• Title, Summary, Keyword: Antibody Production

Search Result 720, Processing Time 0.042 seconds

Production and Characterization of a Monoclonal Antibody against Human ${\beta}_2$-adrenergic receptor

  • Kang, Suk-Jo;Shin, Chan-Young;Song, Mi-Ryoung;Lee, Chung-Jae;Cheong, Jae-Hoon;Lee, Sang-Bong;Ko, Kwang-Ho
    • Biomolecules & Therapeutics
    • /
    • v.5 no.4
    • /
    • pp.344-350
    • /
    • 1997
  • The purpose of the present study was to produce and characterize a monoclonal antibody against human ${\beta}_2$-adrenergic receptor. Male BALB/c mice were immunized with glutathione S-transferase (GST) fusion protein of the C-terminal portion of the human ${\beta}_2$-adrenergic receptor which was expressed in E.Coli. The immunized splenocytes were fused with myeloma SP2/0-Agl4 cells. The resulting hybridomas were screened for the production of a monoclonal antibody which can recognize human ${\beta}_2$-adrenergic receptor, and then subcloned by limiting dilution. The resulting monoclonal antibody was named as mAb$\beta$CO2. The mono-clonal antibody $\beta$CO2 was determined as IgM subtype and then purified by anti-mouse IgM-agarose affinity chromatography. The results of ELISA, Western blot, and immunocytochemistry showed that mAb$\beta$CO2 recognized human ${\beta}_2$-adrenergic receptor in the ${\beta}_2$-adrenergic receptor-GST fusion protein and human spider-moid carcinoma cell line A431 with highly specific immunoreactivity, The monoclonal antibody $\beta$CO2 may provide useful tools for the study of the $\beta$-adrenergic receptor of human and other species including rats.

  • PDF

Production of a Recombinant Anti-Human CD4 Single-Chain Variable-Fragment Antibody Using Phage Display Technology and Its Expression in Escherichia coli

  • Babaei, Arash;Zarkesh-Esfahani, Sayyed Hamid;Gharagozloo, Marjan
    • Journal of Microbiology and Biotechnology
    • /
    • v.21 no.5
    • /
    • pp.529-535
    • /
    • 2011
  • Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.

Expression Vectors for Human-mouse Chimeric Antibodies

  • Xiong, Hua;Ran, Yuliang;Xing, Jinliang;Yang, Xiangmin;Li, Yu;Chen, Zhinan
    • BMB Reports
    • /
    • v.38 no.4
    • /
    • pp.414-419
    • /
    • 2005
  • The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded $30\;pg\;cell^{-1}\;day^{-1}$ at $10^{-6}\;M$ MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

Production of a Monoclonal Antibody and Ultrastructure of the Sporozoite of Cryptosporidium parvum

  • Choi, Young-Sook;Lee, Sung-Tae;Cho, Myung-Hwan
    • Journal of Microbiology
    • /
    • v.34 no.4
    • /
    • pp.379-383
    • /
    • 1996
  • Cryptosporidium parvum causes a life-threatening diarrhea in acquired immunodeficiency syndrome (AIDS) patients. THe sporozoite stage of C. parvum has been known to be a target in treating cryptosporidiosis in AIDS patients as it is an extracellular stage. A sporozoite was ultrastructurally observed. It has a creascent shape with a rounded posterior end and a tapering body. The compact nucleus was located at the posterior end. A monoclonal antibody was produced, which recognized a 43 kDa of sporozoite antigens in a western blot analysis and showed the surface labeling in immunofluorescence.

  • PDF

Effect of Gelatin on the Stability of Heavy Chain Monoclonal Antibody Production from Plant Suspension Cultures

  • Ryland, J.;Robert, P.;Michael, Linzmaier;Lee, James M.
    • Journal of Microbiology and Biotechnology
    • /
    • v.10 no.4
    • /
    • pp.449-454
    • /
    • 2000
  • The heavy chain monoclonal antibody (HC MAb) was produced in suspension cultures of genetically modified Nicotiana tabacum. The HC MAb secreted to the medium was unstable due to unfavorable interactions in the plant cell medium. The addition of gelatin (5g/l) stabilized the extracellular HC MAb and increased its production 10-fold. A kinetic model was developed describing the interaction between the secretedprotein and the stabilizer. The model accounted for the inactivation of the protein by simple aggregation and general instability. It was assumed that the secreted protein and the stabilizer form a stable complex. Culturing the cells semicontinuously could further increase the productivity of HC MAb.

  • PDF

Ammonium Ion Effects and Its In Situ Removal by Using Immobilized Adsorbent in Hybridoma Cell Culture (하이브리도마 세포배양에서 암모늄 이온의 영향 및 고정화 흡착제에 의한 암모늄 이온의 동시제거)

  • 정연호;이해익
    • KSBB Journal
    • /
    • v.11 no.3
    • /
    • pp.329-339
    • /
    • 1996
  • The effects of ammonium ion on cell growth kinetics, monoclonal antibody productivity, and cell metabolism of hybridoma cells were investigated. The mouse-mouse hybridoma cell line VlIIH-8 producing mouse IgG2a was used as a model system. Ammonium ion showed an inhibitory effect on cell growth and monoclonal antibody production. New immobilized adsorbents were developed for the reduction of the inhibitory effect of ammonium ion. The ammonium ion selective zeolite, Phillipsite-Gismondine was entrapped in calcium alginate bead or in dialysis membrane and applied to the hybridoma cell culture system for the in situ removal of ammonium ion from culture media. The effects of ammonium the both serum supplemented and serum free media on the cell growth were studied by applying immobilized adsorbents of calcium alginate bead type. The results demonstrated a substantial enhancement in cell growth. Applying immobilized adsorbents of dialysis membrane type to serum supplemented media also resulted in the stimulation of cell growth, cell viability and monoclonal antibody production.

  • PDF

Independent regulation of antigen processing and presentation on induction of antibody responses to various bacterial antigens in C3H/He mice

  • Kim, Hyung-Su;Jeong, Gajin
    • Journal of Microbiology
    • /
    • v.33 no.4
    • /
    • pp.355-362
    • /
    • 1995
  • Induction of antibody production in C3H/He mice by bacterial infection is regulated through the processing exerted by antigen presenting cells. From the studies with Psudomonas aeruginosa, Salmonella typhimurium, and Micrococcus luteu, lipopolysaccharides (LPS) in Gram negative bacteria, which are known to be T-cell independent B cell mitogen, seem to be the major factor stimulating immune responses via activation of macrophages. Activation of macrophage, however, does not seem to correlate with antibody production. M. luteus was easily eliminatd by activated macrophages, while the processed antigens were immediately releasedd into culture medium before presentation. Nevertheless, antigens from Gram positive bacteria, Staphylococcus aureus and Bacillus subtilis, were very very active in chemotaxis and activation of periotoneal macrophages as well as in antien presnetation, while the very nature of the antigens is not yet clearly understood.

  • PDF

Modelling of a Biomolecular Processing for the Production and Secretion of Monoclounal Antibody (단일콜론항체 생산 및 분비에 대한 생물분자공정의 모델링)

  • 박재성;박선호
    • KSBB Journal
    • /
    • v.13 no.4
    • /
    • pp.369-377
    • /
    • 1998
  • To analyze the unique aspects of biomolecular processing for monoclonal antibody (MAb) production and secretion, the simple working model based on 3-compartment (endoplasmic reticulum, Golgi apparatus, and extracellular medium) was developed. Based on in vitro MAb assembly experimental results, the kinetic model for MAb assembly in the endoplasmic reticulumn was proposed. The dynamics of MAb assembly and secretion was simulated using methematica program. According to the simulation results, the proposed 3-compartment model provides an efficient means to predict the specific MAb productivity as well as intracompartmental concentrations of MAb in endoplasmic reticulum, Golgi apparatus, and extracellular compartment model. In vivo profiles of MAb intermediates gave good agreements with the simulation profiles predicted by the intracellular compartment model. Furthermore, results of such analysis can help in directing the control strategy for optimum biomolecular processing in a mammalian cell culture system.

  • PDF

Effects of high Cell Density on growth-Associated Monoclonal Antibody Production by Hybridoma T0405 Cells Immobilized in Macroporous Cellulose carriers

  • Hideki Mochoda;Wang, Pi-Chao;Fr Jr. Nayve;Ryuji Sato;Minoru Harige;Nakao Nomura;Masatoshi Matsumura
    • Biotechnology and Bioprocess Engineering:BBE
    • /
    • v.5 no.2
    • /
    • pp.110-117
    • /
    • 2000
  • Relationship between monoclonal antibody (MAb) productivity and growth rate, and effects of high cell density on MAb production rate increased with increasing specifis growth rate in both suspended and immobilized continuous cultures indicate a positively growth-associated relationship between MAb productivity and growth rate. moreover, the specific production rate was higher in the immobilized cell culture than that in suspended one at all dilution rates. In order to clarify these phenomana, MAb mPNA experession and cell cycle distribution were investigated in bacth cultures with immobilized cells and suspended cells. RT-PCR was used for observation of MAb mRNA expression and a two-color bromodeoxyuridine (BrdU)/propidium iodide (PI) flow cytometry method for determination of cell cycle distribution. The results revealed that MAb nRNA expression until dead phase, which was longer than in suspended cell. The cell cycle distribution patterns were observed almost the same for both immobilized and suspended cells. Such results may imply that a high cell density state has positive influence on the mRNA expression and on growth-associated Mab productivity of T0405 cells.

  • PDF

Effects of aqua-acupuncture with Panax Ginseng on immune response induced by Glucocorticoid in mouse (인삼약침(人蔘藥鍼)이 glucocorticoid투여 Mouse의 면역반응에 미치는 영향)

  • Lee, Eun-Hong;Moon, Jin-Young;Lim, Jong-Kook
    • The Journal of Korean Medicine
    • /
    • v.18 no.1
    • /
    • pp.326-336
    • /
    • 1997
  • In order to investigate the effect of aqua-acupuncture solution with Panax Ginseng into $Qihai(CV_6)$ and $Shenshu(BL_{23})$ on immune response induced by glucocorticoid in mouse, Panax Ginseng aqua-acupuncture solution was injected into $Qihai(CV_6)$ and $Shenshu(BL_{23})$ for seven days after injection with glucocorticoid. And then MA-HRP (methamphetamine-horseradish peroxidase) induced antibody production, numbers and lysozyme activity in macrophage were measured. The results were as follows: 1. The antibody production in mouse immunized with MA-HRP was decreased in control group as compared with normal group. Although $Qihai(CV_6)$ group showed slight increasement, $Shenshu(BL_{23})$ group indicated great increasement compared with normal group. However in Aa-BL group, antibody production was almost increased to normal group. 2. In control group, the numbers of macrophage were decreased about 14% as compared with normal group. And in the pretreated groups of $Qihai(CV_6)$ and $Shenshu(BL_{23})$ were respectively increased 3.0 times and 2.9 times as compared with normal group. 3. Effect of Panax Ginseng-aqua acupuncture solution on the lysozyme activity in macrophage was increased gradually in the pretreated groups of $Qihai(CV_6)$ and $Shenshu(BL_{23})$ as compared with control group. These results suggest that Panax Ginseng aqua-acupuncture at $Qihai(CV_6)$ and $Shenshu(BL_{23})$ may increase antibody production and lysozyme activity in macrophage that is suppressed by glucocorticoid, and Panax Ginseng aqua-acupuncture will have immuno adjuvant effects on the cells which concerned with immunomechanisms.

  • PDF