracted MC on the mast cell-mediated inflammatory responses. Method : We observed the effect of MC on compound 48/80-induced histamine release of rat peritoneal mast cells and the effect of administering MC on PCA in rat. We measured the amount of inflammatory cytokine production induced by the phorbol myristate acetate (PMA) plus calcium ionophore(A23187) in the human mast cell line (HMC-1) incubated with various concentrations of MC. The TNF-$\alpha$ protein levels were analysised by Western blot. The TNF-$\alpha$, IL-6 and IL-8 secreted protein levels were measured by the ELISA assay. The TNF-$\alpha$, IL-6 and IL-8 mRNA levels were measured by the RT-PCR analysis. NF-$\kappa$B, phospho-I$\kappa$B and MAPKs were exmined by Western blot analysis. The NF-$\kappa$B promoter activity was examined by luciferase assay. Result : 1. Enzyme immunoassay indicated that MC suppressed histamine secretion of rat peritoneal mast cells. 2. In PCA dependent on IgE, MC had anti-allergic effect of the internal surface of rat skin. 3. Western blot indicated that MC decreased TNF-$\alpha$ protein levels. 4. ELISA indicated that MC decreased TNF-$\alpha$, IL-6 but MC had no significant effect on IL-8 in HMC-1 cells. 5. RT-PCR indicated that MC decreased TNF-$\alpha$, IL-8 but MC had no significant effect on IL-6 in HMC-l cells. 6. Western blot indicated that MC suppressed the induction of MAPKs, NF-$\kappa$B & phospho-I$\kappa$B activity in HMC-1 cells. 7. Luciferase assay indicated that MC suppressed the PMA plus A23187-induced NF-$\kappa$B promoting activityin HMC-1 cells. Conclusion : In this study, we have found that MC is an inhibitor of NF-$\kappa$B, MAPKs & cytokines on the mast cell-mediated inflammatory responses.

• Journal of Food Hygiene and Safety
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• v.33 no.3
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• pp.193-199
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• 2018

In this study, PAT protein of genetically modified maize was prepared from the recombinant E. coli strain BL21 (DE3), and mice were immunized with the recombinant PAT protein. After cell fusion and cloning, two hybridoma cells (PATmAb-7 and PATmAb-12) were chosen since the monoclonal antibodies (Mabs) produced by them were confirmed to be specific to PAT protein in the indirect enzyme-linked immunsorbent assay (ELISA) and western blot tests. There were no cross-reactions of either Mabs to other GM proteins or to the extracts of non-GM maize. The ELISA based on the PATmAb-7 can sensitively detect 0.3 ng/g PAT protein in corn. These results indicate that the developed Mabs can be used as bio-receptors in the development of immunosensors and biosensors for the rapid and simple detection of GM corn adulterated in foods.

• The Korea Journal of Herbology
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• v.23 no.2
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• pp.111-121
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• 2008

Objectives : The present study was carried out to investigate the effects of Astragali radix on the asthma treatment. EAR-I is an extract of astragali radix cultured for one year and EAR-III is an extract of astragali radix cultured for three years. Methods : Two asthma-induced groups mice group was treated respectively with EAR- I and EAR- III. The other group was not. Each group was analyzed in vivo and in vitro experiments. Results : In asthma-induced mice by OVA treatment, the weight of lung, total cell number of leukocyte and eosinophil in BALF, both EAR- I and EAR- III treated groups were significantly decreased compared to control group. IL-4, IL-5, IL-13, histamine, IgE in BLAF of group treated with both EAR-I and EM-III were significantly decreased compared to control group. In FACS analyzing, granulocytes, $CD3e^-$/$CCR3^+$, $CD3^+$/$CD19^$, $CD3e^+$/$CD69^+$, $CD4^+$, $CD8^+$ and $GR-1^+$/$CD11b^+$ were significantly increased in asthma induced mice group by OVA treatment compared to control group, and decreased in group treated with both EAR- I and EAR-III. Conclusions : The present data proves that extract of astragali radix has an effect on the inhibiting asthma. Moreover, astragali radix cultured for three years was proved to be superior to the one cultured for one year.

• The Journal of Pediatrics of Korean Medicine
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• v.26 no.1
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• pp.36-45
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• 2012

Objectives Perillae Folium (PF) has been widely used in Korean herbal medicines used for treatment of acute and chronic inflammatory diseases, such as rhinitis, asthma, and enteritis. In this study, to investigate the protective effect of PF on type 1 allergic response, we determined whether PF inhibits early or late allergic responses. Methods The effect of PF was analyzed by ELISA,. RT-PCR and Western blot in RBL-2H3 cells. Levels of ${\beta}$-hexosaminidase, interleukin (IL)-4 and TNF-${\alpha}$ were measured using enzyme-linked immunosorbent assays (ELISAs). mRNA levels of cytokines and enzymes were analyzed with RT-PCR. Signal transduction was analyzed with Western blot. Results We found that PF suppressed ${\beta}$-hexosaminidase release in RBL-2H3 by the IgE-DNP-HSA stimulation. PF also significantly inhibited enzymes level, such as COX-1, COX-2, iNOS, and HDC2, along with reduced cytokine levels, such as IL-2, IL-3, IL-4, IL-6, IL-13, and TNF-${\alpha}$ in RBL-2H3. In addition, PF suppressed the phospholyation of ERK1/2, JNK1/2, and $I{\kappa}B{\alpha}$. Conclusions Our results indicate that PF protects against type 1 allergic response and exert an anti-inflammatory effect through the inhibition of degranulation and expression of cytokines and enzymes via the suppression of signal transduction.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.24 no.1
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• pp.86-95
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• 2011

Objectives : Ulmus davidiana (UD) has been widely used in Korean herbal medicines used for treatment of acute and chronic inflammatory diseases, such as rhinitis, asthma, and abscess. In this study, To investigated the protective effect of UD on type 1 allergic response, we determined whether UD inhibits early and late allergic response. Methods : The effect of UD was analyzed by ELISA and RT-PCR in RBL-2H3 cells. Levels of ${\beta}$ -hexosaminidase, interleukin (IL)-4 and TNF-${\alpha}$ were measured using enzyme-linked immunosorbent assays (ELISAs). mRNA levels of COX-2 and T-helper type 2(Th2) cytokines were analyzed with RT-PCR. Results : We found that UD suppressed ${\beta}$-hexosaminidase release in RBL-2H3 not only by the PMA plus A23187 stimulation, but also by the IgE-DNP-HSA stimulation at the antigen-antibody binding stage and antibody-receptor binding stage. UD also significantly inhibited COX2 level, along with reduced Th2 cytokine levels, such as IL-3, IL-4, IL-5, IL-13, GM-CSF, and TNF-${\alpha}$ in RBL-2H3. Conclusions : Our results indicate that UD protects against type 1 allergic response and exerts an anti-inflammatory effect through the inhibition of degranulation and expression of COX2 and Th2 cytokines.

• Journal of Yeungnam Medical Science
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• v.29 no.2
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• pp.89-95
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• 2012

Background: As Mycoplasma pneumoniae pneumonia has increased in Korea, its relevance to infants, toddlers, and adolescents has magnified as well as. However, it is difficult to perform the serological test and PCR test routinely for diagnosis in actual clinical practice. Thus, the authors conducted this study to help clinicians do presumptive diagnosis of Mycoplasma pneumoniae pneumonia using clinical, radiological, and hematological findings. Methods: The study population consisted of 224 children between 1 month and 14 years old, hospitalized for radiographically confirmed pneumonia. Patients were divided into two groups of 100 children with Mycoplasma pneumoniae pneumonia, as diagnosed using the ELISA method. Groups with negative result in Mycoplasma IgM antibody test were classified into the viral group (98 patients with respiratory virus) and the bacterial group (46 patients with the bacteria detected in the blood sputum culture or antibiotic treatment except macrolide improved the patient's condition). These groups were compared and analyzed using clinical, hematological, and radiographic differences and scoring system. Results: Clinical, hematological, and radiographic characteristics of Mycoplasma pneumoniae pneumonia have shown the intermediate level results between bacterial pneumonia and viral pneumonia. In terms of scoring system, the mean score of Mycoplasma pneumoniae pneumonia was 4.23, which was the intermediate level between bacterial pneumonia (mean score=6.67) and viral pneumonia (mean score=1.48). Conclusion: Results suggest that the combination of the scoring system information can increase the accuracy in the diagnosis even if they may have difficulties on diagnosis, because clinical manifestations, hematological, and radiographic findings are nonspecific.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.583-593
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• 1995

The present study was conducted to develop a toxoplasma latex agglutination test antigen(Test-MT) and evaluate the toxoplasma latex agglutination(LA) test using a newly-made "Test-MT kit" by comparing with the Toxo-MT kit(Eiken chemical co, Tokyo). Also, the specifity and sensitivity test were made by comparing with IFA test and IgG-ELISA. Tachyzoite suspensions of Toxoplasma gondii(RH strain) were ultracentrifuged for 30min at $60,000{\times}g(4^{\circ}C)$ and the supernatant was used as a water-lysate antigen. Polystyrene latex particles of $1.0{\mu}m$ in diameter(Polyscience co) were used for the preparation of sensitized latex-antigen supension(Test-MT). The frequency distribution of LA titers in Test-MT showed two peaks at <1:32 and 1:128. The borderline titer for positive test in Test-MT was determined to be 1:64. But the frequency distribution of LA tites in Toxo-MT showed two peaks at <1:16 and 1:64. The positive borderline was determined to be 1:32. Agreement of reactions between Test-MT and Toxo-MT kit by LA test was shown 92.5% in bovine sera and 97.0% in swine sera, respectively. From the results obtained here it was determined that the sensitized latex-antigen, Test-MT kit, for the microtiter agglutination test prepared as same as by the procedure described in the previous paper(Suh and Lee, 1993) was useful as a highly specific, sensitive and stable immunotiteration reagent for serodiagnosis of toxoplasma infection in animal sera.

• Journal of Microbiology and Biotechnology
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• v.29 no.4
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• pp.651-657
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• 2019

Although smallpox was eradicated in 1980, it is still considered a potential agent of biowarfare and bioterrorism. Smallpox has the potential for high mortality rates along with a major public health impact, eventually causing public panic and social disruption. Passive administration of neutralizing monoclonal antibodies (mAbs) is an effective intervention for various adverse reactions caused by vaccination and the unpredictable nature of emerging and bioterrorist-related infections. Currently, vaccinia immune globulin (VIG) is manufactured from vaccinia vaccine-boosted plasma; however, this production method is not ideal because of its limited availability, low specific activity, and risk of contamination with blood-borne infectious agents. To overcome the limitations of VIG production from human plasma, we isolated two human single-chain variable fragments (scFvs), (SC34 and SC212), bound to vaccinia virus (VACV), from a scFv phage library constructed from the B cells of VACV vaccine-boosted volunteers. The scFvs were converted to human IgG1 (VC34 and VC212). These two anti-VACV mAbs were produced in Chinese Hamster Ovary (CHO) DG44 cells. The binding affinities of VC34 and VC212 were estimated by competition ELISA to $IC_{50}$ values of $2{\mu}g/ml$ (13.33 nM) and $22{\mu}g/ml$ (146.67 nM), respectively. Only the VC212 mAb was proven to neutralize the VACV, as evidenced by the plaque reduction neutralization test (PRNT) result with a $PRNT_{50}$ of ~0.16 mg/ml (${\sim}1.07{\mu}M$). This VC212 could serve as a valuable starting material for further development of VACV-neutralizing human immunoglobulin for a prophylactic measure against post-vaccination complications and for post-exposure treatment against smallpox.