• Title/Summary/Keyword: hybridoma

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A Kinetic Modeling for the Dynamics of Hybridoma Cells in Suspension Culture (현탁배양 하이브리도마 세포의 속도론적 모델링)

  • 정연호;박현규최정우
    • KSBB Journal
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    • v.11 no.3
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    • pp.276-287
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    • 1996
  • Batch suspension cultures of hybridoma cell were performed with various initial glutamine concentrations to investigate the effects of glutamine on cell growth and death, monoclonal antibody production, glucose and glutamine consumption, and the production of lactate and ammonium ion. An mathematical kinetic model was formulated to describe the kinetics of cell growth, the consumption of nutrients (glucose and glutamine), and the production of monoclonal antibody and waste metabolites (lactate and ammonium ion) based on experimental data. An equation for the specific growth rate was developed such that superimposed Monod equation in glucose and glutamine, with non-competitive type inhibition relations in ammonium ion and lactate. The inhibition constant for lactate was inversely proportional to the lactate concentration. The specific death rate was considered to be a function of glucose, glutamine, ammonium ion and lactate concentration.

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Production of Monoclonal Antibodies Specific to FimA of Porphyromonas gingivalis and Their Inhibitory Activity on Bacterial Binding

  • Koh, Eun-Mi;Kim, Ju;Lee, Jin-Yong;Kim, Tae-Geum
    • IMMUNE NETWORK
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    • v.9 no.5
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    • pp.203-207
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    • 2009
  • Background: The FimA of Porphyromonas gingivalis is a crucial pathogenic component of the bacteria and has been implicated as a target for vaccine development against the periodontal diseases. Methods: In this study, the purified fimbriae (FimA subunit polymers) protein was used for immunization in their native form and B hybridoma clones producing antibodies specific to FimA were established. Results: The monoclonal antibodies prepared from selected two clones, designated #123 (IgG2b/ kappa) and #265 (IgG1/kappa), displayed different patterns of binding activity against the cognate antigen. Both antibodies reacted with conformational epitopes expressed by partially dissociated oligomers, but not with monomer as elucidated by Western blot analysis. Ascites fluid containing the monoclonal antibodies showed the inhibitory activity against P. gingivalis to saliva-coated hydroxyapatite beads, an in vitro model for the pellicle-coated tooth surface. Conclusion: These results suggest that the monoclonal antibodies could be used as vaccine material against the periodontal diseases through passive immunization.

Production of Monoclonal Antibody to Avian Infectious Bronchitis Virus (닭 전염성 기관지염 바이러스에 대한 단클론 항체 생산)

  • Lee, Chung-Gil;An, Soo-Hwan;Kwon, Joon-Hun;Park, Chung-Ok
    • Korean Journal of Poultry Science
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    • v.19 no.1
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    • pp.13-16
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    • 1992
  • Avian infectious bronchitis virus(IBV) was propagated in SPF eggs and purified by sucrose density gradient centrifugation in order to prepare the antigen. Several fusions were made between mouse myeloma cells and spleen cells from BALB/c mouse immunized with IBV antigen and two hybridoma clones producing specific monoclonal antibody(MCA) against the IBV were established. The MCAs were classified as IgG type and revealed no neutralizing and hemagglutination inhibition activity. Using the MCA IBV antigen was detected by IFA method in tracheal smears made from chickens infected with IBV during the experimental period of 10 days.

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Differential Activation of T Cells by T-Cell Receptor Ligand Analogs

  • Choi, Yun-Hi;Suh, Yu-Jin;Kim, Kil-Hyoun
    • BMB Reports
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    • v.30 no.6
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    • pp.415-420
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    • 1997
  • Although $CD4^+$ T cell responses to protein-derived antigen have well been understood, the epitopes recognized by hapten-specific $CD4^+$ T cells have not been fully defined. In this study, we characterized the response of a T cell hybridoma (5Di0.1B8) which is specific for a hapten. N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) restricted by MHC class II $I-A^d$. Using three different antigen presenting cells (APCs) expressing $I-A^d$, the role of class II MHC proteins in haptenic antigen presentation and subsequent activation of 5D10.1B8 has been examined. Activation of 5D10.1B8 T cells by HSAB analogs was also performed. Our results show that each APC activated T cells differentially and that interleukin-2 (IL-2) augmented antigen-presenting ability of all the APCs, suggesting that increased expression of class II MHC protein by IL-2 played an important role in HSAB presentation and T cell activation. Finally, early T cell receptor-dependent signals induced by HSAB or its analogs were examined by phosphotyrosine immunoblot analysis, and showed that tyrosine phosphorylation level of a 18-20 kD protein increased upon stimulation.

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Production of Monoclonal Antibodies by Hybridomas Sensitized to Sporozoites of Cryptosporidium parvum (Cryptosporidium parvum Sporozoites 에 감작된 Hybridomas 에서의 Monoclonal Antibody 생산)

  • Cho, Myung-Hwan
    • Microbiology and Biotechnology Letters
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    • v.17 no.5
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    • pp.494-498
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    • 1989
  • Hybridoma cell lines, which secrete monoclonal antibodies (mAbs) against the surface antigens of Cryptosporidium parvum Sporozoites, were produced by fusing spleen cells of C. parvum Sporozoite-immunized mice with P3-X63-Ag8 myeloma cells. Two cloned antibody-secreting cell lines, Kor1 and Ea2, were established and produced IgG1 and IgG2a antibodies, respectively. Percoll-purified sporozoites were solubilized and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Western blot assay demonstrates that an antigen of 20-kDa was bound by monoclonals. By indirect immunofluorescence microscopy, mAb exhibited uniform binding to the sporozoite surface.

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Controlling Mammalian Cell Metabolism in Bioreactors

  • Hu, Wei-Shou;Weichang, Zhou;Lilith F. Europa
    • Journal of Microbiology and Biotechnology
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    • v.8 no.1
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    • pp.8-13
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    • 1998
  • Animal cells in culture typically convert most of the glucose they consume into lactate. The accumulation of lactate, however, is commonly cited as one of the factors that inhibit cell growth and limit the maximum cell concentration that can be achieved in culture. The specific production of lactate and the amount of glucose converted to lactate can be reduced when cells are grown in a fed-batch culture in which the residual glucose concentration is maintained at low levels. Such a fed-batch culture was used to grow and adapt hybridoma cells into a low-lactate-producing state before changing into continuous culture. The cells reached and maintained a high viable cell concentration at steady state. In a similar manner, cells that were initially grown in batch culture and a glucose-rich environment reached a steady state with a cell concentration that is much lower. The feed composition and dilution rates for both cultures were similar, suggesting steady state multiplicity. From a processing perspective the desired steady state among those is the one with the least metabolite production. At such seady state nutrient concentration in the feed can be further increased to increase cell and product concentrations without causing the metabolite inhibitory effect typically seen in a cell culture. Controlling cell metabolism in a continuous culture to reduce or eliminate waste metabolite production may significantly improve the productivity of mammalian cell culture processes.

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High Density Culture of Hybridima Using Cell sedimentation System (세포 침전장치를 이용한 하아브리도마 세포의 고농도 배양)

  • 최대부;조보연
    • KSBB Journal
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    • v.4 no.2
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    • pp.143-149
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    • 1989
  • A cell sedimentaion system was designed and employed for the high culture of hydridoma. An upward divering cell settler allowed a good sedimentation of hybridoma but the accumulation of cell mass on the settler's wall side was a potential prolem. Although a cyinderical cell settler was useful to solve this problem, this device was employable only at low dilytion rate. A modified cell settler could support the high density culture of hybridoma at a concentration of $5{\times}10^6$ cell/ ml during 1 week, producting 380mg of monclonal antibody.

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Pulse-Feeding of Serum Free Media for Enhancing Monoclonal Antibody Production under Perfusion Operation (연속배양에서 단일항체 생산성 향상을 위한 무혈청 배지의 단계적 유입)

  • 강재구;박형환;이현용
    • Microbiology and Biotechnology Letters
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    • v.18 no.1
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    • pp.61-65
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    • 1990
  • Lectin related inducer can enhance IgG$_1$ production rate from murine hybridoma cells by employing step-feeding of serum free media with producing about 40 mg/$\ell$ of monoclonal antibodies. This step-feeding perfusion process also proves to be able to cutivate animal cells when serum free media can not support the growth of these cells in perfusion process, as well as to improve production rate. This process yields about 28 x 10$^{-10}$ mg of MAb/cells/h compared to 11.1 x 10$^{-10}$ and 4.0 x 10$^{-11}$ mg/cells/h for perfusion process and batch cultivation with 10% serum containing media, respectively.

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Development of Assay Methods for Enterotoxin of Escherichia coli Employing the Hybridoma Technology (잡종세포종기법을 이용한 대장균의 장독소 측정법 개발)

  • Kim, Moon-Kyo;Cho, Myung-Je;Park, Kyung-Hee;Lee, Woo-Kon;Kim, Yoon-Won;Choi, Myung-Sik;Park, Joong-Soo;Cha, Chang-Yong;Chang, Woo-Hyun;Chung, Hong-Keun
    • 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.

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Characterization of Immunogens of Infectious Hematopoietic Necrosis Virus Isolated in Korea (전염성 조혈기 괴사 바이러스(IHNV)의 항원 유도 단백질 특성)

  • Park, Myoung-Ae;Sohn, Sang-Gyu;Park, Jeong-Woo;Jeong, Young-Kee
    • Journal of fish pathology
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    • v.7 no.1
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    • pp.13-22
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    • 1994
  • To identify the immunogens of a PRT strain of Infectious Hematopoietci Necrosis Virus (IHNV) isolated from cultrued fish in Korea (Park et al, 1993). a panel of 4 monoclonal antibodies (MAbs) against IHNV-PRT strain and two polyclonal antisera from rainbow trout survived IHN disease were prepared. Proteins of purified IHNV-PRT strain were analysed on 10% SDS-PAGE and transferred onto NC paper and were incubated with the antibody solutions. With the polyclonal antibodies, four bands ($M_1$, $M_2$, G and 90Kd) were detected and the band density was in the order of $M_2$ > 90Kd > $M_1$ > G. However, with the MAbs, only two bands(G and 90Kd) were detected. The origin of 90Kd protein was not clear but maybe cell. All the results represented that among the five proteins of IHNV-PRT strain (Park et al., 1993), $M_2$, $M_1$ and G proteins were immunogens and $M_2$ protein was the strongest one.

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