• Korean Journal of Microbiology
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• v.23 no.1
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• pp.64-68
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• 1985

In this study, hybridoma techniques were applied to produce monoclonal antobodies to Paragonimus westermani, commonly known as lung distoma. Balb/c mice were immunized intraperitoneally every week with increasing doses of purfied protein of Paragonimus westermani adult worms begining with 0.3/mg/mouse/7 days and ending with 0.5mg at 28th day. Spleen cells from these immunized mice were hybridized with myeloma cells (NS-1) and the hybridized cells were selected in HAT media. The antibody secreting cells among the hybrid cells were initially selected by ELISA. Those initially selected cells were further screened by the criteria of antibody producing activity, and seneral cell lines among them were further tested with immunodiffusion method. One hybridoma clone produced IgM and another clone produced IgG1. The supernatant of the hybridoma clone producing IgM had titer 1:64 and the hybridoma clone producing IgG1 had titer 1:256 measured by immunofluorescence technique.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.515-519
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• 2004

Listeria spp. were isolated from a total of 402 naturally contaminated domestic ready-to-eat (RTE) vegetable samples by the conventional Food and Drug Administration protocol and confinned by API-Listeria kit. Also, the susceptibility to 12 antibiotics, polymerase chain reaction (PCR) assay for virulence gene of pathogenic Listeria monocytogenes isolates, and in vitro virulence assay using myeloma and hybridoma cells from murine and human sources were tested. Among the samples, 17 samples (4.2％) were found to be contaminated with Listeria species. Among the 17 strains of Listeria spp. isolates, only 2 strains (11.8％) of L. monocytogenes and 15 strains (88.2％) of L. innocua were identified. Antibiotic susceptibility test showed that the Listeria spp. isolates were very susceptible to the antibiotics tested, except for nalidixic acid. Among 17 strains of Listeria spp., PCR analysis showed that 2 strains of L. monocytogenes isolates proved to have a virulence hly gene, but none of L. innocua had the hly gene. Also, hybridoma Ped-2E9 cells assay showed that only L. monocytogenes isolates killed approximately 95-99％ hybridoma cells after 6 h, but L. innocua isolates had about 0-5％ lethal effect. These results indicate that PCR assay with hly primer or hybridoma Ped-2E9 cells assay could be used as a good monitoring tool or in vitro virulence test for L. monocytogenes.

• Journal of Microbiology
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• v.37 no.3
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• pp.180-184
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• 1999

hybridoma cells producing IgM anti-pneuococcal 6B polysaccharide antibodies were induced to switch to IgG-producing cells in vitro by treating with acridine orange. Treating 0.5 $\mu\textrm{g}$/ml of acridine orange for 24 hours generated maximal number of variant cells. The maximal isotype switch frequency was 3${\times}$10-5, which is about 30-fold higher than the frequency of spontaneous switching. Resulting IgG-producing variants were enriched by sib selection and ELISA spot assay. Two IgG3-producing variant cells were finally cloned by limiting dilution. The variant cells produced similar amounts of antibodies as their parental cells did. The two switched antibodies had similar reactivity to pneumococcal 6B polysaccharide. When compared to their parental IgM antibodies, the switched IgG3 than that of IgM antibody. The antibodies will be useful as essential tools for comparative study of the role of heavy chain isotypes in protection against Streptococcus pneumoniae.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.398-403
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• 1999

In our previous studies, we reported that hybridoma B-lymphocytes can be used to determine the virulence of Listeria species in an in vitro cytotoxicity assay. Here, we examined the cytopathic effect, i.e., membrane damage and the nature of cell death induced by Listeria monocytogenes on murine hybridoma B-lymphocytes (Ped-2E9). Membrane damage was assessed by microscopic analyses and by measuring the release of intracellular alkaline phosphatase(AP) and lactate dehydrogenase (LDH). Cell death was determined by DNA fragmentation analyses using agarose gel electrophoresis. Infection by listeriolysin O (LLO)-producing L. monocytogenes strains induced substantial amounts of AP and LDH release from Ped-2E9 hybridoma B-cells, suggesting severe membrane damage in these cells, while an LLO-negative L. monocytogenes mutant strain had no effect. An LLO-producing recombinant L. innocua ($prifA^+hly^+$) strain also induced high AP and LDH release and cytopathic changes in Ped-2E9 cells. Light or scanning electron microscopic examination revealed L. monocytogenes mediated membrane destabilization, pore formation, intense cytoplasmic granulation, bleb formation, and lysis of Ped-2E9 cells. LLO-producing L. monocytogenes and L. innocua ($prifA^{+}hly{^}+$) also induced ladder-like DNA fragmentation in Ped-2E9 cells. Collectively, these results suggest that L. monocytogenes, specifically LLO-producing strains, can induce a severe cytopathic effect leading to apoptosis in hybridoma B-lymphocytes (Ped-2E9).

• Animal cells and systems
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• v.4 no.4
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• pp.381-388
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• 2000

Using a murine T cell hybridoma, activation-induced cell death (AICD) was studied. As an in vitro model system for the AICD, 1 cell hybridoma expressing TCR/CD3 complex was incubated onto the immobilized purified anti-CD3 antibody. The immobilized anti-CD3 antibody induced AICD effectively up to 40%. At 1-100 $\mu$M range of SNP, an exogenous source of nitric oxide (NO), the cell proliferation was not affected, but at 1 mM SNP, cell proliferation was significantly reduced. The AICD of T cell hybridoma was inhibited by exogenous NO at non-cytotoxic concentration, In the cells undergoing AICD, the expressions of caspase-3 and FasL were detected, but not iNOS. Similar result was recognized in the apoptosis induced by dexamethasone, an apoptosis-inducing agent. However, the conversion from the inactive form of caspase-3 (32 kDa) to the active form (17 kDa) was significantly reduced in the cells in AICD induced by anti-CD3 antibody, With the result of increased PARP cleavage in the cells, we propose that another PARP cleavage pathway not involving caspase-3 may function in the anti-CD3 antibody induced AICD in the T cell hybridoma.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.141-151
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• 1992

Physiological changes of the murine hybridoma cell line $S3H5/\gamma{2bA2}$ during adaptation to RPMI 1640 medium with 1%(v/v) fetal bovine serum were characterized in terms of cell growth, antibody production, morphology, and metabolic quotients. Cells adapted to 1% serum medium in T-flasks became sensitive to shear induced by mechanical agitation and required at least 5% serum in the medium or spent medium for cell growth in spinner flasks, while cells adapted to 10% serum medium in T-flasks could grow in 1% serum medium in spinner flasks. Consequently, long-term adaptation to low serum media may not give the expected growth enhancement. After adaptation to 1% serum medium, changes in cell morphology were observed. The cells in 10% serum medium were uniform and circular, while cells in 1% medium were irregularly shaped. The DNA contents, which were measured by flow cytometry, were almost constant among the cells in the range of 1% to 10%. Further, no significant changes in energy metabolism and specific monoclonal antibody production rate were observed among these cells.

• Applied Biological Chemistry
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• v.34 no.3
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• pp.231-234
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• 1991

In the present investigation we have studied the effect of bear's gall on mammalian cells and demonstrated that COS-7 cells, which were derived Monkey kidney cells, had shown almost same extent of growth with 78 hrs in 10% FCS Dulbecco's Modified Eagle's medium with bear's gall and without bear's gall. But the hybridoma cells which were fused murine myeloma cells and the rat spleen cells for monoclonal antibody production died almost within 48 hrs. To investigate the effect of biosynthetic mechanism, cDNA were transfected to COS-7 cells, and it was shown that cDNA-transfected COS-7 cell had produced 30-40% less the amount of recombinant protein than the medium without bear's gall.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.2
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• pp.117-120
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• 2002

The effect of cell density on cell growth was investigated in a suspension batch culture of hybridoma cells. The specific growth rate was found to increase with increasing initial cell density and then to decrease with further increases in initial cell density. In order to quantitatively describe the dependence of specific growth rate on cell density, a kinetic model is proposed, which satisfactorily represents the experimental data.

• Korean Journal of Food Science and Technology
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• v.30 no.6
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• pp.1409-1414
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• 1998

To develop zearalenone-specific monoclonal antibodies, hybridoma cells were produced by fusion of myeloma cells $(P3{\times}63Ag\;V653)$ and spleen cells from BALB/c female mice immunized with zearalenone-oxime coupled to bovine serum albumin (BSA). After screening of antibody titer of them with a sandwich type enzyme-linked immunosorbent assay (ELISA), 5 hybridomas which could produced monoclonal antibodies with a high affinity for zearalenone were selected. The monoclonal antibody produced by Z-2-M26 hybridoma exhibited the high sensitivity to zearalenone and a little cross-reactivity to ${\alpha}-zearalenol$ (11%), but did not react with ${\beta}-zearalenol,\;{\alpha}-zearalenol,\;{\beta}-zearalenol$ and DON. In conclusion, the developed monoclonal antibody appeared to be a very promising immunoreagent for the future development of a specific and sensitive quantitative ELISA for zearalenone.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.374-377
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• 2003

Hybridoma cells were adapted in media containing up to 80 mM $K^+$. The adapted cells obtained tolerance at high osmotic pressure and low pH. The adapted cells showed a maximum viable cell density of $1.1{\times}10^6$ cells/ml when a batch culture was progressed in a nutrient-fortified medium with Erlenmeyer flask at 450 $mOsm/kgH_2O,$ as compared to $4.8{\times}10^5$ cells/ml for non-adapted cells grown under the same conditions. The adapted cells also showed a maximum viable cell density of $7.8{\times}10^5$ cells/ml with the same method at initial pH 7.0 as compared to $5.3{\times}10^5$ cells/ml for non-adapted cells. Adaptation of animal cells at high $K^+$ levels may therefore lead to an improvement of their performance at limited conditions.