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Sample records for abrogates antibody production

  1. Antibody-mediated neutralization of virus is abrogated by mycoplasma.

    Dickson, C; Elkington, J; Hales, A.; Weiss, R.

    1980-01-01

    The ability of a mouse mammary tumor cell line to abrogate antibody neutralization of vesicular stomatitis virus was shown to be due to the presence of mycoplasma. The mycoplasma was isolated from the cell line and typed as Mycoplasma orale. Colonies of this mycoplasma were used to deliberately infect cell cultures which then gained the capacity to reactivate antibody-neutralized virus. The extent of the reactivation depended on the source of neutralizing antiserum. Other species of mycoplasm...

  2. Monoclonal Antibody Therapy does not Abrogate Rejection Risk in Renal Transplant Recipients

    Sanjeev Goswami

    2013-01-01

    Monoclonal antibodies are being increasingly used as therapeutic agents in medicine. Rituximab (anti-CD20) and Daclizumab (anti-IL2Rα) are two such monoclonal antibodies used to prevent organ rejection, but are not fail-safe. We have analyzed the pre and post-transplant antibody profile in serum of renal transplant recipients receiving Rituximab and /or Daclizumab. Study Group: Kidney recipients with acute rejection and having PRA > 10% pre-transplant were selected for the study (n=11). Those...

  3. PRODUCTION OF MONOCLONAL ANTIBODIES

    TOLKOVA E.S.

    2015-01-01

    Full Text Available The article considers the use of monoclonal antibodies in immunotherapy and immunodiagnostics of oncological diseases and their production using hybridoma technolody with flow diagram and technological scheme of manufacturing process

  4. PRODUCTION OF MONOCLONAL ANTIBODIES

    TOLKOVA E.S.

    2015-01-01

    The article considers the use of monoclonal antibodies in immunotherapy and immunodiagnostics of oncological diseases and their production using hybridoma technolody with flow diagram and technological scheme of manufacturing process

  5. A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells.

    Yigit, Burcu; Halibozek, Peter J; Chen, Shih-Shih; O'Keeffe, Michael S; Arnason, Jon; Avigan, David; Gattei, Valter; Bhan, Atul; Cen, Osman; Longnecker, Richard; Chiorazzi, Nicholas; Wang, Ninghai; Engel, Pablo; Terhorst, Cox

    2016-05-01

    The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220+CD5+ Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo, we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors. PMID:27029059

  6. Production Of Human Antibodies

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  7. DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction.

    Rodriguez, F.; Zhang, J.; Whitton, J L

    1997-01-01

    DNA immunization can induce cytotoxic T lymphocytes (CTL), antibodies, and protection against microbial challenge. The underlying mechanisms remain obscure and must be understood to permit rational manipulation and optimization of the technique. We set out to enhance the intracellular degradation of a viral antigen, with the intent of improving antigen entry into, and presentation by, the class I major histocompatibility complex pathway. We achieved this goal by cotranslational ubiquitination...

  8. Production and characterization of peptide antibodies

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are...... powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as......, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody...

  9. Technological progresses in monoclonal antibody production systems

    Rodrigues, E.; Costa, A R; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2009-01-01

    Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large-scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the op...

  10. PRODUCTION OF MONOCLONAL ANTIBODY AGAINST HUMAN IMMUNOGLOBULIN

    J. Majidi

    2000-04-01

    Full Text Available Immunoglobulin E is one of the five classes of immonoglobulins that plays an important role in allergic diseases. Production of monoclonal antibodies by a single clonotype against different epitopes of immunoglobulin E has high priority in development of diagnostic kits.In this study, an attempt was made to produce monoclonal antibodies against human immunoglobulin E. Balb/c mice were immunized with semipurified immunoglobulin E and spleen cells fused with SP2.0 mouse myeloma eel! line in the presence of polyethylene glycol. Supernatant of hybridoma cells was screened for detection of antibody by enzyme linked immonosorbent assay method. Cloning of selective high absorbance wells were done with limiting dilution method. The suitable clone (monoclone was selected by enzyme linked immunosorbent assay and confirmed by immunoblot. The subclass of the chosen monoclonal antibodies was determined and the clones freezed and kept in liquid nitrogen.During this study three successful fusions were carried out, which resulted in development of 156 clones with high production of anti-IgE. Fourteen clones with the highest titres were selected for cloning. After limiting dilution more than 100 monoclonal antibodies were produced and the suitable (me (GJ0F7, i.e.; the clone which displayed the high absorbance in reaction with purified immunoglobulin E and the lowest cross-reactivity with immunoglobulin M, immunoglobulin G and immoglobulin A was chosen. In immunoblotting, presence of high density band in reaction with immunoglobulin E was confirmed. The suitable mab was shown to be IgG 1 subclass with kappa light chain. It seems that, this mab could be successfully used in diagnostic kits.

  11. Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

    We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases

  12. Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

    Newsom-Davis, J.; Willcox, N.; Calder, L.

    1981-11-26

    We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.

  13. Pan-HER-An antibody mixture targeting EGFR, HER2 and HER3 abrogates preformed and ligand-induced EGFR homo- and heterodimers.

    Ellebaek, Sofie; Brix, Susanne; Grandal, Michael; Lantto, Johan; Horak, Ivan D; Kragh, Michael; Poulsen, Thomas Tuxen

    2016-11-01

    The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR. The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment with Pan-HER were investigated by in situ proximity ligation assay and co-immunoprecipitation, demonstrating that Pan-HER and the EGFR-targeting mAb mixture efficiently down-regulate basal EGFR homo- and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan-HER in resistant settings. PMID:27342948

  14. Production of monoclonal antibodies for radioimmunoassays

    Specific antibodies (Abs) have proven most useful and versatile tools for the identification, quantification, and localization of minute amounts of small and large molecules in biologic materials, e.g., body fluids, specific cells, and other body components. So far the most widely used technique for the production of specific Abs consists in immunization of animals like rabbits, goats, or horses, monitoring of Ab formation in the serum, and selection of animals which produce serum containing Abs sufficiently specific for the use envisaged. Although this approach has yielded many valuable results, it has some deficiencies

  15. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

    Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas

    2013-01-01

    Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable...

  16. Production of recombinant antibodies using bacteriophages

    Shukra, A. M.; Sridevi, N. V.; Dev Chandran,; Kapil Maithal,

    2014-01-01

    Recombinant antibody fragments such as Fab, scFv, diabodies, triabodies, single domain antibodies and minibodies have recently emerged as potential alternatives to monoclonal antibodies, which can be engineered using phage display technology. These antibodies match the strengths of conventionally produced monoclonal antibodies and offer advantages for the development of immunodiagnostic kits and assays. These fragments not only retain the specificity of the whole monoclonal ...

  17. Technological progresses in monoclonal antibody production systems.

    Rodrigues, Maria Elisa; Costa, Ana Rita; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2010-01-01

    Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large-scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the optimization of cell culture productivity in normal bioreactors appears as a better strategy. This review describes the main technological progresses made with this intent, presenting the advantages and limitations of each production system, as well as suggestions for improvements. New and upgraded bioreactors have emerged both for adherent and suspension cell culture, with disposable reactors attracting increased interest in the last years. Furthermore, the strategies and technologies used to control culture parameters are in constant evolution, aiming at the on-line multiparameter monitoring and considering now parameters not seen as relevant for process optimization in the past. All progresses being made have as primary goal the development of highly productive and economic mAb manufacturing processes that will allow the rapid introduction of the product in the biopharmaceutical market at more accessible prices. PMID:20043321

  18. The production of antibodies for radioimmunoassay

    Three factors which affect the outcome of any immunisation schedule designed to produce antisera for radio-immunoassay, the antigen, the method of immunisation and the choice of animal are considered. Several factors concerning the nature of the antigen are dealt with, for example, the molecular size and immunogenicity of the antigen. It is noted that the larger polypeptide and proteins are sufficiently immunogenic to elicit a useful antibody response alone and that whilst substances with molecular weights of less than 2000 may produce a response alone they will probably produce a better one if they are conjugated (chemically coupled) to a much larger molecule. The method of immunisation is discussed including a consideration of the use of adjuvant and the route and timing of injections. It is noted that antisera showing the relevant properties for radio-immunoassay are rarely produced without emulsification of the immunogen in Freund's adjuvant although this is not an absolute requirement for antibody production. Data are presented comparing the intramuscular and multiple intradermal routes of injection. The results, however, fail to demonstrate any major advantage for either method although the latter may be more economical, producing high titre antisera with relatively small amounts of immunogen. Because of their convenience rabbits are generally the first choice of animal for raising antisera for radioimmunoassay although guinea pigs, chickens and sheep have been used successfully in many cases

  19. Cell-Free Synthesis Meets Antibody Production: A Review

    Marlitt Stech; Stefan Kubick

    2015-01-01

    Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufactu...

  20. Production of Monoclonal Antibody against Human Nestin.

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad Mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-04-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays. PMID:23407796

  1. Production of Monoclonal Antibody against Human Nestin

    Hadavi, Reza; Zarnani, Amir Hassan; Ahmadvand, Negah; Mahmoudi, Ahmad Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Sadeghi, Mohammad-Reza; Soltanghoraee, Haleh; Akhondi, Mohammad mehdi; Tarahomi, Majid; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2010-01-01

    We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140–250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such a...

  2. Production of antibodies which recognize opiate receptors on murine leukocytes

    Carr, D.J.J.; Bost, K.L.; Blalock, J.E.

    1988-01-01

    An antibody has been developed which recognizes opiate receptors on cells of the immune system. This antibody blocks specific binding of the radiolabeled opiate receptor ligand, /sup 3/H-dihydromorphine, to receptors on murine splenocytes. Additionally, the anti-receptor antibody competes with ..beta..-endorphin, meta-enkephalin, and naloxone for the same binding site on the leukocytes. Moreover, the anti-receptor antibody possesses agonist activity similar to ..beta..-endorphin in suppressing cAMP production by lymphocytes. These results suggest the development of an antibody which recognizes classical opiate receptors on cells of the immune system.

  3. Antibody Production in Plants and Green Algae.

    Yusibov, Vidadi; Kushnir, Natasha; Streatfield, Stephen J

    2016-04-29

    Monoclonal antibodies (mAbs) have a wide range of modern applications, including research, diagnostic, therapeutic, and industrial uses. Market demand for mAbs is high and continues to grow. Although mammalian systems, which currently dominate the biomanufacturing industry, produce effective and safe recombinant mAbs, they have a limited manufacturing capacity and high costs. Bacteria, yeast, and insect cell systems are highly scalable and cost effective but vary in their ability to produce appropriate posttranslationally modified mAbs. Plants and green algae are emerging as promising production platforms because of their time and cost efficiencies, scalability, lack of mammalian pathogens, and eukaryotic posttranslational protein modification machinery. So far, plant- and algae-derived mAbs have been produced predominantly as candidate therapeutics for infectious diseases and cancer. These candidates have been extensively evaluated in animal models, and some have shown efficacy in clinical trials. Here, we review ongoing efforts to advance the production of mAbs in plants and algae. PMID:26905655

  4. Production and assay of forskolin antibodies

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using 3H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added 3H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC50 was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound

  5. Production and Purification of Rabbit's Polyclonal Antibody Against Factor VIII.

    Sohrabi, Simin; Akbarzadeh, Azim; Norouzian, Dariush; Farhangi, Ali; Mortazavi, Mehri; Mehrabi, Mohammad Reza; Chiani, Mohsen; Saffari, Zahra; Ghassemi, Soheil

    2011-01-01

    The attempt is made to produce recombinant factor VIII but the first step in producing such product is production and purification of rabbit's polyclonal antibody against factor VIII. The second and third steps involve monoclonal antibody and recombinant factor VIII production. Factor VIII is one of the most important coagulating factor where its deficiency leads to diseases like hemophilia type A or classic. It is an inherited disease. Previously, it was obtained through fractionation of blo...

  6. High throughput production of mouse monoclonal antibodies using antigen microarrays

    De Masi, Federico; Chiarella, P.; Wilhelm, H.;

    2005-01-01

    Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification...

  7. Production of monoclonal antibody with Celline-350 bioreactor

    Monoclonal antibodies are protein that are highly specific and sensitive in their reaction with specific sites on target molecules that they have become reagents of central importance in the diagnostic and treatment of human diseases. This paper reports the use of CELLine-350 bioreactor to produce continuous supply of serum-free breast cancer monoclonal antibody. Initial volume of 5ml (1.5 x 106 viable cells/ml) is inoculated into the bioreactor and harvesting is done every 5 days to obtain high yield monoclonal antibody. The serum-free supernatant is precipitated with 50% saturated ammonia sulfate and the antibody is purified by protein-G affinity chromatography. The concentration of monoclonal antibody successfully produced by the bioreactor is 0.91mg/ml respectively and it is measured by the Lowry method. This result shows that bioreactor Celline-350 is easy to handle and cost effective for the continuous production of serum free monoclonal antibody. (Author)

  8. Cell-Free Synthesis Meets Antibody Production: A Review

    Marlitt Stech

    2015-01-01

    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  9. Production of monoclonal antibodies against canine leukocytes.

    Aguiar, Paulo Henrique Palis; Borges dos Santos, Roberto Robson; Lima, Carla Andrade; Rios de Sousa Gomes, Hilton; Larangeira, Daniela Farias; Santos, Patrícia Meira; Barrouin-Melo, Stella Maria; Conrado dos-Santos, Washington Luis; Pontes-de-Carvalho, Lain

    2004-04-01

    A panel of anti-canine leukocyte monoclonal antibodies (MAbs) was produced by immunizing BALB/c mice with canine peripheral blood mononuclear cells (PBMC), either resting or stimulated with concanavalin A (ConA). Three out of 28 clones-IH1, AB6, and HG6-screened by ELISA and producing antibody with the highest specificity for canine cell immunostaining, were subjected to three subsequent subcloning steps by limiting dilution, and selected for further characterization. These MAbs belonged to IgG1 (HG6 and IH1) and IgG2a (AB6) isotypes. The distribution of cell populations expressing the antigen recognized by the antibodies was identified by indirect immunoflorescence on canine PBMC and on tissue sections of lymph node, spleen, liver and skin. The possible crossreactivity with human PBMC was also examined in immunocytochemistry. One of the antibodies specifically recognized macrophages. The MAbs presented here can be foreseen as possible valuable diagnostic and research tools to study immune functions in dogs. PMID:15165486

  10. Heterohybridoma for the production of non murine monoclonal antibodies

    Kh.Victoria Chanu and M. Ayub Ali

    Full Text Available Hybridoma technology described by kohler and Milstein produce only mouse immunoglobulins. Such immunoglobulins have limited use due to its negative side effects such as the recipient’s immune response. The production of a non murine monoclonal antibody to combat the problems of murine monoclonal antibody is again difficult due to the lack of a suitable myeloma cell line. Heterohybridoma formed by the fusion of lymphocyte of one species with the myeloma cell of a different species is the solution, which can be used for the production of non murine monoclonal antibodies. [Veterinary World 2010; 3(8.000: 390-392

  11. [Continuous perfusion culture hybridoma cells for production of monoclonal antibody].

    Mi, Li; Li, Ling; Feng, Qiang; Yu, Xiao-Ling; Chen, Zhi-Nan

    2002-05-01

    Hybridoma cells were cultured by continuous perfusion in Fibra-Cel of 5L packed-bed bioreactor for 22 days in low serum or serum-free media. The corresponded amino acids were fed and serum concentration was decreased by analyzing glucose concentration, oxygen uptake rate, secretary antibody amount and amino acids concentration in culture supernatant. Comparing with continuous perfusion culture that amino acids were not fed, antibody amount of production was increased about 2-3 times. The inoculated cell density was 2.5 x 10(5) cells/mL, while the final cell density was 8.79 x 10(8) cells/mL. Antibody production was reached 295 mg/L/d at average level, and the highest level was reached 532 mg/L/d. These results provided a primary mode of enlarge culture for monoclonal antibody industralization. PMID:12192875

  12. Production of recombinant antibody fragments in Bacillus megaterium

    Jahn Dieter; Schirrmann Thomas; Biedendieck Rebekka; Roth Andreas; Hust Michael; Jordan Eva; Dübel Stefan

    2007-01-01

    Abstract Background Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. ...

  13. Developments in the production of mucosal antibodies in plants.

    Vasilev, Nikolay; Smales, C Mark; Schillberg, Stefan; Fischer, Rainer; Schiermeyer, Andreas

    2016-01-01

    Recombinant mucosal antibodies represent attractive target molecules for the development of next generation biopharmaceuticals for passive immunization against various infectious diseases and treatment of patients suffering from mucosal antibody deficiencies. As these polymeric antibodies require complex post-translational modifications and correct subunit assembly, they are considered as difficult-to-produce recombinant proteins. Beside the traditional, mammalian-based production platforms, plants are emerging as alternative expression hosts for this type of complex macromolecule. Plant cells are able to produce high-quality mucosal antibodies as shown by the successful expression of the secretory immunoglobulins A (IgA) and M (IgM) in various antibody formats in different plant species including tobacco and its close relative Nicotiana benthamiana, maize, tomato and Arabidopsis thaliana. Importantly for biotherapeutic application, transgenic plants are capable of synthesizing functional IgA and IgM molecules with biological activity and safety profiles comparable with their native mammalian counterparts. This article reviews the structure and function of mucosal IgA and IgM antibodies and summarizes the current knowledge of their production and processing in plant host systems. Specific emphasis is given to consideration of intracellular transport processes as these affect assembly of the mature immunoglobulins, their secretion rates, proteolysis/degradation and glycosylation patterns. Furthermore, this review provides an outline of glycoengineering efforts that have been undertaken so far to produce antibodies with homogenous human-like glycan decoration. We believe that the continued development of our understanding of the plant cellular machinery related to the heterologous expression of immunoglobulins will further improve the production levels, quality and control of post-translational modifications that are 'human-like' from plant systems and enhance the

  14. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2013-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel ...

  15. Guidelines to cell engineering for monoclonal antibody production

    Costa, A.; Rodrigues, E; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2010-01-01

    Monoclonal antibodies (mAbs) are currently used for many diagnostic and therapeutic applications. The high demand for these biopharmaceuticals has led to the development of large-scale manufacturing processes, with productivity improvements being mainly achieved by optimization of bioreactor systems. However, more recently, the early steps of production, previous to bioreactor culture, have been presented as alternative areas where productivity enhancements can be achieved. Thus, ...

  16. Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy

    Ghislain Moussavou; Kisung Ko; Jeong-Hwan Lee; Young-Kug Choo

    2015-01-01

    Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the produ...

  17. Production of a diagnostic monoclonal antibody in perennial alfalfa plants.

    Khoudi, H; Laberge, S; Ferullo, J M; Bazin, R; Darveau, A; Castonguay, Y; Allard, G; Lemieux, R; Vézina, L P

    1999-07-20

    The increasing use of monoclonal antibodies (mAbs) in diagnostic reagents necessitates efficient and cost-effective mAb production methods. In blood banks, one of the most routinely used reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Here we report the production of a functional, purified anti-human IgG, through the expression of its encoding genes in perennial transgenic alfalfa. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained, and plants from crosses were found to express fully assembled C5-1. The purification procedure yielded mainly the H2L2 form with specificity and affinity identical to those of hybridoma-derived C5-1. The ability to accumulate the antibody was maintained both in parental F1 lines during repeated harvesting and in clonal material; the antibody was stable in the drying hay as in extracts made in pure water. Also, plant and hybridoma-derived C5-1 had similar in vivo half-lives in mice. These results indicate that plant C5-1 could be used in a diagnostic reagent as effectively as hybridoma-derived C5-1, and demonstrates the usefulness of perennial systems for the cost-effective, stable, and reliable production of large amounts of mAbs. PMID:10397849

  18. Antibody

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  19. Production of biological reagents for radioimmunoassay second antibody

    The experimental production of second antibody to be used in hormonal assays, in which the first antibody is raised in rabbits, is described. Four sheep were immunized with the rabbit immunoglobulin prepared at IPEN-CNEN laboratory. Their antisera were evaluated by the human thyrotropin radioimmunoassay employing materials provided by the National Hormone and Pituitary Program (USA), in comparison with a reference antiserum of known quality, produced in goat by the Radioassay Systems Laboratories - RSL (USA). From the fourth booster injection the animals developed antiserum with titer similar to that exhibited by the commercial product, even presenting higher values. These antisera are now being examinated for the optimal conditions of precipitation before be packed for future use and distribution. (author)

  20. Production and purification of polyclonal antibody against melatonin hormone

    Fooladsaz K

    1999-09-01

    Full Text Available Nowadays immunochemical techniques have played a very important and valuable role in quantitative and qualitative assays of liquid compounds of the body. Producing antibody against immunogenes is the first step to make immunochemical kits. In this study production and purification of polyclonal antibody against melatonin has been considered. This hormone which has several important functions in physiological conditions such as migraine, cirrhosis, mammary gland cancer and other diseases, is the most important pineal gland secretion. This gland is a circumventricular organ of brain and according to histological and anatomical studies, it is a high secretory organ, that secretes active biological substances like melatonin, oxytocin, serotonin and ect. In this study, melatonin has been considered as hapten and has become an immunogen by being linked to the bovine serum Albumin. Then, by the immunization of three white New Zeland rabbits that had the booster injections in regular intervals, the antibody titer was detected to be 1/2000, by using checkboard curves, and with the use of melatonin linked to penicillinase as a labeled antigen, the titer was detected 1/200. Finally an antibody with high purification rate has been obtained, which can be used in immunochemical assays like RIA, ELISA, and EIA.

  1. Production, isolation, and characterization of rabbit anti-idiotypic antibodies directed against human antithyrotrophin receptor antibodies.

    Baker, J. R.; Lukes, Y G; Burman, K. D.

    1984-01-01

    Previous studies have shown that anti-idiotypic antibodies can be developed in vivo through animal immunization with idiotype, and that these antibodies can be isolated from other anti-immunoglobulin antibodies by affinity purification. These techniques have relied on large amounts of idiotype, which were produced either by hyperimmunization or by monoclonal antibodies, to serve as the affinity adsorbent. In the present study, we produced anti-idiotypic antibodies to human anti-thyroid-stimul...

  2. Cell line profiling to improve monoclonal antibody production.

    Kang, Sohye; Ren, Da; Xiao, Gang; Daris, Kristi; Buck, Lynette; Enyenihi, Atim A; Zubarev, Roman; Bondarenko, Pavel V; Deshpande, Rohini

    2014-04-01

    Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest

  3. Antibody Production From Immunized Rabbits By Brucella Abortus

    In this research Brucella abortus was used as antigen which was made by killing the bacteria in boiling water for 1 hour and then add 0.5% phenol. The suspension of bacteria of 6x108 cells/mm3 was used as antigen. Rabbits of about 3 months old were injected with 0.50 mI of the antigen by intradermal route with an interval of two weeks. The animals were divided in three groups i.e. A (control group), B (immunization group) and C (immunization and irradiation group). In C group, the animals were first immunized by the antigen and then 2 days later were irradiated by a low dose of 0.50 Gy of gamma rays. Each group consisted of 3 animals. Parameters were observed by weighing the animals, counting leucocyte and lymphocyte cells, and anaIysing the antisera. The research were done two times, included immunization I x, boostered 4 x and analysed 5x. The results obtained were as follows: A (control group) yielded 2.34 g/dl of non specific antibody, B (immunization group) yielded 3.22 g/dI of specific antibody, C (immunization and irradiation group) yielded 3.50 g/dl of spesific antibody. The leucocyte cells of A, B , and C group were 8.240, 7.887, and 8.120 cells/mm3, respectively. The lymphocyte cells of A, B, and C group were 69%, respectively. The weigh of A, B, and C group were 1.44; 1.53; and l.41 kg, respectively. The purpose of this research was prepared to produce the diagnostic reagen (RIA Kit) for a rapid detection of animals disease especially brucellosis. It seemed that C group (the combination of immunization and irradiation treatments) yielded the highest value of antibody production compared to another group

  4. NCI Requests Targets for Monoclonal Antibody Production and Characterization - Office of Cancer Clinical Proteomics Research

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  5. Production and radioiodination of monoclonal antibodies and its applications in nuclear medicine

    The basis of the monoclonal antibody production methodology, some immunological concepts which are important for the understanding of what is a Monoclonal Antibody, its radioiodination and acceptance as receptor-specific radiopharmaceuticals in nuclear medicine are reviewed. (author)

  6. A multi-Fc-species system for recombinant antibody production

    Nizak Clément; Vielemeyer Ole; El Marjou Ahmed; Moutel Sandrine; Benaroch Philippe; Dübel Stefan; Perez Franck

    2009-01-01

    Abstract Background Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural anti...

  7. Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

    Hiroyuki Tanaka

    2012-01-01

    Full Text Available We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb. Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was observed. The detection limit was 1.6 ng of solasonine. The hydrolysed products of solamargine were determined by fingerprint of eastern blotting compared to their Rf values depending on the sugar number. Fingerprint by eastern blotting using anti-ginsenoside Rb1 MAb distinguished the formula containing ginseng prescribed in traditional Chinese medicine. By double-staining of ginsenosides it is possible to suggest that the staining color shows the pharmacological activity, such as the purple bands indicate ginsenosides having stimulation activity, and the blue color indicated compound like ginsenosides possessed the depression affect for the central nervous system (CNS, respectively.

  8. Production and characterization of a hybridoma-derived antibody to Blastomyces dermatitidis.

    Young, K D; Larsh, H W

    1982-01-01

    A hybridoma cell line was isolated which produced monoclonal antibody to one protein component of a yeast-phase cytoplasmic antigenic complex of Blastomyces dermatitidis. The immunoglobulin M antibody product was characterized by immunodiffusion, autoradiography of polyacrylamide gels, and cellulose acetate electrophoresis. By attaching the antibody to an affinity gel, one major protein band was identified by polyacrylamide gel electrophoresis as the antigen for which the antibody was specific.

  9. Interleukin-6 Enhances Production of Anti-OspC Immunoglobulin G2b Borreliacidal Antibody

    Remington, Monica C.; Munson, Erik L.; Callister, Steven M.; Molitor, Melanie L.; Christopherson, John A.; DeCoster, David J.; Lovrich, Steven D.; Schell, Ronald F.

    2001-01-01

    Protection against infection with Borrelia burgdorferi is dependent primarily on induction of complement-dependent antibody that can kill the spirochete. Measuring the production of sustained high levels of borreliacidal antibody is thus paramount for determining potential vaccine efficacy. We investigated the borreliacidal antibody response in sera and the amount of antibody produced by cultured lymph node cells of C3H/HeJ mice vaccinated with outer surface protein C (OspC). We showed that r...

  10. Monoclonal antibodies in animal production; their use in diagnostics and passive immunization.

    Booman, P.

    1989-01-01

    One of the landmarks in immunology was the invention and development of monoclonal antibody-secreting hybridomas by Milstein and his coworkers. The enormous promise of monoclonal antibody technology, which became apparent soon after its discovery, may explain the unusual speed with which monoclonal antibodies have been applied to biological and medical sciences.In animal production monoclonal antibodies are increasingly finding application in the areas of diagnostics, passive immunization and...

  11. Production and characterization of antibody against aflatoxin Q1.

    Fan, T. S.; Zhang, G S; Chu, F. S.

    1984-01-01

    Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization w...

  12. Production and characterization of monoclonal antibodies against mink leukocytes

    Chen, W.S.; Pedersen, Mikael; Gram-Nielsen, S.;

    1997-01-01

    Three monoclonal antibodies (mAbs) were generated against mink leukocytes. One antibody reacted with all T lymphocytes, one with all monocytes and one had platelet reactivity. Under reducing conditions, the T lymphocyte reactive antibody immunoprecipitated 18 kDa, 23 kDa, 25 kDa and 32-40 kDa pol...

  13. TIM-1 signaling in B cells regulates antibody production

    Highlights: → TIM-1 is highly expressed on anti-IgM + anti-CD40-stimulated B cells. → Anti-TIM-1 mAb enhanced proliferation and Ig production on activated B cell in vitro. → TIM-1 signaling regulates Ab production by response to TI-2 and TD antigens in vivo. -- Abstract: Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3+ anti-CD28-stimulated CD4+ T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.

  14. Interleukin-6 Promotes Anti-OspA Borreliacidal Antibody Production In Vitro

    Munson, Erik L.; Dean T. Nardelli; Luk, K. H. Kevin; Remington, Monica C.; Callister, Steven M.; Schell, Ronald F.

    2006-01-01

    Determination of the immunological mediators responsible for promoting the production of borreliacidal antibody may facilitate the development of an improved borreliosis vaccine for human and veterinary use. Previously, we developed an in vitro assay to determine if borreliacidal antibody production could be augmented by treatment with different cytokines. In this study, in vitro treatment of lymph node cells producing borreliacidal antibody with recombinant interleukin-6 (rIL-6) resulted in ...

  15. Production of Borreliacidal Antibody to Outer Surface Protein A In Vitro and Modulation by Interleukin-4

    Munson, Erik L.; Du Chateau, Brian K.; Jobe, Dean A.; Lovrich, Steven D.; Callister, Steven M.; Schell, Ronald F.

    2000-01-01

    Borreliacidal antibody production is one of several parameters for establishing the effectiveness of Borrelia burgdorferi vaccines. The production of borreliacidal antibody was studied in vitro by culturing immune lymph node cells with macrophages and B. burgdorferi. We showed that borreliacidal antibody, directed primarily against outer surface protein A (OspA), was readily produced by lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi in aluminum...

  16. Production of anti-Candida antibodies in mice with gut colonization of Candida albicans.

    Yasuo Ono; Osamu Koshio; Nobuo Suegara; Tatsuo Ikeda; Kayoko Wada; Masayasu Mitsuya; Hiroko Ishibashi; Shigeru Abe; Shigeru Tansho; Hideyo Yamaguchi

    2004-01-01

    BACKGROUND: Production of antibodies that are specific for allergens is an important pathological process in inflammatory allergic diseases. These contain the antibodies against antigens of Candida albicans, one of the normal microbial flora in an intestinal tract. We studied the effects of the prednisolone administration on the production of anti-Candida antibodies in the gastrointestinally C. albicans-colonized mice. METHODS AND MATERIALS: BALB/c mice, treated with antibacterial antibiotics...

  17. A tetravalent dengue nanoparticle stimulates antibody production in mice

    Silva Elisângela F

    2012-03-01

    Full Text Available Abstract Background Dengue is a major public health problem worldwide, especially in the tropical and subtropical regions of the world. Infection with a single Dengue virus (DENV serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing secondary infection with a different serotype progresses to the severe form of the disease, dengue hemorrhagic fever/dengue shock syndrome. Currently, there are no licensed vaccines or antiviral drugs to prevent or treat dengue infections. Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines. Findings Here, we used a murine model to evaluate the IgG production after administration of inactivated DENV corresponding to all four serotypes adsorbed to bovine serum albumin nanoparticles. This formulation induced a production of anti-DENV IgG antibodies (p Conclusions Our results show that while the nanoparticle system induces humoral responses against DENV, further investigation with different DENV antigens will be required to improve immunogenicity, epitope specicity, and functional activity to make this platform a viable option for DENV vaccines.

  18. Production of monoclonal antibodies reactive with ovine eosinophils

    Meeusen Els NT

    2007-09-01

    Full Text Available Abstract Background There is strong evidence implicating eosinophils in host defence against parasites as well as allergic disease pathologies. However, a lack of reagents such as monoclonal antibodies (mAbs specific for eosinophils has made it difficult to confirm the functional role of eosinophils in such disease conditions. Using an established mammary model of allergic inflammation in sheep, large numbers of inflammatory cells enriched for eosinophils were collected from parasite-stimulated mammary glands and used for the generation of mAbs against ovine eosinophils. Results A panel of mAbs was raised against ovine eosinophils of which two were shown to be highly specific for eosinophils. The reactivity of mAbs 3.252 and 1.2 identified eosinophils from various cell and tissue preparations with no detectable reactivity on cells of myeloid or lymphoid lineage, tissue mast cells, dendritic cells, epithelial cells or other connective tissues. Two other mAbs generated in this study (mAbs 4.4 and 4.10 were found to have reactivity for both eosinophils and neutrophils. Conclusion This study describes the production of new reagents to identify eosinophils (as well as granulocytes in sheep that will be useful in studying the role of eosinophils in disease pathologies in parasite and allergy models.

  19. Production, isolation, and characterization of rabbit anti-idiotypic antibodies directed against human antithyrotrophin receptor antibodies.

    Baker, J R; Lukes, Y G; Burman, K D

    1984-01-01

    Previous studies have shown that anti-idiotypic antibodies can be developed in vivo through animal immunization with idiotype, and that these antibodies can be isolated from other anti-immunoglobulin antibodies by affinity purification. These techniques have relied on large amounts of idiotype, which were produced either by hyperimmunization or by monoclonal antibodies, to serve as the affinity adsorbent. In the present study, we produced anti-idiotypic antibodies to human anti-thyroid-stimulating hormone (TSH) receptor antibodies by first injecting rabbits with (TSH receptor purified) IgG from Graves' patients. The resulting antiserum was then adsorbed with Sepharose-coupled TSH in an attempt to specifically bind and isolate the anti-idiotype. The antibody obtained from this process was shown to bind specifically to TSH receptor-binding antibodies from Graves' patients, and this binding could be inhibited by 56% with the addition of 10(-4) M TSH but not by HCG (10(-2) M). The anti-idiotype also bound to TSH, and this binding could be specifically inhibited by receptor-purified Graves' IgG (60% inhibition at 10 micrograms/ml IgG), but not by IgG from normal subjects (no inhibition at 50 micrograms/ml IgG). In a TSH receptor binding assay, the anti-idiotype could inhibit TSH receptor binding in Graves' sera at a 1,000-fold lower concentration than could anti-kappa/lambda antiserum; the anti-idiotypic antiserum also inhibited in vitro TSH-mediated adenylate cyclase stimulation at an IgG concentration of 5 micrograms/ml, while heterologous anti-TSH antisera and normal IgG at similar concentrations had no effect. Finally, despite being generated against a single patient's TSH receptor binding antibody, the anti-idiotype was able to block TSH receptor binding in the serum of six other Graves' patients, thus suggesting that there may be conformational conservation in the antigen that is recognized by different individuals' TSH receptor-binding immunoglobulins. PMID

  20. Production and characterization of monoclonal antibodies specific for Pseudorabies virus

    Marković Ljiljana; Ašanin Ružica; Radojičić Sonja; Isenović Esma

    2007-01-01

    Monoclonal antibodies (MAbs) against Pseudorabies virus (PrV) were obtained by the fusion of P3x-Ag8.653 myeloma and spleen cells from immunized BALB/c mice with a suspension of Pseudorabies (PrV) virus strains: MAVE (Morbus Aujeszk'y virus Ercegovac) and NS 257 (Novosadski virus strain). A total of 95 antibody-secreting hybridoma cells against the virus strain (MAVE and NS 257) of Pseudorabies virus have been isolated. Ten of these monoclonal antibodies were found by ELISA (Enzy...

  1. Enhancement of antibody production by growth factor addition in perfusion and hollow-fiber culture systems.

    Omasa, T; Kobayashi, M; Nishikawa, T; Shioya, S; Suga, K; Uemura, S; Kitani, Y; Imamura, Y

    1995-12-20

    The effects of the high-molecular-weight growth factors, transferrin and bovine serum albumin (BSA), on antibody production were analyzed quantitatively in continuous hollow-fiber cultivation over a period of 60 days. Transferrin enhanced cell growth but had no significant effect on the specific antibody production rate, whereas BSA significantly enhanced antibody production. The antibody production rate was increased 4- and 14-fold respectively by feeding BSA at 2 and 5 g L(-1) into the EC side of the system (the side connected to the cell-containing outer part of the hollow-fiber unit) compared with the production achieved without BSA. Addition of 5 g L(1) BSA into the IC side of the system (the side connected to the inner part of the hollow-fiber unit) resulted in a 2.5-fold increase in the antibody production rate. The effect of BSA was also analyzed using the perfusion culture system with a separation unit. When fresh medium containing either 2 or 5 g L(-1) BSA was fed into the reactor, both the specific growth rate and specific death rate increased, while the specific antibody production rate was increased 2- and 25-fold, respectively, by feeding BSA at these two concentrations compared with no addition. Comparing the two systems, the increase in the antibody production rate achieved with the hollow-fiber system was threefold greater than that in the perfusion culture system with the same concentration of BSA feeding. (c) 1995 John Wiley & Sons, Inc. PMID:18623537

  2. Gamma Interferon Inhibits Production of Anti-OspA Borreliacidal Antibody In Vitro

    Munson, Erik L.; Du Chateau, Brian K.; Jensen, Jani R.; Callister, Steven M.; DeCoster, David J.; Schell, Ronald F.

    2002-01-01

    The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. b...

  3. The production of recombinant single chain antibody fragments for the detection of illicit drug residues

    Brennan, Joanne

    2005-01-01

    Recombinant antibodies represent a more sensitive and specific detection tool for immunoanalysis. The research carried out for this thesis describes the production of genetically-derived single chain antibody fragments to detect illicit drugs. A variety of novel recombinant antibody fragments against morphine-3-glucuronide, a metabolite of heroin has been produced. A monomeric, dimeric and enzyme-labelled scFv were characterised with respect to their binding abilities and cross reactiviti...

  4. Hybridoma growth in a new generation hollow fibre bioreactor: antibody productivity and consistency

    Kessler, Nicole; Thomas, Ghislaine; Gerentes, Lionel; Delfosse, Gaelle; Aymard, Michèle

    1997-01-01

    This paper analyses the performance of MAbMaxTM/TricentricTM, a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process parameters were optimised using a mouse hybridoma, IgG1K secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long period of ...

  5. Impaired Antigen-Specific Immune Response to Vaccines in Children with Antibody Production Defects

    Szczawinska-Poplonyk, Aleksandra; Breborowicz, Anna; Samara, Husam; Ossowska, Lidia; Dworacki, Grzegorz

    2015-01-01

    The impaired synthesis of antigen-specific antibodies, which is indispensable for an adaptive immune response to infections, is a fundamental pathomechanism that leads to clinical manifestations in children with antibody production defects. The aim of this study was to evaluate the synthesis of antigen-specific antibodies following immunization in relation to peripheral blood B cell subsets in young children with hypogammaglobulinemia. Twenty-two children, aged from 8 to 61 months, with a def...

  6. "Abrogation of Rulings” Methodology: A Critique

    Gasser Auda

    2004-01-01

    Surveying the subject of abrogation (naskh) in the Qur’ān, ḥādīth and Islamic literature, it is clear that most abrogation cases were introduced after the Prophetic era in order to interpret certain Qur’ānic verses and Prophetic narrations (aḥādīth) that some scholars perceived as “conflicting.” Two striking examples are “The Verse of the Sword” (āyat al-saif) and “The Verse of the Barrier” (āyat al-ḥijāb). The Qur’ānic verses and aḥādīth,...

  7. Production of antibodies against measles virions by use of the mouse hybridoma technique

    Togashi, T.; Oervell, C.; Norrby, E. (Kungliga Karolinska Mediko-Kirurgiska Inst., Stockholm (Sweden)); Vartdal, F. (Rikshospitalet, Oslo (Norway))

    1981-01-01

    Mouse hybridoma cell lines were produced by fusion of P3 x 63 Ag8 mycloma cells with spleen cells from BALB/c mice immunized with purified measles virions. About 60 per cent of single cell colonies in wells were found to produce measles antibodies as determined by a radioimmune assay. Selected measles antibody producing hybridoma cell lines were passaged intraperitoncally in mice and ascites fluids were collected. This material contained 20 - 200 times higher antibody titers than unconcentrated medium from hybridoma cell lines propagated in tissue culture. The ascites fluid antibody products of 23 hybridoma cell lines were characterized by different measles serological tests. Seventeen lines produced high titers of hemagglutination inhibiting (HI) and hemolysis-inhibition (HLI) antibodies. One hybridoma cell line produced Ig with low HI but high HLI activity and the remaining 5 hybridoma cell line products only carried HLI activity. Unexepctedly it was found in radioimmune precipitation assays that all hybridomas studied, including those showing HLI but no HI antibody activity, gave a selective precipitation of the 79 K measles hemagglutinin polypeptide. Radioimmune precipitation assays with sera from immunized animals showed that they contained high titers of antibodies precipitating the 79 K polypeptide but in addition also somewhat lower titers of antibodies precipitating the 60 K nucleoprotein, 40 K fusion and 36 K matrix polypeptides. Homogeneous Ig products carrying measles antibody activity were demonstrated by imprint immunoelectrophoresis of ascites materials.

  8. Production of antibodies against measles virions by use of the mouse hybridoma technique

    Mouse hybridoma cell lines were produced by fusion of P3 x 63 Ag8 mycloma cells with spleen cells from BALB/c mice immunized with purified measles virions. About 60 per cent of single cell colonies in wells were found to produce measles antibodies as determined by a radioimmune assay. Selected measles antibody producing hybridoma cell lines were passaged intraperitoncally in mice and ascites fluids were collected. This material contained 20 - 200 times higher antibody titers than unconcentrated medium from hybridoma cell lines propagated in tissue culture. The ascites fluid antibody products of 23 hybridoma cell lines were characterized by different measles serological tests. Seventeen lines produced high titers of hemagglutination inhibiting (HI) and hemolysis-inhibition (HLI) antibodies. One hybridoma cell line produced Ig with low HI but high HLI activity and the remaining 5 hybridoma cell line products only carried HLI activity. Unexepctedly it was found in radioimmune precipitation assays that all hybridomas studied, including those showing HLI but no HI antibody activity, gave a selective precipitation of the 79 K measles hemagglutinin polypeptide. Radioimmune precipitation assays with sera from immunized animals showed that they contained high titers of antibodies precipitating the 79 K polypeptide but in addition also somewhat lower titers of antibodies precipitating the 60 K nucleoprotein, 40 K fusion and 36 K matrix polypeptides. Homogeneous Ig products carrying measles antibody activity were demonstrated by imprint immunoelectrophoresis of ascites materials. (Author)

  9. Recombinant mouse interferon-gamma regulation of antibody production.

    Johnson, H M; Torres, B A

    1983-01-01

    Interferon-gamma produced in monkey cells by transfection with mouse interferon-gamma cDNA suppressed the mouse in vitro antibody response in a manner similar to that of natural mouse interferon-gamma. Significant suppression was obtained with as little as 1 U of interferon. Recombinant human interferon-gamma produced by cloning in a similar fashion was not suppressive. Both the suppressive and the antiviral activities of recombinant interferon-gamma were neutralized by antibodies to mouse na...

  10. Production and characterization of yeast killer toxin monoclonal antibodies

    Polonelli, L; Morace, G

    1987-01-01

    Monoclonal antibodies were obtained after fusion of mouse myeloma cells with spleen cells isolated from mice primed with a crude extract of yeast killer toxin produced by a strain of Hansenula anomala. Hybridomas were selected by specific immunoassay reaction of their fluid with crude yeast killer toxin extract. Among the monoclonal antibodies, which were characterized by the Western blot technique, one (designated KT4) proved to have precipitating properties, thus permitting the neutralizati...

  11. Production of Bartonella Genus-Specific Monoclonal Antibodies

    Liang, Zhongxing; La Scola, Bernard; Lepidi, Hubert; Raoult, Didier

    2001-01-01

    Monoclonal antibodies (MAbs) which react with heat-resistant proteins with molecular masses of 32 to 33 kDa of 14 different Bartonella species were produced. These antibodies did not react with antigens of 26 diverse bacterial strains by microimmunofluorescence assay except MAb B3D4, which reacted with Chlamydia psittaci and Chlamydia trachomatis at low titers. The identification of a common Bartonella antigenic protein will make it possible to later produce a diagnostic antigen by cloning an...

  12. Control of IgE and IgGl antibody production in mice

    The production of IgE and IgCl was studied in untreated, thymectomized, splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl, Ascaris, and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgGl antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgGl production. A single dose of rabbit antithymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgGl production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgGl production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgGl antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgGl production in mice

  13. Production and Purification of Rabbit’s Polyclonal Antibody Against Factor VIII

    Sohrabi, Simin; Akbarzadeh, Azim; Norouzian, Dariush; Farhangi, Ali; Mortazavi, Mehri; Mehrabi, Mohammad Reza; Chiani, Mohsen; Saffari, Zahra; Ghassemi, Soheil

    2011-01-01

    The attempt is made to produce recombinant factor VIII but the first step in producing such product is production and purification of rabbit’s polyclonal antibody against factor VIII. The second and third steps involve monoclonal antibody and recombinant factor VIII production. Factor VIII is one of the most important coagulating factor where its deficiency leads to diseases like hemophilia type A or classic. It is an inherited disease. Previously, it was obtained through fractionation of blo...

  14. Production in vitro of antibodies directed against alloantigen-specific recognition sites on T cells and on lymphocytotoxic HLA antibodies.

    Singal, D P; Blajchman, M A; Joseph, S; Roberge, B; Smith, E K; Ludwin, D

    1988-01-01

    We have examined the mechanism of immunological unresponsiveness in a recipient (P.S.) with a long-term functioning renal allograft. P.S., whose HLA type is A1, A30; B14, B18; DR1, w8; DRw52; DQw1 and in whose serum we had earlier demonstrated the presence of antiidiotypic antibodies, received a kidney from a cadaver donor of HLA type A1, A10, B8 in March, 1970. Peripheral blood B lymphocytes from the patient were transformed with Epstein-Barr virus (EBV), and by the cluster-picking technique a B cell line was propagated with continuous production of antibodies. Antiidiotypic antibodies with two distinct biological functions were demonstrable; one specifically inhibiting the lymphocytotoxic activity of anti-HLA-B8, B5, and DR3 reference typing sera, and the other specifically inhibiting proliferative responses in MLC of the recipient's lymphocytes and of third party cells sharing B14, DR1, DQw1 with the patient against stimulator cells carrying B8, DR3 antigens. Immunodepletion experiments demonstrated that the inhibitory activity was associated with the IgM fraction. Absorption experiments suggested that different antibodies may be responsible for the inhibition of lymphocytotoxic activity of anti-HLA sera and of the proliferative responses in MLC. Antiidiotypic antibodies have been postulated to be important in maintaining allograft tolerance in vivo, thereby enhancing renal allograft survival. The availability of such antibodies in large quantities, produced in vitro, could provide antisera for the immunochemical characterization of specific idiotypic receptors on immunoglobulins and T lymphocytes. PMID:2970351

  15. Quantification of antibody production of individual hybridoma cells by Surface Plasmon Resonance imaging.

    Stojanovic, I.; Velden, van der T.J.G.; Mulder, H.W.; Schasfoort, R.B.M.; Terstappen, L.W.M.M.

    2015-01-01

    Surface plasmon resonance imaging (SPRi) is most frequently used for the label-free measurement of biomolecular interactions. Here we explore the potential of SPRi to measure antibody production of individual hybridoma cells. As a model system, cells from a hybridoma, producing monoclonal antibodies

  16. Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression

    Haredy, Ahmad M.; Nishizawa, Akitoshi; Honda, Kohsuke; Ohya, Tomoshi; Ohtake, Hisao; Omasa, Takeshi

    2013-01-01

    To improve antibody production in Chinese hamster ovary (CHO) cells, the humanized antibody-producing CHO DP-12-SF cell line was transfected with the gene encoding activating transcription factor 4 (ATF4), a central factor in the unfolded protein response. Overexpression of ATF4 significantly enhanced the production of antibody in the CHO DP-12-SF cell line. The specific IgG production rate of in the ATF4-overexpressing CHO-ATF4-16 cells was approximately 2.4 times that of the parental host c...

  17. Clearance of injected radioactively labelled antibodies to tumour products by liposome-bound second antibodies

    Liposomes containing anti-goat immunoglobulin were injected 24 h after administration of 125I-labelled goat antibody against the carcinoembryonic antigen (anti-CEA) to groups of nude mice bearing human tumour xenografts, and normal mice. Controls in each group received radioactively labelled anti-CEA only. In liposome-treated mice, blood 125I levels were lower than those of controls 30 min to 24 h after liposome administration, with corresponding accumulation of 125I activity in the liver and spleen for the first 2 h after liposome injection. [14C]Cholesterol or sup(99m)Tc labels in the bilayer were eliminated rapidly from the blood, with uptake in the liver and spleen. In xenograft-bearing mice, 125I activity detected in the tumours up to 6 h after liposome injection was identical to that detected in the tumours of controls. However, 24 h after liposome injection a reduction in the tumour concentration of 125I-labelled anti-CEA was obtained, but the tumour/blood radioactivity was still increased. In two mice given 27 μmol lipid, the blood radioactivity count after 24 h was only 5% of that in the controls. In rabbits, 2 h after administration of liposomes containing anti-goat second antibody, the circulating 125I activity had dropped by 28-40%. The results suggest that administration of liposome-entrapped second antibody approximately 2 h prior to external scintigraphy may clear circulating radioactively labelled primary antibody by up to 50%. (Auth.)

  18. Optimizing selection of large animals for antibody production by screening immune response to standard vaccines.

    Thompson, Mary K; Fridy, Peter C; Keegan, Sarah; Chait, Brian T; Fenyö, David; Rout, Michael P

    2016-03-01

    Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humoral antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these "high level responders" could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals. PMID:26775851

  19. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  20. Enhancement of Monoclonal Antibody Production by Lysine-Containing Peptides

    Franěk, František; Eckschlager, T.; Hermann, K.

    2003-01-01

    Roč. 19, č. 1 (2003), s. 169-174. ISSN 8756-7938 R&D Projects: GA MŠk OC 844.10 Institutional research plan: CEZ:AV0Z5038910; CEZ:MSM 111300005 Keywords : Monoclonal Antibody * Lysine -Containing Peptides Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.488, year: 2003

  1. An innovative approach for the characterization of the isoforms of a monoclonal antibody product

    Sundaram, Shanmuuga; Matathia, Alice; Qian, Jun; Zhang, Jingming; Hsieh, Ming-Ching; Liu, Tun; Crowley, Richard; Parekh, Babita; Zhou, Qinwei

    2011-01-01

    Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the...

  2. Isolation of monoclonal antibodies specific for products of avian oncogene myb.

    Evan, G. I.; Lewis, G K; Bishop, J M

    1984-01-01

    We isolated a series of monoclonal antibodies which were raised against a bacterially expressed protein, bp37v-myb, and coded for by part of the avian v-myb gene. These monoclonal antibodies recognized a range of antigenic specificities on bp37v-myb, and this was reflected in their differing specificities for the gene products of the v-myb, c-myb, and E26 viral oncogenes. One monoclonal antibody recognized, in addition to the v-myb and c-myb gene products, a conserved nuclear protein found in...

  3. Production of monoclonal antibodies for use in immunoassays based on the magnetizable solid phase separation technique

    Monoclonal antibodies to TSH were produced by using mouse-ascites techniques. Various methods for purifying the antibody from the ascetic fluid have been tried in order to obtain an appropriate TSH kit production protocol. The purified antibodies were then immobilized on magnetizable cellulose for developing an IRMA for TSH. A detailed study of the assay system, including the stability of the magnetic adsorbent was made, which showed that the SCIPAc magnetizable cellulose is suitable for the production of TSH - Blood spot IRMA kits for use in the Neonatal hypothyroid screening programme to be launched in Thailand in the near future. (author). 4 refs, 12 figs, 2 tabs

  4. Production of monoclonal antibodies to human glomerular basement membrane.

    Mino,Yasuaki

    1984-10-01

    Full Text Available Using the technique of somatic cell fusion, we produced monoclonal antibodies to collagenase-digested human glomerular basement membrane (GBM. Fourteen monoclonal antibodies which reacted with normal human kidney in indirect immunofluorescence (IIF studies were produced. An analysis of the binding patterns indicated that the antigens recognized could be divided into six broad groups. Monoclonal antibody B3-H10 (Group 1 reacted with only GBM in a fine granular pattern. A5-B12 and B5-C2 (Group 2 reacted with GBM and peritubular capillary in a linear pattern. B2-A12 (Group 3 reacted with only epithelial cells. Al-C9 and A4-E2 (Group 4 showed a mesangial pattern in glomerulus and a lineal pattern in tubular basement membrane (TBM, Bowman's capsule and peritubular capillary. A1-E1, A1-E11, A2-E6, A3-B6, A4-F8 and B5-H2 (Group 5 recognized determinants common to GBM, TBM, Bowman's capsule and/or peritubular capillary. A3-F1 and B5-E10 (Group 6 reacted with TBM and Bowman's capsule. The staining pattern of B3-H10 (Group 1 was characteristic because it was not linear, but finely granular along the GBM. The staining pattern of B2-A12 (Group 3 was also characteristic because only epithelial cells were stained, and processes of epithelial cells were observed as fine fibrils. To the best of our knowledge, these two types of monoclonal antibodies have not been reported previously.

  5. Assessing monoclonal antibody product quality attribute criticality through clinical studies

    Goetze, Andrew M; Schenauer, Matthew R; Flynn, Gregory C.

    2010-01-01

    Recombinant therapeutic proteins, including antibodies, contain a variety of chemical and physical modifications. Great effort is expended during process and formulation development in controlling and minimizing this heterogeneity, which may not affect safety or efficacy and, therefore, may not need to be controlled. Many of the chemical conversions also occur in vivo and knowledge about the alterations can be applied to assessment of the potential impact on characteristics and the biological...

  6. Production and purification of avian antibodies (igys) from inclusion bodies of a recombinant protein central in nad+ metabolism

    Moreno-González, Paula A.; Diaz, Gonzalo J.; María H. Ramírez-Hernández

    2013-01-01

    The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recom...

  7. A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

    Na, Hong; Laver, John D; Jeon, Jouhyun; Singh, Fateh; Ancevicius, Kristin; Fan, Yujie; Cao, Wen Xi; Nie, Kun; Yang, Zhenglin; Luo, Hua; Wang, Miranda; Rissland, Olivia; Westwood, J Timothy; Kim, Philip M; Smibert, Craig A; Lipshitz, Howard D; Sidhu, Sachdev S

    2016-04-01

    Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins. PMID:26847261

  8. ASSOCIATION OF TRYPANOSOME INFECTION WITH SPERM ANTIBODIES PRODUCTION IN RED SOKOTO (MARADI GOATS

    O. FAYEMI

    2006-01-01

    Full Text Available A total of 1021 randomly selected serum samples of adult male goats that had been screened for trypanosome infection were assayed for sperm antibodies using the immunoperoxidase staining technique. The result of the trypanosome screening revealed that 586(57.39% goats were positive for trypanosome infection, while 435(42.61% were negative. The assay for sperm antibodies showed that 482(47.21% animals were positive, while 539(52.79% were negative. In the group that was positive for trypanosome infection, 364(62.12% animals were positive, whereas 222(37.88% were negative for sperm antibodies (P<0.001. The group that was negative for trypanosome infection, had a significantly lower number and proportion 118(27.13% of positive compared to 317(72.87% negative for sperm antibodies. Out of a total 482 goats that were positive for sperm antibodies, a significantly higher number, 364(75.52%, were positive than 118(24.48% that were negative for trypanosome infection (P<0.001. In the group that was found negative for sperm antibodies, a significantly lower proportion, 222(41.19%, was positive compared to 317(58.81% that were negative for trypanosome infection (P<0.001. Seropositivity to sperm antibodies was positively correlated to trypanosome infection (P<0.001. Further work on the pathogenesis of sperm antibody production in trypanosome infection is advocated.

  9. Mycofix ameliorative effect on Newcastle disease antibody production in broiler chickens during aflatoxicosis

    M. T. Gargees

    2008-01-01

    Full Text Available Three experiments were conducted to elucidate the alleviation effects of Mycofix plus 3.0 on Newcastle antibody formation during aflatoxicosis in broiler chickens. Three levels of Mycofix (0.05%, 0.15%, and 0.25% and aflatoxin (2.5ppm, 3.5ppm, and 5ppm were used. Chickens were vaccinated at 8 and 18 days of age. Enzyme-linked immunosorbent assay and Haemagglutination inhibition tests were employed for determination Newcastle antibody titers at 28 days. The results showed that, Mycofix , and only at its high level of addition (0.25% was effective in ameliorating the negative effect of aflatoxin at the rates 2.5ppm and 3.5 ppm levels of inclusion on antibody production but not at the high level of 5ppm on antibody production, comparing with titers in control groups.

  10. Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development.

    Tomar, Dheeraj S; Kumar, Sandeep; Singh, Satish K; Goswami, Sumit; Li, Li

    2016-01-01

    Effective translation of breakthrough discoveries into innovative products in the clinic requires proactive mitigation or elimination of several drug development challenges. These challenges can vary depending upon the type of drug molecule. In the case of therapeutic antibody candidates, a commonly encountered challenge is high viscosity of the concentrated antibody solutions. Concentration-dependent viscosity behaviors of mAbs and other biologic entities may depend on pairwise and higher-order intermolecular interactions, non-native aggregation, and concentration-dependent fluctuations of various antibody regions. This article reviews our current understanding of molecular origins of viscosity behaviors of antibody solutions. We discuss general strategies and guidelines to select low viscosity candidates or optimize lead candidates for lower viscosity at early drug discovery stages. Moreover, strategies for formulation optimization and excipient design are also presented for candidates already in advanced product development stages. Potential future directions for research in this field are also explored. PMID:26736022

  11. Production and Characterization of Monoclonal Antibodies Against Thytoxine

    2001-01-01

    Four hybridoma cell lines (T410D11,T415611, T413A4, T409F6) producing MAbs againstthytoxine(T4) are established by using T4-conjugated bovine serum albumin as an immunogen. These monoclonal antibodies have high affinitiess and specific against T4. The association constants of these MAbs are higher than 108 L/mol. Their cross-reactivities with T3, T2 and rT3 are lower than 0.4%, 0.04% and 0.22%, respectively. The clinical application of the T4 ELISA Kit

  12. Effects of X irradiation on homocytotropic and agglutinating antibody production in mice

    The effect of X irradiation on homocytotropic and agglutinating antibody production was studied in mice exposed to 400 rad either before or after immunization with dinitrophenylated Ascaris plus aluminium hydroxide as adjuvant. The adjuvant effect of irradiation was also determined in animals receiving antigen alone. Irradiation 1 day before immunization with adjuvant enhanced IgE and slightly enhanced IgM-antibody formation, although the onset was delayed, but partially suppressed IgG-antibody formation. When the same treatment followed antigen priming, there was a similar enhancement of IgE production which varied with the time between the two procedures. IgG and IgM production, however, were fairly resistant under the same conditions. Irradiation preceding immunization with soluble antigen had no significant adjuvant effect on IgE-, IgG- or IgM-antibody production. On the contrary, it suppressed production of the latter two classes. The results may indicate that production of IgE and of IgG and IgM antibodies in the mouse is regulated by separate mechanisms. (author)

  13. [Use of leukocyte-filtered, cytomegalovirus-antibody negative and irradiated cellular blood products].

    Solheim, B G; Albrechtsen, D H; Evensen, S A; Leivestad, T

    1990-01-10

    This paper presents both quality requirements and indications for use of leucocyte-filtered, cytomegalovirus antibody negative and irradiated cellular blood products at Rikshospitalet. Emphasis is placed on the use of standardized buffycoat depleted red cells or platelet concentrates for filtration, and the selection of leucocyte filters with high capacity and ease of bedside application. Leucocyte counts as low as 1-2 10(5) per unit are recommended after filtration in order to avoid HLA-antibody production. For bedside filtration, our choice was RC100 and PL100 (Pall) for red cells and platelets respectively. For laboratory use we prefer, for economic reasons, to use Sepacell R500 (Asahi) and Imugard IG500 (Terumo) for red cells and platelets respectively. Leucocyte-filtered blood products are considered indicated in all pre-transplant transfusions, in post-transplant HLA-sensitized patients, in other patients with febrile transfusion reactions, and in patients with an expected protracted platelet requirement. CMV antibody negative products are recommended for all immuno-deficient patients and pregnant women negative for CMV antibody. Irradiated blood products are used in the treatment of immuno-deficient patients receiving large amounts of blood, and in all severely immuno-compromised patients. In emergency situations where CMV antibody negative and/or irradiated blood products cannot be supplied, leucocyte filtration is suggested. PMID:2154061

  14. Production and Characterization of a Murine Monoclonal Antibody Against Human Ferritin

    Bayat, Ali Ahmad; Yeganeh, Omid; Ghods, Roya; Zarnani, Amir Hassan; Ardekani, Reza Bahjati; Mahmoudi, Ahmad Reza; Mahmoudian, Jafar; Haghighat-Noutash, Farzaneh; Jeddi-Tehrani, Mahmood

    2013-01-01

    Background Ferritin is an iron storage protein, which plays a key role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody (mAb) against human ferritin was reported. Methods Balb/c mice were immunized with purified human ferritin ...

  15. Impaired Antigen-Specific Immune Response to Vaccines in Children with Antibody Production Defects.

    Szczawinska-Poplonyk, Aleksandra; Breborowicz, Anna; Samara, Husam; Ossowska, Lidia; Dworacki, Grzegorz

    2015-08-01

    The impaired synthesis of antigen-specific antibodies, which is indispensable for an adaptive immune response to infections, is a fundamental pathomechanism that leads to clinical manifestations in children with antibody production defects. The aim of this study was to evaluate the synthesis of antigen-specific antibodies following immunization in relation to peripheral blood B cell subsets in young children with hypogammaglobulinemia. Twenty-two children, aged from 8 to 61 months, with a deficiency in one or more major immunoglobulin classes participated in the study. Postvaccination antibodies against tetanus and diphtheria toxoids, the surface antigen of the hepatitis B virus, and the capsular Haemophilus influenzae type b polysaccharide antigen were assessed along with an immunophenotypic evaluation of peripheral blood B lymph cell maturation. A deficiency of antibodies against the tetanus toxoid was assessed in 73% of cases and that against the diphtheria toxoid was assessed in 68% of cases, whereas a deficiency of antibodies against the surface antigen of the hepatitis B virus was revealed in 59% of the children included in the study. A defective response to immunization with a conjugate vaccine with the Haemophilus influenzae type b polysaccharide antigen was demonstrated in 55% of hypogammaglobulinemic patients. Increased proportions of transitional B lymph cells and an accumulation of plasmablasts accompanied antibody deficiencies. The defective response to vaccine protein and polysaccharide antigens is a predominating disorder of humoral immunity in children with hypogammaglobulinemia and may result from a dysfunctional state of the cellular elements of the immune system. PMID:26018535

  16. Selective suppression of antibody production with the aid of radiolabelled birch pollen allergen

    In accordance with the clonal selection theory we intended to prevent the development of artificially induced birch pollen allergy in rabbits with the aid of of the radiolabelled pollen allergen (75-1000 μCi125 I-pollen/animal) intravenously administered prior to pollen sensitization. The birch pollen allergen, in accordance with Burnet's working hypothesis, reacts only with a genetically determining B cell subpopulation. The fixation of the radiolabelled birch pollen allergen to the receptors of the competent B cell clone causes the lesion of the latter. Compared with the control group, this group of rabbits showed an extensive suppression of anaphylactic reagin-like PCA-antibodies, and haemagglutinating antibodies in the blood as well as in nasal secretion. In addition, we tried to influence the already ongoing synthesis of the antibodies with the aid of a subsequent intravenously administered radiolabelled birch pollen allergen (750-1000μCi125 I-pollen/animal). An intensive suppression of the synthesis of antibodies could also be proved in this case. The simultaneous immunization of the control rabbits with birch pollen and egg albumin resulted in the production of antibodies against both antigens, as expected. The hot-labelled birch pollen antigen intravenously injected before or after immunization with egg albumin and birch pollen led selectively to suppression of anti-birch-pollen PCA antibodies. The synthesis of anti-egg albumin PCA antibodies was unaffected. (author)

  17. Production of monoclonal antibody against Salmonella typhimurium by hybridoma technique

    In this research S.typhimurium killed by irradiation was used as antigen was prepared by exposing the bacteria to gamma rays from 60Cobalt source with the dose of 2.5 kGy, Specific lymphocyte cell were obtained by immunizing 3 months old Balb-C mice with the antigen. the immunizations were done by subcutan route with the interval of 2 weeks. The hybridoma cells were made by fussing the specific lymphocyte cells with the myeloma cells. It was found that the animals (immunization + irradiation with a low dose of I Gy ) yielded monoclonal antibody with higher value (5.15 mg/ml) than the control animals (3.25 mg/ml). (author)

  18. Rapid production of antigen-specific monoclonal antibodies from a variety of animals

    Kurosawa Nobuyuki

    2012-09-01

    Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

  19. Enhancement of antibody production against rabies virus by uridine 5'-triphosphate in mice.

    Iwaki, Yoshimi; Sakai, Yusuke; Ochiai, Kenji; Umemura, Takashi; Sunden, Yuji

    2014-03-01

    Extracellular nucleotides such as adenosine 5'-triphospate (ATP) and uridine 5'-triphosphate (UTP) interact with P2 purinergic receptors on the surface of phagocytic cells and induce various physiological reactions. In this study, the production of antibody in mice immunized with an inactivated rabies vaccine containing these nucleotides was investigated. Injection of inactivated rabies vaccine with UTP, but not with ATP, induced significantly higher serum antibody production in mice. The enhancement of antibody production by UTP was inhibited by an anti-P2Y4 receptor antibody. In an air pouch experiment, UTP treatment increased the number of monocytes and macrophages infiltrating the pouch and up-regulated the gene expression of IL-4 and IL-13 in the regional lymph nodes. These results suggested that UTP admixed with rabies vaccine activates Th2 cells and induces a humoral immune response. Furthermore, the survival rate of mice immunized with a rabies vaccine admixed with UTP before rabies virus challenge was slightly higher than that of control mice. In conclusion, UTP can act as a vaccine adjuvant to enhance antibody production against the rabies virus in mice. PMID:24309427

  20. Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

    Shah, Kartik A; Clark, John J; Goods, Brittany A; Politano, Timothy J; Mozdzierz, Nicholas J; Zimnisky, Ross M; Leeson, Rachel L; Love, J Christopher; Love, Kerry R

    2015-12-01

    Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV). PMID:26032261

  1. Nutritional abrogation of photoimmunosuppression: in vivo investigations.

    Pilkington, Suzanne M; Gibbs, Neil K; Friedmann, Peter S; Rhodes, Lesley E

    2014-01-01

    Skin cancer is a major public health concern, and the primary aetiological factor in the majority of skin cancers is ultraviolet radiation (UVR) exposure. UVR not only induces potentially mutagenic DNA damage but also suppresses cell-mediated immunity (CMI), allowing cancerous cells to escape destruction and progress to tumours. A considerable proportion of an individual's annual sun exposure is obtained outside the vacation period when topical and physical measures for photoprotection are irregularly used. Certain nutrients could provide an adjunctive protective role, and evidence is accruing from experimental studies to support their use in abrogation of photoimmunosuppression. Moreover, developments in clinical research methods to evaluate impact of solar-simulated radiation on cutaneous CMI allow the immune protective potential of nutritional agents to be examined in humans in vivo. This article summarises the mediation of CMI and its suppression by UVR, evaluates the methodology for quantitative assessment in vivo, reviews the human studies reported on nutritional abrogation of photoimmunosuppression including recent randomized controlled trials and discusses the mechanisms of photoprotection by the nutrients. This includes, in addition to antioxidants, novel studies of omega-3 polyunsaturated fatty acids and nicotinamide. PMID:24283330

  2. Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes.

    Arce, C; Moreno, A; Millán, Y; Martín de las Mulas, J; Llanes, D

    2002-09-01

    A panel of six monoclonal antibodies (mAbs) recognizing antigenic determinants on canine immunoglobulin (Ig) heavy or light chains was produced and characterized. All monoclonals recognized the IgG(2) subclass, although only two were subclass-specific (CA3H1 and CA4F1). The CA3B8 mAb was found to be specific for an epitope on canine immunoglobulin G heavy chain, (IgG(1) and IgG(2) subclasses). Two mAbs (CA2E9 and CA5B2) reacted with an epitope on the heavy chain of canine IgG and IgM and another, CA4E7, bound to canine IgA, IgG and IgM isotypes; CA4E7 recognized an epitope on canine immunoglobulin light chain. CA4E7, CA4F1 and CA5B2 recognized an epitope in the Fab region. Three mAbs, CA3B8, CA4E7 and CA5B2, showed much lower reactivity with canine IgG by ELISA when IgG was periodate-treated, suggesting that they recognized a carbohydrate determinant. Cross-reactivity analysis of these mAbs with sera from horse, goat, cow, sheep, pig, cat, rabbit, hamster, rat, mouse and human indicated that two mAbs, CA3B8 and CA5B2, recognized a canine IgG-specific epitope; two others, CA3H1 and CA4E7, recognized an epitope also present in rabbit and sheep immunoglobulin respectively; and the remaining two (CA2E9 and CA4F1) recognized an epitope broadly present on the Igs of the species analyzed. This panel of antibodies will be a useful tool for future canine immunodiagnosis tests. With the exception of CA2E9, all mAbs were able to recognize plasma cells on paraffin-embedded tissues, and will thus be useful for immunohistochemical assays. PMID:12088642

  3. Production and Characterization of Monoclonal Antibody Against Recombinant Human Erythropoietin

    JIE-BO MI; JIN YAN; XIAO-JIE DING; ZHEN-QUAN GUO; MEI-PING ZHAO; WEN-BAO CHANG

    2007-01-01

    Objective To produce specific monoclonal antibody(mAb)against recombinant human erythropoietin(rHuEPO)for development of higmy efficient methods for erythropoietin detection in biological fluids.Methods rHuEPO was covalently coupled with bovine serum albumin(BSA)and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology.The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA),SDS-PAGE and Western blot.Results The isotype of F3-mAb Was found to be IgM with an affinity constant of 2.1x108 L/mol.The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

  4. Production of radiolabeled monoclonal antibody conjugates by photoaffinity labeling

    Volkert, W.A.; Ketring, A.R.; Kuntz, R.R.; Holmes, R.A.; Mitchell, E.P. (Missouri Univ., Columbia, MO (USA)); Feldbush, T.L. (Harry S. Truman Memorial Veterans Hospital, Columbia, MO (USA))

    1990-06-01

    This report discusses activities and progress that has occurred since initiation of this project on September 1, 1989. We have synthesized ethyl N,N{prime}-bis(benzoylmercaptoacetyl)-2,3-diaminopropanoate, a ligand to be used as a bifunctional chelating agent (BFCA), to form {sup 186}Re or {sup 188}Re ({sup 186}Re/{sup 188}Re) complexes. {sup 186}Re/{sup 188}Re, in reducing media, reacts with this ligand to form {sup 186}Re/{sup 188}Re-CO{sub 2}DADS chelates that will be used to formulate new radiolabeled photoaffinity labels (RPALs). Initial steps have been taken to synthesize R-As-dithiol compounds. This approach will be used to produce {sup 77}As-RPALs or covalently link {sup 77}As directly to monoclonal antibodies (MAbs). The R group will contain a group that can be used for conjugation reactions. Spectral and photochemical properties of various types of photoaffinity labels (PALs) have been studied. Acrylo-azido compounds and 9-azido acridine have been studied as well as several other photoprobes. The binding characteristics of the azido-based PALs to HSA have been studied and progress has been made on developing techniques for efficiently separating of non-covalently sound PALs. The Nd-YAG laser was purchased and arrived in 1990. It has been assembled and tested and is now operational.

  5. Production of monoclonal antibodies specific for Haemophilus ducreyi: a screening method to discriminate specific and cross-reacting antibodies.

    Odumeru, J A; Alfa, M J; Martin, C F; Ronald, A R; Jay, F T

    1989-06-01

    Haemophilus ducreyi is the etiological agent of chancroid. The organism shares extensive immunological cross-reactivity with other Haemophilus species. This presents substantial difficulties for the production of specific monoclonal antibodies (MAbs). A competition ELISA was devised for hybridoma screening which allowed the detection of H. ducreyi-specific antibody-producing hybridoma cultures during the initial screening process. With this screening method, seven MAbs specific for H. ducreyi were obtained in a single cell fusion exercise. The specificities of the 7 MAbs were demonstrated by direct ELISA and dot immunobinding assays against several strains each of H. influenzae, H. parainfluenzae and Neisseria gonorrhoeae. Five of the MAbs reacted against all ten strains of H. ducreyi. These MAbs may permit the development of rapid and efficient immunodiagnostics for chancroid. The principle of the competition ELISA for hybridoma screening should be widely applicable to the development of specific MAbs to other organisms in which immunological cross-reactivity is an impediment to hybridoma screening by conventional methods. PMID:2787274

  6. Production of antibodies with peptide-CpG-DNA-liposome complex without carriers

    Kim Doo-Sik

    2011-05-01

    Full Text Available Abstract Background The screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers. Results We present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells. Conclusions Our overall results show that Lipoplex(O is a potent adjuvant and that complexes of peptide and Lipoplex(O are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

  7. Optimization of a methamphetamine conjugate vaccine for antibody production in mice.

    Stevens, Misty W; Gunnell, Melinda G; Tawney, Rachel; Owens, S Michael

    2016-06-01

    There are still no approved medications for treating patients who abuse methamphetamine. Active vaccines for treating abuse of nicotine and cocaine are in clinical studies, but have not proven effective seemingly due to inadequate anti-drug antibody production. The current studies aimed to optimize the composition, adjuvant and route of administration of a methamphetamine conjugate vaccine, ICKLH-SMO9, in mice with the goal of generating significantly higher antibody levels. A range of hapten epitope densities were compared, as were the adjuvants Alhydrogel and a new Toll-like receptor 4 (TLR4) agonist called GLA-SE. While methamphetamine hapten density did not strongly affect the antibody response, the adjuvant did. Glucopyranosyl lipid A in a stable oil-in-water emulsion (GLA-SE) produced much higher levels of antibody in response to immunization compared with Alhydrogel; immunization with GLA-SE also produced antibodies with higher affinities for methamphetamine. GLA-SE has been used in human studies of vaccines for influenza among others and like some other clinical TLR4 agonists, it is safe and elicits a strong immune response. GLA-SE adjuvanted vaccines are typically administered by intramuscular injection and this also proved effective in these mouse studies. Clinical studies of the ICKLH-SMO9 methamphetamine vaccine adjuvanted with GLA-SE have the potential for demonstrating efficacy by generating much higher levels of antibody than substance abuse vaccines that have unsuccessfully used aluminum-based adjuvants. PMID:27039212

  8. Serum and colostral antibody production in cows immunized with recombinant human tumor necrosis factor.

    Burton, Randall; Kim, Skaison; Patel, Rutvij; Scola, Michele; Hartman, Deborah; Tracey, Daniel; Fox, Barbara S

    2016-06-01

    The use of hyper-immune bovine colostrum as a human therapeutic platform is an emerging technology with potential to deliver the efficacy of antibody therapeutics with the convenience and safety of oral or topical application. It is necessary to understand how the bovine immune system responds to immunization with foreign proteins, both in terms of the serum antibody response and the transfer of antigen-specific antibodies into the colostrum to enable efficient large-scale production of therapeutic antibodies. We have immunized 25 cows with recombinant human tumor necrosis factor (rhTNF) and measured the levels of rhTNF-specific antibodies in the serum and colostrum of these animals. We observed a decline of 84±9% in serum IgG1 concentrations in the final weeks of pregnancy that presumably reflects rapid transport of IgG1 into colostrum. The serum IgG2 levels remained constant, such that the serum IgG1 to IgG2 ratio was 1:20 at parturition. We observed substantial animal-to-animal variability in the levels of anti-rhTNF antibodies in both serum and colostrum samples. In particular, a subset of 4 cows had extraordinarily high colostral anti-rhTNF antibody production. Only a weak correlation was found between the peak serum anti-rhTNF activity and the colostral anti-rhTNF activity in these animals. The 4 cows with high colostral anti-rhTNF activities trended toward higher serum IgG1 loss relative to average colostral anti-rhTNF producers, but this difference was not statistically significant in this small sample. The high-anti-rhTNF-producing cows also exhibited a greater proportion of rhTNF-specific antibodies that bound to bovine IgG1- and IgG2-specific detection antibodies relative to the total anti-rhTNF immunoglobulin population. This finding suggests that the isotype distribution of the anti-rhTNF response is varied between individuals and genetic or environmental factors may increase the yield of antigen-specific colostral antibodies. PMID:27040787

  9. The present state of the art in expression, production and characterization of monoclonal antibodies.

    Gaughan, Christopher L

    2016-02-01

    Monoclonal antibodies (MAb's) have become one the most powerful therapeutic and diagnostic tools in modern medicine. Some estimates target the worldwide market of MAb's on the order of $125 billion in the next four years. Recent advances in molecular biology, immunology, and development of robust production platforms will drive the development of more MAb's suitable to treat an ever increasing number of disease states. This circumstance combined with the fact that many of the original antibody therapies from the 1980 s and 1990 s will soon be coming off patent will attract a great deal of investment in the development of larger industrial facilities to increase monoclonal antibody to meet increasing demand. In this review, the present state of the science that underlies the development of new antibodies therapies in Chinese hamster ovary cells combined with a description of the present challenges facing the industry in terms of the limitations of output and compliance with current good manufacturing practices and FDA regulations. Also addressed are future challenges to overcome production bottlenecks, description of critical quality control attributes particular to antibodies, and detailed treatment of scale-up considerations. PMID:26299798

  10. Production of anti-IgG antibodies in sheep for using in the radioimmunoassays of LH, FSH and prolactin

    In this work described the production of second antibodies in sheep against rabbit IgG for being used in radioimmunoassays for determination LH, FSH and Prolactin. There was made the comparison between the results obtained using the Kits-RIA produced by us and the commercial WHO Kits-RIA, using these antibodies. The results allowed us to use these antibodies for production Kits-RIA of LH, FSH and Prolactin

  11. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

    Joosten, V.; Gouka, R.J.; Hondel, C.A.M.J.J. van den; Verrips, C.T.; Lokman, B.C.

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA level

  12. Effects of Temperature on Production and Specificity of Antibodies in Rainbow Trout (Oncorhynchus mykiss)

    Mikkelsen, Heidi; Nielsen, Michael Engelbrecht; Lindenstrom, Thomas

    2006-01-01

    The effect of temperature on production and affinity of antibodies against antigens from the parasitic ciliate Ichthyophthirius multifiliis were studied in rainbow trout (Oncorhynchus mykiss). Fish were immunized with I. multifiliis antigens and reared at three different temperatures, 5, 12, and ...

  13. Comparison of antibody production against aflatoxin B1 in goats and rabbits.

    Gaur, P K; El-Nakib, O; Chu, F. S.

    1980-01-01

    Antibody production against aflatoxin B1 was compared in three rabbits and one goat. Titers obtained were 20 times higher in the rabbits than in the goat. The goat antiserum appeared to have a higher degree of cross-reactivity for other aflatoxins and related metabolites than did the rabbit antiserum.

  14. An Experimental Study on Production of Egg Yolk Antibody(IgY) against Bee Venom

    Hwang, Tae-Jun; Lee, Seung-bae; Gwon, Gi-Rok

    2001-01-01

    This study was carried out for production of neutral antibody to bee venom(anti-phospholipase A2 IgY). Hen layings were injected repeatedly with bee venom and phospholipase A2 with Freund's adjuvant. Specific antibody in egg yolk from immunized hen laying was separated, and purified, also immunological characteristics of anti-phospholipase A2 IgY was invested. The results were summarized as follows : 1. Phospholipase A2 was showed single band at molecular weight 17,000 in SDS-PAGE and b...

  15. Production of Anti-triiodothyronine sulfate antibody for radioimmunoassay applications

    Triiodothyronine sulfate (T3S) may be an obligatory intermediate metabolic of the metabolism of thyroid gland hormones invertebrates in peripheral during the process of deiodination of the inactive form of the thyroid gland hormones, thyroxine(T4), into the active form triiodothyronine (1,2). Construction of a reliable procedure for the estimation of T3S accurately in blood serum will be of great importance for medical, biochemical and physiological investigations. In this work we developed a robust method for the production of anti-triiodothyronine sulfate polyclonal antiserum with good specifications using a derivatized immuno gen and a modified immunization process and a sensitive radioimmunoassay system was designed and developed

  16. Guardians at the gate: Patent protection of therapeutic monoclonal antibodies through product life cycle management—Part 3

    McCabe, Kevin W; Calvo, Paul A

    2009-01-01

    Product life cycle management, which necessarily utilizes a multi-disciplinary approach, is an essential tool for companies that develop or market therapeutic monoclonal antibodies (mAbs). Too little attention to such a plan, or use of the wrong resources, could substantially curtail a product's life span. The most difficult part of the therapeutic antibody business is the development of high-quality, safe and effective products. Great care should thus be taken to ensure that products with th...

  17. CD20 antibody primes B lymphocytes for type I interferon production.

    Dongsheng Xu

    Full Text Available CD20 is a B cell surface marker that is expressed in various stages in B lymphocytes and certain lymphomas. Clinical administration of CD20 antibody, such as rituximab, is used widely to treat human B-cell lymphomas and other diseases. However, CD20 antibody failed to treat systemic lupus erythematosus (SLE or lupus. The reason for the failure is currently unknown. Type I interferons (IFN are a major component for the host innate immunity, and a key pathogenic factor in lupus. We found that CD20 antibody potentiated human B cells for its production of IFNs in vitro. This function was specific to CD20-expressing cells and the potentiation function seems to be instant. In addition, ectopic expression of CD20 in non-B-lymphocytes increased the IFN promoter reporter activities. Because IFNs are a key pathogenic factor in lupus, our data suggest that, in the presence of virus infection, the CD20-antibody-mediated enhancement of IFN production might be related to its failure in lupus treatments. This work may provide new insights for CD20-Ab therapeutic applications.

  18. Production of the recombinant single chain anti-B cell lymphoma antibody and evaluation of immunoreactivity

    Recombinant ScFv lym-1 was produced, using pET vector system for large scale production. ScFv lym-1 gene inserted pET-22b (+) vector, was expressed in E. coli BL-21 strain. ScFv lym-1 antibody extracted from periplasm, was purified with His-Taq column. To evaluated immunoreactivity with Raji cell, ScFv lym-1 was labeled with I-125 and I-125 ScFv lym-1 was purified with desalting column. Raji cell was injected into the C57BR/cdJ SCID mice. Gamma camera imaging were taken time point at 1, 8, 24 and 48 hr with 8 mm pinhole collimator. An active scFv lym-1 could be produced in E. coli with soluble from using pET vector system. Immunoreactivity and affinity constant of lgG lym-1 were 54% and 1.83 x 109 M-1, respectively, and those of scFv lym-1 were 53.7% and 1.46 x 109 M-1, respectively. Biodistribution of I-125 scFv lym-1 antibody showed faster clearance in blood, spleen, kidney and than I-125 lgG lym-1 antibody. Gamma camera image of I-125 scFv lym-1 antibody showed faster clearance and tumor targeting liver than I-125 lgG lym-1 antibody. In vitro properties of scFv lym-1 were similar to those of lgG lym-1. ScFv lym-1 showed faster blood clearance than lgG lym-1. These results suggest that scFv lym-1 antibody can be useful for tumor imaging agent

  19. Effect of inoculation route on the production of antibodies and histological characteristics of the spleen in laying hens

    SF Eto

    2012-03-01

    Full Text Available Recent studies have reported the use of IgY antibody in the prevention or treatment of diseases in animals. IgY can be obtained in large amounts from the yolk of chicken eggs through a low-cost process. This study evaluated the effect of different routes of inoculation on antibody production and spleen morphological characteristics of laying hens (White Leghorn inoculated with sheep red blood cells. The analysis of the results showed that the intramuscular route is the most efficient for total antibody production in the primary immune response, while the intravenous route is the most efficient in producing IgY antibodies in the secondary immune response. No histological changes were observed in the spleen of laying hens. This study could be useful for developing protocols of antigen inoculation in laying hens for IgY antibody production.

  20. Production of polyclonal antibodies against protein antigens purified by electroelution from SDS-polyacrylamide gel

    sprotocols

    2015-01-01

    This protocol describes the purification of insoluble or moderately soluble proteins using an electro-separation system for elution from SDS-polyacrylamide gel and the use of the purified proteins for the production of polyclonal antibodies. The proteins to be purified must be highly expressed in bacterial cells and visible in polyacrylamide gels following Coomassie Blue staining. The gel should not be stained because non-stained proteins are electroelueted much faster than fixed and stained ...

  1. Prediction of transgenic tobacco plant processing properties by ultra scale down and physical property measurement for monoclonal antibody production.

    Hassan, S. Y.

    2008-01-01

    There are numerous potential advantages of producing significant quantities of a monoclonal antibody (MAb) via transgenic tobacco plants over other heterologous production systems, thus paving the way for new prophylactic and therapeutic applications within global human and animal health. However, current information on the key processing factors for large scale production of antibodies from transgenic plants is limited. This thesis presents the issues involved in the production of monoclonal...

  2. Production and purification of avian antibodies (IgYs from inclusion bodies of a recombinant protein central in NAD+ metabolism

    Paula A. Moreno-González

    2013-08-01

    Full Text Available The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models.  The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies. A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT was expressed in Escherichia coli.  The protein was purified through solubilization from inclusion bodies prior to its renaturalization.  Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT.  IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.

  3. Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis

    Telma M. Alves

    2012-07-01

    Full Text Available Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs anti-C. fetus subsp. venerealis of the IgM (1 and IgG (14 classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs: two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

  4. Production and Quality Control of Technetium-99m Radiolabeled Monoclonal Antibody

    M.H. Babaei, Ph.D.

    2007-01-01

    Full Text Available AbstractBackground and purpose: Binding a monoclonal antibody to tumor associated antigens is an effective method for cancer therapy because these agents can specifically target malignant cells. In fact, monoclonal antibodies are effective agents for diagnosis, grading and treatment of different kinds of cancers. In this research, a new monoclonal antibody against colon cancer cells was prepared and radiolabeling with technetium-99m evaluated.Materials and Methods: This research was done in three parts: preparation of hybridoma cell against colon cancer cell line (HT29, production of monoclonal antibody, determination of its characterizations and radiolabeling with technetium-99m.Results: mAb-D2 is an IgG1 with affinity constant of 7.2 × 109M-1 which can recognize CEA in tumor cells. Radiolabeling showed that 99mTc-HYNIC-mAb-D2 complex is stable, immunoradioactive, and has a desirable biodistribution.Conclusion: In this study, we gained a new radiopharmaceutical that may be a good candidate for radioimmunoscintigraphy.

  5. The production of high affinity monoclonal antibodies to human growth hormone

    The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4x1010 l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay. (Auth.)

  6. Production of immunoglobin G human monoclonal antibodies by cellular fusion method

    The increasing clinical use of immunological detection i.e. radioimmunoassays (RIA) enzyme linked immunosorment assay (ELISA) and immunoscintigraphy, has focused the attention on the important of monoclonal antibodies production. Monoclonal antibody (MAB) has been prepared by the cellular fusion method using immunoglobin G human (IgGh) as the antigen and polyethylene glycol (PEG) as the fusion agent. Selection of hybridoma cells was carried out using hypoxanthine-aminopterin-thymidine (HAT) medium. The immunoreactivity and specificy of the antibodies were determined by ELISA technique using Goat anti Mouse (GAM) polyclonal antibodies conjugated with horse-radish peroxidase. It has been shown that the MAB secreted from hybridoma cells are specific to IgGh, IgGh-, IgGh-k, IgG1h, and IgG2h. It was also observed that the MAB are specific to goat IgG or rabbit IgG. All MAB in the ascities fluid were IgG1-k class as determined by ouchterlony method. (author). 6 refs.; 4 tabs.; 5 figs

  7. Antibody Detection and Kinetics of Antibody Production during Early Stages of Immunization with Hepatitis B Virus Vaccine▿

    Odinsen, Odd; Owusu-Ofori, Shirley; Dompreh, Albert; Sarkodie, Francis; Opare-Sem, Ohene; Parker, David; Allain, Jean-Pierre

    2007-01-01

    Antibodies to influenza virus and human immunodeficiency virus are detectable in B cells during the early stages of the immune response, prior to their occurrence in plasma. To investigate similar phenomena in a model of immunization against hepatitis B virus (HBV) infection, medical students in Ghana were screened for HBV markers, HBV surface (HBs) antigen (HBsAg), and HBV core antibodies (anti-HBc). Consenting volunteers, 24 of whom were seronegative (susceptible) and 2 of whom were positiv...

  8. Immune response in mice to ingested soya protein: antibody production, oral tolerance and maternal transfer.

    Christensen, Hanne R; Brix, Susanne; Frøkiaer, Hanne

    2004-05-01

    While allergic reactions to soya are increasingly investigated, the normal immune response to ingested soya is scarcely described. In the present study, we wanted to characterise the soya-specific immune response in healthy mice ingesting soya protein. Mice fed a soya-containing diet (F0) and mice of the first (F1) and second (F2) offspring generation bred on a soya protein-free diet were used either directly or were transferred between the soya-containing and soya protein-free diet during pregnancy or neonatal life. The mice were compared as to levels of naturally occurring specific antibodies analysed by ELISA, and to the presence of oral tolerance detected as a suppressed antibody and cell-proliferation response upon immunisation with soya protein. F0 mice generated soya-specific antibodies, while oral tolerance to the same soya proteins was also clearly induced. When F0 dams were transferred to soya protein-free feed before mating, the F1 and F2 offspring generations showed no significantly different response, indicating that soya-specific immune components were not maternally transmitted. However, the ingestion of dietary soya protein by F1 mice during late pregnancy and lactation caused a lasting antibody response in the offspring, but in this case in the absence of oral tolerance. This indicates that, under certain conditions, factors involved in spontaneous antibody production can be transmitted from mother to offspring. Understanding the immune response to soya protein ingested under healthy conditions is important in the assessment of adverse effects of soya protein and in the use of animal allergy models. The present results add to this understanding. PMID:15137924

  9. Production of monoclonal and polyclonal antibodies against prostate-specific antigen, a prostate cancer serum marker.

    Chou, Shu-Fen; Chen, Chien-Yuan

    2004-02-01

    The aim of this study was to produce monoclonal and polyclonal antibodies against prostate-specific antigen (PSA), a prostate cancer serum marker. Hyperimmune ICR mice produced polyclonal antibodies (PoAbs) after injection with 0.5 mL of pristane, and were injected with NS-1 myeloma cells 2 weeks later. Hyperimmune Balb/c mice were used for the production of monoclonal antibodies (MAbs). Mice were immunized four times and given a final boost, and their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500. The fused cells were then selected in the HAT-RPMIX medium. Anti-PSA antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS) HT-RPMIX medium. Twelve murine hybridoma producing anti-PSA MAbs were obtained and designated C3m1G11, B3m1E5, C3m1E8, C3m1C5, C3m2F4, C3m1F8, C3m2B3, C3m2E6, B3m2B11, B3m2F2, C3m2C7, and C3m2D9. Isotypes of these MAbs were identified as IgG2a heavy chain and kappa light chain. Hitrap Protein A column was used for the purification of polyclonal and monoclonal antibodies. The purity analysis of MAb was performed by capillary electrophoresis. PMID:15000852

  10. Optimizing conditions of large scale production of monoclonal antibodies for determination of blood group

    Conditions for large scale production of monoclonal antibodies (monoAB) on mice hybrids were determined: irradiation dose, amount of introduced hybridoma cells, possible substitution of imported pristane for Soviet-produced vaseline oil or a mixture thereof. DMSO did not exert toxic action neither on the cell itself, nor on the development of ascytic tumor in mice after thawing of cryopreserved cells. This enabled to abstain from washing of DMSO off the introduced cells. A simple method for enhancing the monoAB yield - acquisition of the washing-out solution for increased efficiency of large scale production - was developed and tested

  11. Enhancement of monoclonal antibody production in CHO cells by exposure to He–Ne laser radiation

    Ghaleb, Rana; Naciri, Mariam; Al-Majmaie, Rasoul; Maki, Amel; Al-Rubeai, Mohamed

    2013-01-01

    This study tested the effectiveness of laser biostimulation in small-scale cultures in vitro. We investigated the response of recombinant CHO cells, which are used for the production of monoclonal antibody, to low level laser radiation. The cells were irradiated using a 632.8 nm He–Ne laser in a continuous wave mode at different energy doses. We incubated the irradiated cells in small batch cultures and assessed their proliferation and productivity at various time intervals. Compared to untre...

  12. Effect of inhibin gene immunization on antibody production and reproductive performance in Partridge Shank hens.

    Mao, Dagan; Bai, Wujiao; Hui, Fengming; Yang, Liguo; Cao, Shaoxian; Xu, Yinxue

    2016-04-01

    To investigate the effect of inhibin gene immunization on antibody production and reproductive performance in broiler breeder females, Partridge Shank hens aged 380 days were immunized with inhibin recombinant plasmid pcISI. One hundred and twenty hens were randomly assigned to four groups and treated intramuscularly with 25, 75, or 125 μg/300-μL inhibin recombinant plasmid pcISI (T1∼T3) or 300-μL saline as control (C), respectively. Booster immunization was given with the same dosage 20 days later. Blood and egg samples were collected to detect the antibody against inhibin by enzyme-linked immunosorbent assay and to evaluate egg performance. The ovaries were collected to classify the follicles and detect the FSH receptor (FSHR) messenger RNA (mRNA) expression by reverse transcription-PCR. The results showed that immunization against pcISI could elicit antibody against inhibin in both plasma and egg yolk compared with the control (P lay during the first 30 days after primary vaccination (P laying performance in Partridge Shank hens, which provides a good foundation for the application of inhibin DNA vaccine in avian production. PMID:26739531

  13. Production of the Polyclonal Anti-human Metallothionein 2A Antibody with Recombinant Protein Technology

    Faiz M.M.T.MARIKAR; Qi-Ming SUN; Zi-Chun HUA

    2006-01-01

    Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the eDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

  14. Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs

    de Marco, Ario

    2016-01-01

    Background Antibodies have been a pillar of basic research, while their relevance in clinical diagnostics and therapy is constantly growing. Consequently, the production of both conventional and fragment antibodies constantly faces more demanding challenges for the improvement of their quantity and quality. The answer to such an increasing need has been the development of a wide array of formats and alternative production platforms. This review offers a critical comparison and evaluation of t...

  15. Benchmarking of commercially available CHO cell culture media for antibody production.

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-06-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable

  16. Development of a multi-product leached protein A assay for bioprocess samples containing recombinant human monoclonal antibodies.

    Ren, Diya; Darlucio, Maria R; Chou, Judy H

    2011-03-01

    The detection of low level of protein A leached from monoclonal antibody downstream purification process is often interfered by the presence of excess amount of product antibody. In order to prevent this interference, we developed a new multi-product leached protein A assay that used acidification to completely dissociate the IgG-ProteinA complex, followed by neutralization under selected condition to prevent re-formation of the IgG-ProteinA complex. The amount of protein A was then determined by a sandwich immunoassay using Meso Scale Discovery technology. The assay takes approximately 3h to complete for one 96-well plate of samples, and this has been successfully applied to samples containing different monoclonal antibody products examined so far. The data demonstrates that this assay is robust and efficient in determining leached protein A contamination during purification of recombinant monoclonal antibodies. PMID:21315722

  17. Production and characterization of monoclonal antibodies that discriminate among individual S100 polypeptides

    The term S100 refers to a heterogeneous fraction of low molecular weight, acidic, calcium binding proteins. The S100 fraction is a mixture of polypeptides, only some of which have been isolated and characterized. The amino acid sequences of two S100 proteins from bovine brain, S100α and S100β, have been determined. The physiological functions of the S100 proteins are not known. Although assay of immunoreactive S100 has been used clinically to screen tumors of neural origin, as an index of cell injury in various disorders, and as an index of malignancy, most of the antisera used in previous studies react with more than one protein in the S100 fraction. Even the currently available monoclonal antibodies against S100 (2-4) do not appear to measure the individual S100α and S100β components. In order to unequivocally interpret studies on the localization of S100 and its potential alterations in various disease states, and on the validity of S100 immunoreactivity as a diagnostic tool for tumor diagnosis, it would be useful to have antibodies that discriminate among the individual S100 components. The authors report here the production of monoclonal antibodies that appear to be specific for S100β

  18. Production and characterization of antibodies against irradiated human erythrocytes membrane proteins

    Gamma irradiation affects people in several situations, with few if any sensitive biological assay of its action. Nucleic acids and proteins are affected by radiation, but only the former was used in most dosimetric techniques. The irradiation of proteins promotes structural modifications attributed to free radicals from water radiolysis. Theoretically, antibodies induced by irradiated proteins could recognize these radical-related new epitopes, allowing their use as a probe. Human erythrocyte membrane proteins (HEMP), few and well defined molecules, are certainly exposed to radiation, being the ideal target. With this rationale, we study the production of antibodies in mice immunized with 60 Co irradiated HEMPs. Menbranes from hypotonic lysis with differential centrifugation of A+ erythrocytes, were irradiated in a Gammacell 220 with 400, 800 and 1600 Gy, and used as immunogen for Balb/c mice, after SDS-PAGE. Irradiated HEMP induced antibodies recognize only irradiated human erthrocytes in an intact cell indirect immunofluorescence assay (ICIIFA). When used in Wester-blot against non-irradiated HEMPs, those sera recognize most proteins, suggesting a pool of abs directed both to native, as detected by Western Blot, or irradiated, as detected by ICIFA, HEMPs. Those data confirmed our assumptions, allowing the use of those abs in the search for a method of biological dosimetry. (author). 18 refs., 3 figs

  19. ZP-binding peptides identified via phage display stimulate production of sperm antibodies in dogs.

    Samoylova, Tatiana I; Cox, Nancy R; Cochran, Anna M; Samoylov, Alexandre M; Griffin, Brenda; Baker, Henry J

    2010-07-01

    Zona pellucida (ZP) glycoproteins play a central role in sperm-oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs. PMID:20434854

  20. Contribution of V(H replacement products in mouse antibody repertoire.

    Lin Huang

    Full Text Available VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS near the 3' end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

  1. Polyclonal antibody production and expression of CREG protein in human vascular smooth muscle cells

    Yaling HAN; Haiwei LIU; Jian KANG; Xiaozeng WANG; Ye HU; Lianyou ZHAO; Shaohua LI

    2005-01-01

    Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.

  2. SSB peptide and DNA co-immunization induces inhibition of anti-dsDNA antibody production in rabbits

    2008-01-01

    Background Patients with systemic lupus erythematosus often have various autoantibodies.The relationship between these antibodies is still poorly understood.The aim of the present study was to observe the anti-SSB antibody and anti-dsDNA antibody production profiles following immunization with synthetic SSB peptide alone,DNA alone or co-immunization with these two antigens.Methods SSB 214-225 aa peptide was synthesized by organic chemistry solid-phase peptide synthesis.Rabbits were immunized with the foliowing antigens:synthetic SSB peptide linked with keyhole limpet hemocyanin (KLH),DNA,SSB plus dsDNA,KLH and PBS.Antibodies were measured by ELISA.Histopathology and direct immufluorescence assays were also applied.Results Ainit-SSB and anti-dsDNA antibodies were produced following immunization with SSB peptide and DNA respectively.The level of SSB antibody in the co-immunization group was higher than that of the SSB peptide immunization group.The level of anti-dsDNA antibody in the co-immunization group was,however,lower than that in the DNA immunization group.Meanwhile,the level of anti-SSB antibody was higher than that of anti-DNA antibody in the co-immunization group.No morphological or immunological abnormalities were found in the heart,liver,kidney,spleen or skin tissues.Conclusion Inhibition of anti-dsDNA-antibody was induced by co-immunization with synthesized SSB peptide and DNA,which might explain,at least partly,the mild disease in some LE subsets associated with SSB antibody.

  3. PRODUCTION OF A HUMAN RECOMBINANT ANTIBODY AGAINST SEROTYPE A CANDIDA ALBICANS

    Jafari, A. A.

    2005-01-01

    After using 3 different generations of antibodies including human and non-human hyperimmune sera, monoclonal antibodies and chimeric antibodies, more recently a newer approach has been developed in which the antibody genes are cloned directly from a patient peripheral B-lymphocytes and expressed in a host like E. coli. In this study the Candida albicans serotype A (NCTC 3153) mannan was purified using a modified Fehling method and used for selection of human recombinant antibody from a C. alb...

  4. Production, Characterization and Use of Monoclonal Antibodies Recognizing IgY Epitopes Shared by Chicken, Turkey, Pheasant, Peafowl and Sparrow

    Narat, Mojca; BIČEK, Ajda; Vadnjal, Robert; Benčina, Dušan

    2004-01-01

    Chicken antibodies are not only a part of immune defense but are more and more popular commercial products in form of chicken polyclonal, monoclonal or recombinant antibodies. We produced and characterized mouse monoclonal antibodies (mAbs) that recognize epitopes located on heavy or light chain of chicken immunoglobulin Y (chIgY) shared also by some other Phasianidae birds. The use of mAbs 1F5 and 2F10 that recognize heavy chain on chIgY common epitopes was demonstrated on immunoglobulins of...

  5. Designed Amino Acid Feed in Improvement of Production and Quality Targets of a Therapeutic Monoclonal Antibody.

    Fatemeh Torkashvand

    Full Text Available Cell culture feeds optimization is a critical step in process development of pharmaceutical recombinant protein production. Amino acids are the basic supplements of mammalian cell culture feeds with known effect on their growth promotion and productivity. In this study, we reported the implementation of the Plackett-Burman (PB multifactorial design to screen the effects of amino acids on the growth promotion and productivity of a Chinese hamster ovary DG-44 (CHO-DG44 cell line producing bevacizumab. After this screening, the amino acid combinations were optimized by the response surface methodology (RSM to determine the most effective concentration in feeds. Through this strategy, the final monoclonal antibody (mAb titre was enhanced by 70%, compared to the control group. For this particular cell line, aspartic acid, glutamic acid, arginine and glycine had the highest positive effects on the final mAb titre. Simultaneously, the impact of the designed amino acid feed on some critical quality attributes of bevacizumab was examined in the group with highest productivity. The product was analysed for N-glycan profiles, charge variant distribution, and low molecular weight forms. The results showed that the target product quality has been improved using this feeding strategy. It was shown how this strategy could significantly diminish the time and number of experiments in identifying the most effective amino acids and related concentrations in target product enhancement. This model could be successfully applied to other components of culture media and feeds.

  6. Abnormal antigens in breast cancer tissues and production of monoclonal antibodies against one of these antigens

    Breast cancer is associated with up regulation, down regulation of normal antigens or abnormal antigens. These antigens are very useful candidates as targets for the different breast cancer therapies and for vaccination trials. This study was done to characterize abnormal antigens, extract one of them and to produce monoclonal antibodies against the extracted antigen. One hundred and twenty Sudanese female patients were included in this study after informed consent. The mean age was 47. 2 years (16-80). Two tissue samples were obtained from each patient and they were confirmed as normal and cancerous breast tissues microscopically. 2D PAGE was used to analyze the protein content of samples. LC/MS and nr. fast a database search were used for separation and indentification of the abnormal proteins. Three different patterns of 2D Page results were obtained, the first pattern involved detection of four abnormal proteins in 26.7% of the patient cancerous tissues while they were undetected in the normal tissues of the same patients. In the second 2D PAGE result pattern the cancerous and the normal tissues of 67.5% patients were identical and they did not contain the four abnormal proteins while the third 2D PAGE pattern involved the presence of two abnormal antigens (from the four) in the cancerous tissues of 5.8% of the patients and they were absent from the normal tissues of the same patients. The four abnormal proteins were identified as, human Thioredoxin (D60nmutant), x-ray crystal structure of human galectin-1, retrocopy of tropomyosin 3(rc TPM3) and beta-tropomyosin (isoform 2). The primary and the secondary structures were obtained from the SWISSPROT and the PDB databases. Beta tropomyosin spot was extracted and used as antigen for monoclonal antibody production. Monoclonal antibody against beta- tropomyosin with a concentration of 0.35 mg/ml and a G11 anti beta-tropomyosin hybridoma cell line were produced. The monoclonal antibody was with single bad and

  7. THE STUDY OF PRODUCTION AND MECHANISM OF ANTIPHOSPHOLIPID ANTIBODIES IN PATIENTS WITH CORONARY HEART DISEASE

    2001-01-01

    Objective To assess whether there was strong association between antiphospholipid antibodies(APA) and coronary heart disease(CHD), to study the environmental factors of APA production and APA pathogenic mechanism in patients with CHD.Methods Blood samples from 76 patients with CHD and 30 controls were tested for anticardiolipin antibodies IgG(ACA-IgG),human cytomegalovirus IgG,IgM(HCMV-IgG,IgM) by enzyme-link immunosorbant assay(ELISA) and 6-keto-PGF1a,endothelin(ET) by radioimmunoassay(RIA).Results A total of 27 patients(35.53%) were ACA positive in 76, as compared to 2 of 30(6.67%) healthy individuals, P<0.05. There was no difference in ACA among acute myocardial infarction(AMI,39.13%), old myocardial infarction(OMI,26.53%), unstable angina pectoris(UA,38.40%), P>0.05. The number of ACA positive subjects was higher in HCMV infection patients with CHD than no HCMV infectious patients with CHD. There was no PGI2 and ET level difference between ACA-IgG positive and negative CHD.Conclusion There are strong association between APA and CHD. The HCMV infection may be an environmental factor of APA production in CHD patients with raised ACA. The alteration of PGI2 and ET are not the pathogenic mechanism of ACA in patients with CHD.

  8. Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

    Leili Aghebati

    2013-02-01

    Full Text Available Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells.

  9. Production of double antibody for radioimmunoassay (sheep anti-rabbit IgG antiserum)

    A second antibody (sheep anti-rabbit IgG antiserum) to be used in RIAs in which the first antibody is raised in rabbits was produced. For this production, initially the IgG was isolated from rabbit serum and purified by sodium sulphate precipitation followed by ion exchange chromatography on DEAE-cellulose. Four sheep were immunized with 500 u g of purified rabbit IgG, emulsified in Freund Complete Adjuvant and administered by multisite subcutaneous injections. These injections were repeated at 20-days intervals and blood samples (40 ml) were taken from the jugular vein 10 days after the boosts for the evaluation of the antisera title. After each four boosts a great bleeding was done by the same route. Approximately 500 ml of serum were obtained in each bleeding per animal. The antisera were evaluated by the human thyrotropin RIA developed at IPEN laboratories employing reagents provided by NIDDKD, USA. These evaluations referred to the determination of the antisera title and of the ideal concentration of carrier IgG, to the study of the kinetic of precipitation and to the confirmation of the inexistent cross-reactivity with human IgG, in comparison with a reference antiserum of know precipitation characteristics supplied by the Radioassay System Laboratories. Approximately 3,6 l of antiserum (sheep anti-rabbit IgG serum) were produced from the four sheep, which presented title and precipitation characteristics very similar to those exhibited by the imported commercial product, even presenting higher titles. The results obtained in this work indicated that it was created enough experience for the production of this biological reagent for RIA, that could be done integrally in the country in greater scale, and at a very reduced cost. (author). 81 refs, 36 figs, 33 tabs

  10. Antibody-independent Targeted Quantification of TMPRSS2-ERG Fusion Protein Products in Prostate Cancer

    He, Jintang; Sun, Xuefei; Shi, Tujin; Schepmoes, Athena A.; Fillmore, Thomas L.; Petyuk, Vladislav A.; Xie, Fang; Zhao, Rui; Gritsenko, Marina A.; Yang, Feng; Kitabayashi, Naoki; Chae, Sung Suk; Rubin, Mark; Siddiqui, Javed; Wei, John; Chinnaiyan, Arul M.; Qian, Weijun; Smith, Richard D.; Kagan, Jacob; Srivastava, Sudhir; Rodland, Karin D.; Liu, Tao; Camp, David G.

    2014-10-01

    Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. The studies on TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies or an antibody-independent method that is sufficiently sensitive for detecting the truncated ERG protein products resulting from TMPRSS2-ERG gene fusions and alternative splicing. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays led to confident detection of 6 unique ERG peptides in either the TMPRSS2-ERG positive cell lines or tissues but not in the negative controls, indicating that ERG protein expression is highly correlated with TMPRSS2-ERG gene rearrangements. Significantly, our results demonstrated for the first time that at least two groups of ERG protein isoforms were simultaneously expressed at variable levels in TMPRSS2-ERG positive samples as evidenced by concomitant detection of two mutually exclusive peptides. Three peptides shared across almost all fusion protein products were determined to be the most abundant peptides, and hence can be used as “signature” peptides for detecting ERG overexpression resulting from TMPRSS2-ERG gene fusion. These PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products, thus improving our understanding of the role of TMPRSS2-ERG gene fusion in the biology of prostate cancer.

  11. Production of the TSH immunoradiometric assay kit using antibody coated magnetizable iron particles technique

    To produce TSH immunoradiometric Assay (TSH-IRMA) test kit, three kinds of magnetizable iron particles were tested. The best one was the cellulose coated iron particles with the particle size range 1-10 um. The particles were coated with monoclonal antibody to α-TSH using 1,1'-carbonyldiimidazole method. The iodine 125-monoclonal antibody to β-TSH was produced by N-bromo-succinimide method for use as a radiotracer. Both reagents were successfully developed for TSH-IRMA kit. The advantage of the TSH magnetic kit was the same sensitivity (0.04 mu/l) could be approached within 4 hours of incubation period whereas the cellulose system needed 18 hours incubation period. Moreover, the separation process was done by using simple magnet block instead of high cost refrigerated centrifuge. The production cost was 10 times cheaper than the commercial available one. These kits will be distributed to Ministry of Public Health Laboratories all over the country, especially in iodine deficiency disorder control programme laboratories

  12. Selenium and vitamin E effect on antibody production of sheep vaccinated against enzootic abortion (Chlamydia psittaci).

    Giadinis, N; Koptopoulos, G; Roubles, N; Siarkou, V; Papasteriades, A

    2000-03-01

    The effect of selenium (Se) and vitamin E (vit E) on antibody production of sheep vaccinated against Chlamydia psittaci (ovis) was investigated. Thirty-two sheep, one year old, seronegative to Chlamydia infection, vaccinated against enterotoxemia and dewormed were used. Injectable sodium selenite (0.1 mg/kg b.w.) was given twice to animals of the first group (gSe), with a three week interval. The sheep of the second group (gE) received 1 g vit E each orally, six times at weekly intervals. The animals of the third group (gSeE) were given Se and vit E in doses and routes of administration as in gSe and gE. The animals of the fourth group served as controls (gC) and injected normal saline. The first vaccination was made at the time that the second Se injection was given. Revaccination was made two weeks later. The experiment lasted 29 weeks. The results indicated that Se alone led to a significant increase of Chlamydia antibody response (P < 0.05), but not when it was given in combination with vit E. Animals that received vit E (gE) had much lower titres, just above of those of the controls. PMID:10670702

  13. Optimization of monoclonal antibody production in mouse ascites by single whole-body irradiation

    Hybridoma cells injected intraperitoneally into mice induce formation of ascites tumors producing ascites fluid with high levels of monoclonal antibodies. Several parameters affect the growth of the immunoglobulin-producing tumors in vivo. In 10 different hybridomas the average ascites tumor formation rate could be increased from 32% (n = 338 mice) to 77% (n = 112 mice) by only one whole-body irradiation of paraffin-pretreated Balb/c mice. Production of monoclonal antibodies was better in males because of the significantly (p < 0.01) increased volume of ascites fluid. From the increased tumor formation rate in irradiated mice it is suggested that in non-irradiated recipients the tumor growth rate was lowered by immunological reactions against hybridoma cells provoked by cell surface neoantigens revealed by cell fusion and/or tumor-associated antigens of the myeloma parent cells as well as by altered antigen pattern caused by possible mutations in the myeloma cell line and/or Balb/c/K strain. (author)

  14. Prebiotic and antimicrobials on performance, carcass characteristics, and antibody production in broilers

    Maíra Fomentini

    2016-06-01

    Full Text Available ABSTRACT: To evaluate the effect of supplementation with mannan oligosaccharides, avilamycin and halquinol, alone or in combination, on the performance, carcass characteristics and antibody production in broilers (1-49 days old, male broiler chicks (n=1440; Cobb 500; one day old were housed and distributed into a completely randomized design into six treatments (eight replicates; 30 animals per pen. To produce the experimental diets, three types of performance enhancer additives were used. Halquinol (HAL, avilamycin (AVI and mannan oligosaccharides (MOS were included (alone or in combination in the basal diet (instead of corn starch. Effects of diet were observed on results of animal performance in the period 1-21 and 1-42 days old. Broilers fed with a diet without growth promoter showed lower weight gain in relation to those fed with diets with antimicrobials, MOS or a combination of them. In the period 1-49 days old, feed conversion increased in broilers fed with rations without promoter. At the end of the experimental period no influence of diets was observed on the carcass yield and cuts, and titles of specific antibodies to avian infectious bronchitis. The use of MOS and/or antimicrobials (AVI or HAL, alone or in combination, improves feed conversion of broilers reared until 49 days of age.

  15. Production of Mouse Monoclonal Antibody against Morphine without Cross Reactivity with Heroin

    S Kashaninan

    2014-12-01

    Conclusion: The study findings revealed that the produced antibody against morphine was comparable with other antibodies for specificity and affinity; therefore it is usable in design of diagnostic immunoassay in biologic fluids.

  16. Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli.

    Miller, Keith D.; Feldhaus, Jane M.; Gray, Sean A.; Siegel, Robert W.; Feldhaus, Michael J.

    2005-08-01

    Single chain (scFv) antibodies are used as affinity reagents for diagnostics, therapeutics, and proteomic analyses. The antibody discovery platform we use to identify novel antigen binders involves discovery, characterization, and production. The discovery and characterization components have previously been characterized but in order to fully utilize the capabilities of affinity reagents from our yeast surface display library, efforts were focused on developing a production component to obtain purified, soluble, and active scFvs. Instead of optimizing conditions to achieve maximum yield, efforts were focused on using a system that could quickly and easily produce and process hundreds of scFv antibodies. Heterologous protein expression in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli were evaluated for their ability to rapidly, efficaciously, and consistently produce scFv antibodies for use in downstream proteomic applications. Following purification, the binding activity of several scFv antibodies were quantified using a novel Biacore assay. All three systems produced soluble scFv antibodies which ranged in activity from 0-99%. scFv antibody yields from Saccharomyces, Pichia, and E. coli were 1.5-4.2, 0.4-7.3, and 0.63-16.4 mg L-1 culture, respectively. For our purposes, expression in E. coli proved to be the quickest and most consistent way to obtain and characterize purified scFv for downstream applications. The E. coli expression system was also used to compare scFv production levels from the periplasm, inclusion bodies, and culture media. The E. coli production system was then used to produce variants of several scFv to determine structure function relationships.

  17. Microarray-based MALDI-TOF mass spectrometry enables monitoring of monoclonal antibody production in batch and perfusion cell cultures.

    Steinhoff, Robert F; Karst, Daniel J; Steinebach, Fabian; Kopp, Marie R G; Schmidt, Gregor W; Stettler, Alexander; Krismer, Jasmin; Soos, Miroslav; Pabst, Martin; Hierlemann, Andreas; Morbidelli, Massimo; Zenobi, Renato

    2016-07-15

    Cell culture process monitoring in monoclonal antibody (mAb) production is essential for efficient process development and process optimization. Currently employed online, at line and offline methods for monitoring productivity as well as process reproducibility have their individual strengths and limitations. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based on a microarray for mass spectrometry (MAMS) technology to rapidly monitor a broad panel of analytes, including metabolites and proteins directly from the unpurified cell supernatant or from host cell culture lysates. The antibody titer is determined from the intact antibody mass spectra signal intensity relative to an internal protein standard spiked into the supernatant. The method allows a semi-quantitative determination of light and heavy chains. Intracellular mass profiles for metabolites and proteins can be used to track cellular growth and cell productivity. PMID:26707204

  18. Production of monoclonal antibodies to Legionella pneumophila serogroups 1 and 6.

    Para, M F; Plouffe, J F

    1983-01-01

    To better define the surface antigens of Legionella pneumophila for clinical and experimental purposes, we have produced monoclonal antibodies to L. pneumophila serogroups 1 and 6. Two hybridomas were produced in serogroup 1. One antibody, LP-I-17, recognized a serogroup-common antigen. The second antibody, LP-I-81, was specific for serogroup 1. This antibody was able to agglutinate bacterial cells belonging to the serogroup 1 reference strains. Philadelphia and Knoxville. Microagglutination ...

  19. Obtention of antibodies anti prolactin from human prolactin of national production

    In this work was studied the use of the the Prolactin hormone as immuno gen, which is obtained in Cuba by the pharmaceutical institute Mario Munoz, to produce the antibody antiprolactin. Was made the validation of obtained antibody (tritatium, specificity and affinity) The produced antibody had necessary quality to be use as a component of the Kits-RIA Prolactin

  20. Chemo-enzymatic production of O-glycopeptides for the detection of serum glycopeptide antibodies

    Nøstdal, Alexander; Wandall, Hans H

    Protein microarray is a highly sensitive tool for antibody detection in serum. Monitoring of patients' antibody titers to specific antigens is increasingly employed in the diagnosis of several conditions, ranging from infectious diseases, allergies, autoimmune diseases, and cancer. In this protoc...... we present a detailed method for enzymatic generation of disease-specific O-glycopeptides and how to monitor the antibody response to these in serum using microarray technology.......Protein microarray is a highly sensitive tool for antibody detection in serum. Monitoring of patients' antibody titers to specific antigens is increasingly employed in the diagnosis of several conditions, ranging from infectious diseases, allergies, autoimmune diseases, and cancer. In this protocol...

  1. Mammalian Cell Culture Process for Monoclonal Antibody Production: Nonlinear Modelling and Parameter Estimation

    Dan Selişteanu

    2015-01-01

    Full Text Available Monoclonal antibodies (mAbs are at present one of the fastest growing products of pharmaceutical industry, with widespread applications in biochemistry, biology, and medicine. The operation of mAbs production processes is predominantly based on empirical knowledge, the improvements being achieved by using trial-and-error experiments and precedent practices. The nonlinearity of these processes and the absence of suitable instrumentation require an enhanced modelling effort and modern kinetic parameter estimation strategies. The present work is dedicated to nonlinear dynamic modelling and parameter estimation for a mammalian cell culture process used for mAb production. By using a dynamical model of such kind of processes, an optimization-based technique for estimation of kinetic parameters in the model of mammalian cell culture process is developed. The estimation is achieved as a result of minimizing an error function by a particle swarm optimization (PSO algorithm. The proposed estimation approach is analyzed in this work by using a particular model of mammalian cell culture, as a case study, but is generic for this class of bioprocesses. The presented case study shows that the proposed parameter estimation technique provides a more accurate simulation of the experimentally observed process behaviour than reported in previous studies.

  2. Mammalian cell culture process for monoclonal antibody production: nonlinear modelling and parameter estimation.

    Selişteanu, Dan; Șendrescu, Dorin; Georgeanu, Vlad; Roman, Monica

    2015-01-01

    Monoclonal antibodies (mAbs) are at present one of the fastest growing products of pharmaceutical industry, with widespread applications in biochemistry, biology, and medicine. The operation of mAbs production processes is predominantly based on empirical knowledge, the improvements being achieved by using trial-and-error experiments and precedent practices. The nonlinearity of these processes and the absence of suitable instrumentation require an enhanced modelling effort and modern kinetic parameter estimation strategies. The present work is dedicated to nonlinear dynamic modelling and parameter estimation for a mammalian cell culture process used for mAb production. By using a dynamical model of such kind of processes, an optimization-based technique for estimation of kinetic parameters in the model of mammalian cell culture process is developed. The estimation is achieved as a result of minimizing an error function by a particle swarm optimization (PSO) algorithm. The proposed estimation approach is analyzed in this work by using a particular model of mammalian cell culture, as a case study, but is generic for this class of bioprocesses. The presented case study shows that the proposed parameter estimation technique provides a more accurate simulation of the experimentally observed process behaviour than reported in previous studies. PMID:25685797

  3. Differential effects of immunosuppressants and antibiotics on human monoclonal antibody production in SCID mouse ascites by five heterohybridomas.

    Yoshinari, K; Arai, K

    1998-02-01

    SCID mice were inoculated with five human-mouse heterohybridomas derived by fusion of human lymph node lymphocytes from lung cancer patients with murine myeloma cells or human-mouse heteromyeloma cells, and the production of their human monoclonal antibodies (MAb) in the mouse ascites was investigated. In a comparison of the effects of pretreatment by i.p. (intraperitoneal) injection of pristane and anti-asialo GM1 serum on the antibody production of three of the hybridomas, pristane pretreatment resulted in substantial antibody production by all three, and pretreatment with anti-asialo GM1 serum resulted in similar or slightly lower levels of antibody production by two of the hybridomas but none by the third. In a second series of experiments using four of the hybridomas with pristane pretreatment, the co-injection of either penicillin G and streptomycin or kanamycin together with the hybridoma at the time of i.p. inoculation resulted in enhanced MAb production by the two heterohybridomas that had been propagated in medium containing hypoxanthine-aminopterin-thymidine (HAT) but not by the two that had been propagated in HAT-free medium. PMID:9523236

  4. Detailed analyses of antibodies recognizing mitochondrial antigens suggest similar or identical mechanism for production of natural antibodies and natural autoantibodies.

    Czömpöly, Tamás; Olasz, Katalin; Nyárády, Zoltán; Simon, Diána; Bovári, Judit; Németh, Péter

    2008-06-01

    Because of their endosymbiotic evolutionary origin, proteins compartmentalized into mitochondria represent an interesting transition from prokaryotic foreign to essential self molecules. We investigated the presence of naturally occurring antibodies (nAbs) recognizing mitochondrial inner membrane enzymes. Epitope mapping analysis of a mitochondrial inner membrane enzyme, citrate synthase (CS) by synthetic overlapping peptides and phage display libraries using sera from healthy individuals and from patients having systemic autoimmune disease revealed CS recognizing nAbs with IgM isotype. We analyzed cross-reactive epitopes on human CS, bacterial CS, and various standard autoantigens. We have found that the fine epitope pattern on CS is different under physiological and pathological conditions. Moreover sera affinity purified on CS cross reacts with nucleosome antigen, which cross-reactivity could be mapped to a short epitope on human CS. These data indicate that in theory, nAbs "specific" for a given self antigen could fulfill the function of participating in innate defense mechanisms and at the same time recognize a target antigen in a systemic autoimmune disease. Thus, at the level of recognized epitopes there is a possible link between the innate like part and the adaptive-autoimmune arm of the humoral immune system. PMID:18558363

  5. PRODUCTION OF A HUMAN RECOMBINANT ANTIBODY AGAINST SEROTYPE A CANDIDA ALBICANS

    A A. Jafari

    2005-07-01

    Full Text Available After using 3 different generations of antibodies including human and non-human hyperimmune sera, monoclonal antibodies and chimeric antibodies, more recently a newer approach has been developed in which the antibody genes are cloned directly from a patient peripheral B-lymphocytes and expressed in a host like E. coli. In this study the Candida albicans serotype A (NCTC 3153 mannan was purified using a modified Fehling method and used for selection of human recombinant antibody from a C. albicans phage antibody library. After four rounds of affinity selecting (panning, 2 predominant clones were chosen by DNA fingerprinting and ELISA. A 248 amino acid DNA fragment coding for anti-C. albicans mannan scFv was sequenced and cloned in a pBAD-TOPO cloning vector to produce a soluble and phage free antibody. The analysis of antibody sequences by V base Index (DNAPLOT confirmed the human antibody origin with the VH4 family in V segment of heavy variable chain and VL3 (Lambda 3 in J segment of the light variable chain. This antibody fragment was purified using immobilized metal affinity chromatography and inmmunoblotted as a 31kDa recombinant protein.

  6. Evaluation of host humoral antibody production against Plasmodium falciparum recombinant circumsporozoite antigen in Nigerian children

    Olalubi Adewole Oluwasogo , Ogunlana Oluseyi Ebenezer & Anumudu Chiaka

    2012-09-01

    Full Text Available Background & objectives: The challenge of malaria and efforts targeted at developing malaria vaccines triggeredthis study on the reactivity of IgG and its subclasses in the test serum specific to CSP. This work was directed atassessing the influence of age and gender on host humoral antibody against Plasmodium falciparum recombinantcircumsporozoite antigen in Nigerian children.Methods: In all, 67 serum samples (>10,000 parasites/μl of blood collected from malaria-infected children atthe University College Hospital, Ibadan during the transmission season were analyzed by ELISA.Results: The mean absorbance values of IgG subclasses reactive against P. falciparum CSP appeared to be agedependent and ranged from 0.01 for IgG4 in younger children to 0.95 for IgG3 in older children. The sixty-sevensubjects investigated in this study had significantly higher mean IgG1 and IgG3 than the uninfected controls(p IgG1>IgG2>IgG4 which confirmed the prevalence of the cytophilicantibodies (IgG1 and IgG3 in 65% of the malaria infected children over the non-cytophilic subclasses (IgG2and IgG4. Similarly, there was low production of IgG4 and IgG2 levels in 35% of the subjects compared withcontrol. IgG was detected in the serum of North American Subjects (NAS which served as negative control forCSP-specific IgG subclasses. Although the NAS titre was lower than that of the malaria subjects in Nigeria, itsIgG2 was, however, higher (0.16 than that of other subclasses. The mean absorbance values of total serum IgGsubclass were higher than those of IgG subclasses specific to P. falciparum circumsporozoite antigen. The meanabsorbance values of the total serum IgG subclass follows the order IgG2>IgG1>IgG4>IgG3.Interpretation & conclusion: Age and gender-dependent correlations of results suggest that acquired immunitycould play a significant role in protection from malaria. Antibody levels are higher in male than female childrenof the same age group. Antibody levels also

  7. Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.

    Li, Jin; Stoddard, Thomas J; Demorest, Zachary L; Lavoie, Pierre-Olivier; Luo, Song; Clasen, Benjamin M; Cedrone, Frederic; Ray, Erin E; Coffman, Andrew P; Daulhac, Aurelie; Yabandith, Ann; Retterath, Adam J; Mathis, Luc; Voytas, Daniel F; D'Aoust, Marc-André; Zhang, Feng

    2016-02-01

    Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products. PMID:26011187

  8. New adjuvant design using layered double hydroxide for production of polyclonal antibodies in radioimmunoassay techniques

    Various adjuvants have been used to enhance the immune response against specific antigens. So the objective of this work describes the immune stimulating activity of layered double hydroxide (LDH) particles incorporate with mineral oil as a new formulation of adjuvant as compared to known Freund's adjuvant for production of alpha-fetoprotein polyclonal antibody (anti-AFP) for estimation of alpha fetoprotein (AFP) in human serum by radioimmunoassay technique. In this concern, the study comprised two groups of white New Zealand rabbits, 2-2.5 kg body weight and each group comprised three rabbits. The first group vaccinated with AFP antigen emulsified with Freund's adjuvant and the second group vaccinated with AFP antigen emulsified with LDH formulation. The obtained data show that the highest displacement using LDH adjuvant reached (74.2, 61.7 and 66.5 %) while the corresponding values with Freund's adjuvant recorded (64.8, 60.3 and 54.6 %) which indicates that the use of LDH adjuvant as a cellular vehicle is a more suitable choice. Also, the preparation of AFP tracer using lactoperoxidase oxidation method and its purification using gel chromatography on PD-10 column were carried out. Different factors affecting the optimization of the assay process were studied. Validation testes of the assay were carried out. The reproducibility as measured by the intra- and inter- assay variations is satisfactory. The recovery and dilution testes indicated accurate calibration and appropriate matrix. The present technique agreed well with the currently used commercial kit (Siemens, IRMA kit). In conclusion, the liquid phase double antibody RIA technique proved to be sensitive, specific, precis and accurate for routine laboratory use. (author)

  9. Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation

    Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian;

    2015-01-01

    Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream proces...

  10. Improving food and agricultural production. Thailand. Application on monoclonal antibodies for progesterone measurement

    The duties of the mission were to provide instructions on the maintenance of hybridoma cell lines and their culture and the harvesting of monoclonal antibodies; to assist the counterparts in Thailand to develop work plans for the use of monoclonal antibodies in radioimmunoassay measurements of progesterone; and to assess the need for and feasibility of establishing a laboratory for producing monoclonal antibodies directed against progesterone. The report contains a summary of the activities performed in fulfillment of these duties

  11. Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

    Wan-gang GU; Jun-fa YUAN; Ge-lin XU; Li-juan LI; Ni LIU; Cong ZHANG; Jian-hong ZHANG; Zheng-li SHI

    2007-01-01

    BALB/c mice were immunized with purified White spot syndrome virus (WSSV).Six monoclonal antibody cell lines were selected by ELISA with VP28 protein expressed in E.coll in vitro neutralization experiments showed that 4 of them could inhibit the virus infection in crayfish.Westernblot suggested that all these monoclonal antibodies were against the conformational structure of VP28.The monoclonal antibody 7B4 was labeled with colloidal gold particles and used to locate the VP28 on virus envelope by immunogold labeling.These monoclonal antibodies could be used to develop immunological diagnosis methods for WSSV infection.

  12. Production of IgM antibody to HHV6 in reactivation and primary infection.

    Fox, J D; Ward, P; Briggs, M; Irving, W; Stammers, T. G.; Tedder, R S

    1990-01-01

    The cross-reaction of HHV6 antibody with that to the other herpesviruses was studied in 96 blood donors whose sera were tested for IgG antibody to human herpesvirus type 6 (HHV6), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zostervirus (VZV) and herpes simplex virus (HSV). No correlation was found between IgG antibody to HHV6 and that to any of the other herpesviruses in these individuals. Antibodies to HHV6 and CMV were measured in patients undergoing documented serological re...

  13. Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

    Emma J Mead

    Full Text Available Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based and engineering (nonlinear models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.

  14. Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

    Mead, Emma J; Chiverton, Lesley M; Spurgeon, Sarah K; Martin, Elaine B; Montague, Gary A; Smales, C Mark; von der Haar, Tobias

    2012-01-01

    Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway. PMID:23071804

  15. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans

    Both, L.; van Dolleweerd, C.; Wright, E.; Banyard, A. C.; Bulmer-Thomas, B.; Selden, D.; Altmann, F.; Fooks, A.R.; Ma, J. K.- C.

    2013-01-01

    Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed ...

  16. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Nazila Amini

    2014-06-01

    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  17. Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

    Meagher, Richard B.; Laterza, Vince

    2006-12-12

    The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

  18. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  19. A Novel Scheme for Production of Polyclonal Antibody against Estrogenic Bisphenols

    2002-01-01

    A polyclonal antibody against the currently concerned estrogenic bisphcnol compoundswas produced according to a new scheme. 4,4-Bis (4-hydroxyphenyl) valeric acid was used tosynthesize the complete antigen in which the characteristic bisphenol structure was exposed to thelargest extent. The produced polyclonal antibody showed high specificity and affinity forbisphenol A.

  20. Production, purification and characterization of polyclonal antibody against the truncated gK of the duck enteritis virus

    Zhang Shunchuan

    2010-09-01

    Full Text Available Abstract Duck virus enteritis (DVE is an acute, contagious herpesvirus infection of ducks, geese, and swans, which has produced significant economic losses in domestic and wild waterfowl. With the purpose of decreasing economic losses in the commercial duck industry, studying the unknown glycoprotein K (gK of DEV may be a new method for preferably preventing and curing this disease. So this is the first time to product and purify the rabbit anti-tgK polyclonal antibody. Through the western blot and ELISA assay, the truncated glycoprotein K (tgK has good antigenicity, also the antibody possesses high specificity and affinity. Meanwhile the rabbit anti-tgK polyclonal antibody has the potential to produce subunit vaccines and the functions of neutralizing DEV and anti-DEV infection because of its neutralization titer. Indirect immunofluorescent microscopy using the purified rabbit anti-tgK polyclonal antibody as diagnostic antibody was susceptive to detect a small quantity of antigen in tissues or cells. This approach also provides effective experimental technology for epidemiological investigation and retrospective diagnose of the preservative paraffin blocks.

  1. Characterization of a monoclonal antibody cell culture production process using a quality by design approach.

    Horvath, Brian; Mun, Melissa; Laird, Michael W

    2010-07-01

    The goal of quality by design (QbD) in cell culture manufacturing is to develop manufacturing processes which deliver products with consistent critical quality attributes (CQAs). QbD approaches can lead to better process understanding through the use of process parameter risk ranking and statistical design of experiments (DOE). The QbD process starts with an analysis of process parameter risk with respect to CQAs and key performance indicators (KPIs). Initial DOE study designs and their factor test ranges are based on the outcomes of the process parameter risk ranking exercises. Initial DOE studies screen factors for significant influences on CQAs as well as characterize responses for process KPIs. In the case study provided here, multifactor process characterization studies using a scale-down model resulted in significant variation in charge heterogeneity of a monoclonal antibody (MAb) as measured by ion-exchange chromatography (IEC). Iterative DOE studies, using both screening and response surface designs, were used to narrow the operating parameter ranges so that charge heterogeneity could be controlled to an acceptable level. The data from the DOE studies were used to predict worst-case conditions, which were then verified by testing at those conditions. Using the approach described here, multivariate process parameter ranges were identified that yield acceptable CQA levels and that still provide operational flexibility for manufacturing. PMID:20300882

  2. Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies.

    Corrêa, Arthur Luiz; Senna, José Procópio Moreno; de Sousa, Álvaro Paiva Braga

    2016-05-01

    Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction

  3. Production and characterisation of a monoclonal antibody to human papillomavirus type 16 using recombinant vaccinia virus.

    McLean, C S; Churcher, M J; Meinke, J.; Smith, G.L.; Higgins, G; Stanley, M.; Minson, A C

    1990-01-01

    A monoclonal antibody was raised against the major capsid protein L1 of human papillomavirus type 16, using a recombinant vaccinia virus that expresses the L1 protein, as a target for screening. This antibody, designated CAMVIR-1, reacted with a 56 kilodalton protein in cells infected with L1-vaccinia virus, and the protein was present in a predominantly nuclear location. The antibody also detects the HPV-16 L1 antigen in formalin fixed, paraffin wax embedded biopsy specimens and on routine c...

  4. Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR)

    A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qr) has been produced by fusion of the P3 x 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 x 107 cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: a) this antibody competes for C1q binding sites on C1qR-bearing cells; b) the molecule recognized by this MAb is the C1qR; and c) cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125I-MAb detected a major protein band of approximately 85000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125I-II/D1 binding experiments revealed that the antibody bound to Raji cells or u937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant K/sub D/ is 2.9 x 10-10 M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 x 106 cells, giving an estimated 7.8 x 103 antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific dose-dependent manner

  5. Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

    Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders;

    2015-01-01

    eradication program, initiated in 1994. During the last decade, the cattle herd size has increased while the prevalence of BVDV has decreased. In this study, we investigated how these changes could affect the performance of the Danish blocking ELISA and of the SVANOVIR® BVDV-Ab indirect ELISA. The latter has...... successfully been used to eradicate BVD in Sweden. Data (2003–2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples...... BVDV infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level...

  6. Overexpression of CHOP alone and in combination with chaperones is effective in improving antibody production in mammalian cells.

    Nishimiya, Daisuke; Mano, Takashi; Miyadai, Kenji; Yoshida, Hiroko; Takahashi, Tohru

    2013-03-01

    Secretory capacities including folding and assembly are believed to be limiting factors in the establishment of mammalian cell lines producing high levels of recombinant therapeutic proteins. To achieve industrial success, it is also important to improve protein folding, assembly, and secretory processes in combination with increasing transcription and translation. Here, we identified the expression of CHOP/Gadd153 and GRP78, which are unfolded protein response (UPR)-related genes, correlated with recombinant antibody production in stable CHO cells. Subsequently, CHOP overexpression resulted in increasing recombinant antibody production in some mammalian cell lines, and in addition a threefold further enhancement was obtained by combining expression with UPR-related genes or ER chaperones in transient assays. Overexpression of CHOP had no effect on the biochemical characteristics of the product. These results suggest overexpression of CHOP and its combinations may be an effective method to efficiently select a single cell line with a high level of antibody production in the development of cell lines for manufacturing. PMID:22926643

  7. Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans

    Caterson, B; Christner, J E; Baker, J R;

    1985-01-01

    Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs...... attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate......-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential...

  8. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Nazila Amini; Mohadeseh Naghi Vishteh; Omid Zarei; Reza Hadavi; Negah Ahmadvand; Hodjattallah Rabbani; Mahmood Jeddi-Tehrani

    2014-01-01

    Objective(s):Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protei...

  9. [Correlation between Staphylococcus carriage, specific antibody-production and AB0-blood grouping in plasma donors].

    Nemyrovs'ka, L M; Patoka, V V

    2002-01-01

    Interaction peculiarities of three components of the immune human homeostasis-antigens of blood groups AB0, staphylococcus antigens and antistaphylococcus antibodies have been investigated. Donors (85) of antistaphylococcus plasma immunized by staphylococcus anatoxin have been investigated. It is found that the nasal staphylococcus carriage in donors depends on the level of specific and natural antibodies and on the coincidence between the staphylococcus antigen structure and the protein substance of the specific blood group factors. PMID:12190026

  10. Production and characterization of a monoclonal antibody to the O-acetylated peptidoglycan of Proteus mirabilis.

    Gyorffy, S; Clarke, A J

    1992-01-01

    A monoclonal antibody (PmPG5-3) specific for the O-acetylated peptidoglycan of Proteus mirabilis 19 was produced by an NS-1 myeloma cell line and purified from ascites fluid by a combination of ammonium sulfate precipitation and affinity chromatography. The monoclonal antibody (an immunoglobulin M) was characterized by a competition enzyme-linked immunosorbent assay to be equally specific for both insoluble and soluble O-acetylated peptidoglycan but weakly recognized chemically de-O-acetylate...

  11. Production of Antibodies against Multipass Membrane Proteins Expressed in Human Tumor Cells Using Dendritic Cell Immunization

    Takahiko Tamura; Joe Chiba

    2009-01-01

    Antibody mediated therapeutic strategies against human malignant tumors have been widely authorized and clinically applied to cancer patients. In order to develop methods to generate antibodies reactive to the extracellular domains of multipass plasma membrane proteins specifically expressed in malignant tumors, we examined the use of dendritic cells (DCs) for immunization. DCs were transduced with genes encoding the human six transmembrane epithelial antigen of prostate 1 (STEAP1), STEAP4, a...

  12. RECOMBINANT PRODUCTION OF HORSERADISH PEROXIDASE CONJUGATES WITH FAB ANTIBODIES IN PICHIA PASTORIS FOR ANALYTICAL APPLICATIONS

    Koliasnikov, O.; Grigorenko, V.; Egorov, A.; S. Lange(Justus Liebig-Universität Gie\\ssen II Physikalisches Institut, Germany); Schmid, R

    2011-01-01

    Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e. they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody. Based on the pPICZαB shuttle vector, we first managed to obtain a recombinant conjugate of key marker enzyme horseradish peroxidase (HRP) with Fab fragments of anti...

  13. Production and characterisation of monoclonal antibodies specific for chicken interleukin-2

    Rothwell, L.; Hamblin, A; Kaiser, P

    2001-01-01

    Using genetic immunisation of mice, we produced antibodies against chicken interleukin-2 (ChIL-2), the first produced against a non-mammalian interleukin. After a final injection with a recombinant ChIL-2 protein, two stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of the two MAbs to recombinant ChIL-2 produced by Escherichia coli and COS-7 cells was demonstrated in an indirect ELISA, Western blotting and dot blo...

  14. Effects of probiotic bacteria at different concentrations on production of immunomodulatory antibodies against rabies virus in vaccinated cattle

    Renata Maria Bottino Vizzotto-Martino

    2016-02-01

    Full Text Available This study evaluated the effects of supplementation with a combination of probiotic microorganisms, added at different concentrations to the mineral mixture, on the production of serum antibodies against rabies virus in cattle vaccinated with a single dose of rabies vaccine. Forty-two male Nellore cattle, aged 12 months, were randomly divided into three groups (n = 14: the control group (CG received 70 grams of mineral mixture/animal/day; and the 2 gram probiotic group (G2P and 8 gram probiotic group (G8P received 70 grams of mineral mix/animal/day with 2 and 8 grams added, respectively, of a combination of probiotic microorganisms (Lactobacillus acidophilus, Streptococcus faecium, Bifidobacterium thermophilum and Bifidobacterium longum. Individual antibody titers were determined using a neutralization in cell-based rapid fluorescent focus inhibition test (RFFIT technique. One-way analysis of variance (one-way ANOVA was used with contrasts using the Tukey method to determine whether the experimental groups differed within each time point, and the paired t-test was used to determine whether differences occurred between time points within each group. The level of significance was set at 5%. There were statistically significant differences between the mean serum concentrations of the CG and G8P groups at 30 and 60 days after the first vaccination, and at 60 days, 100% of the animals maintained minimum titers of protective antibodies only in the G8P group. There was also improvement in the production of antibodies in the G2P group compared with the CG after 30 and 60 days, but this difference was not statistically significant. In conclusion, increasing doses of probiotic added to the mineral mix beneficially affected the rabies humoral immune response, as determined by serum antibodies, and enabled the maintenance of minimum protective titers for a longer period in previously vaccinated cattle.

  15. Effects of probiotic bacteria at different concentrations on production of immunomodulatory antibodies against rabies virus in vaccinated cattle

    Renata Maria Bottino Vizzotto-Martino; Cristina Cecilia Augusto Vella Bonancéa; Thaís Cristina de Souza Geroti; Neuza Maria Frazati-Gallina; Paulo Eduardo Pardo; Hermann Bremer-Neto

    2016-01-01

    This study evaluated the effects of supplementation with a combination of probiotic microorganisms, added at different concentrations to the mineral mixture, on the production of serum antibodies against rabies virus in cattle vaccinated with a single dose of rabies vaccine. Forty-two male Nellore cattle, aged 12 months, were randomly divided into three groups (n = 14): the control group (CG) received 70 grams of mineral mixture/animal/day; and the 2 gram probiotic group (G2P) and 8 gram prob...

  16. Optimisation of recombinant protein production in Pichia pastoris:single-chain antibody fragment model protein

    Khatri, N. K. (Narendar Kumar)

    2011-01-01

    Abstract Potential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is one of the most common diseases in young pigs. It can be cured by single-chain antibody fragments (scFv), which can be produced in recombinant microorganisms. Pichia pastoris, a methylotrophic yeast, is generally considered an interesting production system candidate, as it can secrete properly folded proteins. These proteins accumulate in high concentrations during fermentation, reducing the cost for...

  17. Proteomic Profiling of Recombinant Escherichia coli in High-Cell- Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

    Aldor, Ilana S.; Krawitz, Denise C.; Forrest, William; Chen, Christina; Nishihara, Julie C.; Joly, John C.; Champion, Kathleen M.

    2005-01-01

    By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein sp...

  18. EB66 cell line, a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity

    Olivier, Stéphane; Jacoby, Marine; Brillon, Cédric; Bouletreau, Sylvana; Mollet, Thomas; Nerriere, Olivier; Angel, Audrey; Danet, Sévérine; Souttou, Boussad; Guehenneux, Fabienne; Gauthier, Laurent; Berthomé, Mathilde; Vié, Henri; Beltraminelli, Nicola; Mehtali, Majid

    2010-01-01

    Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutic proteins. The increasing demand for mAb manufacturing and the associated high production costs call for the pharmaceutical industry to improve its current production processes or develop more efficient alternative production platforms. The experimental control of IgG fucosylation to enhance antibody dependent cell cytotoxicity (ADCC) activity constitutes one of the promising strategies to improve the efficacy of m...

  19. Hapten synthesis and antibody production for the development of a melamine immunoassay

    Lei Hongtao; Shen Yudong; Song Lijun; Yang Jinyi [Key Laboratory of Food Safety of Guangdong Province/Institute of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, Guangdong (China); Chevallier, Olivier P.; Haughey, Simon A. [Institute of Agri-Food and Land Use, Queen' s University Belfast, Belfast BT9 5AG, Northern Ireland (United Kingdom); Wang Hong [Key Laboratory of Food Safety of Guangdong Province/Institute of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, Guangdong (China); Sun Yuanming, E-mail: ymsun@scau.edu.cn [Key Laboratory of Food Safety of Guangdong Province/Institute of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, Guangdong (China); Elliott, Christopher T., E-mail: chris.elliott@qub.ac.uk [Institute of Agri-Food and Land Use, Queen' s University Belfast, Belfast BT9 5AG, Northern Ireland (United Kingdom)

    2010-04-14

    The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by {sup 1}H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC{sub 50} of 70.6 ng mL{sup -1}, a LOD of 2.6 ng mL{sup -1} and a LOQ of 7.6 ng mL{sup -1}. Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical.

  20. Production of antibody labeled gold nanoparticles for influenza virus H5N1 diagnosis kit development

    Preparation of colloidal gold conjugated antibodies specific for influenza A/H5N1 and its use in developing a virus A/H5N1 rapid diagnostic kit is presented. Colloidal gold nanoparticles (AuNPs) were prepared through citrate reduction. Single chain antibodies specific to H5N1 (scFv7 and scFv24) were produced using pTI2 + vector and E. coli strain HB2151. These antibodies were purified by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + . The method for preparation of antibody–colloidal gold conjugate was based on electrostatic force binding antibody with colloidal gold. The effect of factors such as pH and concentration of antibody has been quantitatively analyzed using spectroscopic methods after adding 1 wt% NaCl which induced AuNP aggregation. The morphological study by scanning electron microscopy (SEM) showed that the average size of the spherical AuNPs was 23 nm with uniform sizes. The spectroscopic properties of colloidal AuNPs showed the typical surface plasmon resonance band at 523 nm in UV-visible spectrum. The optimal pH of conjugated colloidal gold was found between 8.0 and 10.0. The activity of synthesized antibody labeled AuNPs for detection of H5N1 flu virus was checked by dot blot immunological method. The results confirmed the ability in detection of the A/H5N1 virus of the prepared antibody labeled gold particles and opened up the possibility of using them in manufacturing rapid detection kit for this virus. (paper)

  1. Continuous production of monoclonal antibody in a packed-bed bioreactor.

    Golmakany, Naghmeh; Rasaee, Mohammad Javad; Furouzandeh, Mehdi; Shojaosadati, Seyed Abbas; Kashanian, Soheila; Omidfar, Kobra

    2005-06-01

    In the present study the growth and MAb (monoclonal antibody) production of a mouse x mouse hybridoma cell producing anti-digoxin MAb was evaluated. The hybridoma cells entrapped within the support matrix Fibra-Cel were cultured in batch and continuous mode following special protocols. Cell-culture studies were performed in a 1-litre spinner basket containing 3 g.litre-1 support matrix. Batch culture was operated with the cell density of 42x10(6) cells. During the 7 days of culture, the medium was sampled daily in order to assess glucose and MAb concentrations and the lactate dehydrogenase released into the culture medium. After a culture period of 72 h, the cell density and MAb concentration were found to be 10.4x10(7) cells/3 g of NWPF (non-woven polyester fibre) discs and 250 microg/ml respectively. This yield gradually decreased to 0.55x10(6) cells/3 g of packaging material and 60 microg/ml respectively at the end of the batch culture. In the continuous-culture studies, the batch culture was initially operated for 64.5 h and then continuous flow was started at the dilution rates of 0.15, 0.2, 0.25 and 0.3 day-1 and finally stabilized at 0.25 day-1 within 288 h (12 days). The MAb concentration at steady state was found to be 116-120 microg/day per ml, and the yield of operation was 62.5 mg/day per ml, which was 3.5 times higher than that of batch culture. In conclusion, a packed-bed bioreactor with the support matrix Fibra-Cel, operated in continuous-feeding mode, is more efficient for large-scale MAb production than a batch culture. On the other hand, by using a continuous-culture system, a better supply of nutrients and removal of inhibitory metabolites and proteolytic enzymes was obtained. PMID:15506916

  2. Production of a macromomycin (MCR-monoclonal antibody conjugate and its biological activity.

    Manabe,Yuichi

    1984-04-01

    Full Text Available Macromomycin (MCR, an unique membrane-reactive anticancer antibiotic, was incubated with murine monoclonal anti-HLA IgG1 antibody (H-1 in the presence of carbodiimide. The resulting mixture was fractionated with a Sephadex G-200 column. The first and second fractions were shown to contain MCR-(H-1 conjugate by the elution profile, as well as by the Sarcina lutea growth inhibition assay and Ouchterlony double-diffusion method. A membrane immunofluorescence test with anti-MCR and anti-mouse IgG antibodies demonstrated specific localization of MCR-(H-1 on the surface of HLA-bearing NALL -1 cells. MCR-(H-1 inhibited the growth of HLA-lacking NS-1 cells statistically less effectively than MCR alone (p less than 0.01. On the other hand, the conjugate and free MCR equally inhibited the growth and 3H-TdR incorporation of HLA-bearing NALL -1 cells. These results indicate that the antibody-bound MCR retained both MCR and antibody activities, and thus exerted antibody-targeting MCR cytotoxicity in vitro.

  3. Evaluation of Hollow Fiber And Miniperm Bioreactors as An Alternative to Murine Ascites for Small Scale Monoclonal Antibody Production

    The objective of this study was to compare monoclonal antibody production in hollow fiber, miniPERM bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice. One hybridoma cell line was grown in hollow fiber, miniPERM bioreactor systems and in groups of 5 mice. Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1X107 hybridoma cells. Each mouse was tapped a maximum of three times for collection of ascites. Bioreactors were harvested three times weekly for 30 days and were monitored by cell counts, cell viability and media consumption. Time and materials logs were maintained. The total quantity of monoclonal antibody produced in 5 mice versus the total production for the two different bioreactors (hollow fiber and miniPERM) in 30 days was as follows: cell line 2AC10E6C7 produce 158 mg vs.97.5 mg; vs 21.54 mg respectively. Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, from 0.71 to 3.8 mg/ml in hollow fiber bioreactor system, and from 0.035 to 1.06 in miniPERM. Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber and miniPERM bioreactor systems merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (< 1 g) monoclonal antibody production.

  4. Rapid transient production in plants by replicating and non-replicating vectors yields high quality functional anti-HIV antibody.

    Frank Sainsbury

    Full Text Available The capacity of plants and plant cells to produce large amounts of recombinant protein has been well established. Due to advantages in terms of speed and yield, attention has recently turned towards the use of transient expression systems, including viral vectors, to produce proteins of pharmaceutical interest in plants. However, the effects of such high level expression from viral vectors and concomitant effects on host cells may affect the quality of the recombinant product.To assess the quality of antibodies transiently expressed to high levels in plants, we have expressed and characterised the human anti-HIV monoclonal antibody, 2G12, using both replicating and non-replicating systems based on deleted versions of Cowpea mosaic virus (CPMV RNA-2. The highest yield (approximately 100 mg/kg wet weight leaf tissue of affinity purified 2G12 was obtained when the non-replicating CPMV-HT system was used and the antibody was retained in the endoplasmic reticulum (ER. Glycan analysis by mass-spectrometry showed that the glycosylation pattern was determined exclusively by whether the antibody was retained in the ER and did not depend on whether a replicating or non-replicating system was used. Characterisation of the binding and neutralisation properties of all the purified 2G12 variants from plants showed that these were generally similar to those of the Chinese hamster ovary (CHO cell-produced 2G12.Overall, the results demonstrate that replicating and non-replicating CPMV-based vectors are able to direct the production of a recombinant IgG similar in activity to the CHO-produced control. Thus, a complex recombinant protein was produced with no apparent effect on its biochemical properties using either high-level expression or viral replication. The speed with which a recombinant pharmaceutical with excellent biochemical characteristics can be produced transiently in plants makes CPMV-based expression vectors an attractive option for

  5. Evaluation of hollow fiber and mini perm bioreactors as an alternative to murine ascites for small scale monoclonal antibody production

    The objective of this study was to compare monoclonal antibody production in hollow fiber, mini perm bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice. One hybridoma cell line was grown in hollow fiber, mini perm bioreactor systems and in groups of 5 mice. Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1x107 hybridoma cells. Each mouse was tapped a maximum of three times for collection of ascites. Bioreactors were harvested three times weekly for 30 days and were monitored by cell counts, cell viability and media consumption. Time and materials logs were maintained. The total quantity of monoclonal antibody produced in 5 mice versus the total production for the two different bioreactors (hollow fiber and mini perm) in 30 days was as follows: cell line 2AC10E6C7 produce 158 mg vs.97.5 mg, vs 21.54 mg respectively. Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, from 0.71 to 3.8 mg/ml in hollow fiber bioreactor system, and from 0.035 to 1.06 in mini perm. Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber and mini perm bioreactor systems merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (<1mg) monoclonal antibody production.(Author)

  6. Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

    Lilian Terezinha de Queiroz Leite

    1996-10-01

    Full Text Available Monoclonal antibodies (MABs ivere produced against an etbylenediaminetetraacetate (EDTA extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b. The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.

  7. CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production.

    Khairnar, Vishal; Duhan, Vikas; Maney, Sathish Kumar; Honke, Nadine; Shaabani, Namir; Pandyra, Aleksandra A; Seifert, Marc; Pozdeev, Vitaly; Xu, Haifeng C; Sharma, Piyush; Baldin, Fabian; Marquardsen, Florian; Merches, Katja; Lang, Elisabeth; Kirschning, Carsten; Westendorf, Astrid M; Häussinger, Dieter; Lang, Florian; Dittmer, Ulf; Küppers, Ralf; Recher, Mike; Hardt, Cornelia; Scheffrahn, Inka; Beauchemin, Nicole; Göthert, Joachim R; Singer, Bernhard B; Lang, Philipp A; Lang, Karl S

    2015-01-01

    B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1(-/-) mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1(-/-) mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses. PMID:25692415

  8. CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production

    Khairnar, Vishal; Duhan, Vikas; Maney, Sathish Kumar; Honke, Nadine; Shaabani, Namir; Pandyra, Aleksandra A.; Seifert, Marc; Pozdeev, Vitaly; Xu, Haifeng C.; Sharma, Piyush; Baldin, Fabian; Marquardsen, Florian; Merches, Katja; Lang, Elisabeth; Kirschning, Carsten; Westendorf, Astrid M.; Häussinger, Dieter; Lang, Florian; Dittmer, Ulf; Küppers, Ralf; Recher, Mike; Hardt, Cornelia; Scheffrahn, Inka; Beauchemin, Nicole; Göthert, Joachim R.; Singer, Bernhard B.; Lang, Philipp A.; Lang, Karl S.

    2015-01-01

    B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1−/− mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1−/− mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses. PMID:25692415

  9. Isolation and purification of G immunoglobulin from guinea-pig for the production of a second antibody for radioimmunoassay

    The IgG of guinea-pig was isolated and purified by precipitation with caprylic acid and the batch absorption with DEAE-cellulose. The efficiency of the operating was verified by the determination of total proteins, during the purification stages. The purity of the end product was proved by immuno electrophoresis face to rabbit serum total antiserum of guinea-pig. it was obtained 240 mg of purity IgG to be used in the production of second specific antibody for radioimmunoassay. (C.G.C.)

  10. Isolation and purification of rabbit imunoglobulin (IgG) for the production of a second antibody for radioimunoassay

    Immunoglobulin (IgG) from rabbit serum was isolated and purified by sodium sulphate precipitation followed by ion exchange chromatography on DEAE-cellulose. The efficiency of the procedure was followed by total protein determination during all purification steps. The purity of the final product was verified through immunoelectroforesis of IgG with sheep serum anti-rabbit whole serum. Were obtained 850 mg of pure IgG, enough for the immunization of several sheeps to be used in the production of a second antibody for radioimmunoassay. (author)

  11. Production and characterization of a peptide-based monoclonal antibody against CD44 variant 6.

    Zarei, Saeed; Bayat, Ali Ahmad; Hadavi, Reza; Mahmoudi, Ahmad R; Tavangar, Banafsheh; Vojgani, Yasaman; Jeddi-Tehrani, Mahmood; Amirghofran, Zahra

    2015-02-01

    The gene that codes for the CD44 family members consists of 20 exons, nine of which encode the standard form of the molecule. The other exons can be inserted in various combinations into the membrane proximal region of the extracellular domain of the protein, giving rise to variant isoforms (CD44v). CD44 variants, especially the CD44v6, have been reported to regulate tumor invasion, progression, and metastasis of carcinomas. Producing a high affinity monoclonal antibody against human CD44v6 provides a powerful tool to monitor and trace CD44v6 function in different biological fluids. In this study, a synthetic peptide from CD44v6 was conjugated to keyhole limpet hemocyanin (KLH) and injected into BALB/c mice. Splenocytes from the immunized mice were fused with murine SP2/0 myeloma cells followed by selection of antibody producing hybridoma cells. After screening of hybridoma colonies by ELISA, high affinity antibodies were selected and purified by affinity chromatography. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibodies. Six stable hybridoma cell lines, designated as 1H1, 1H2, 2A12, 2G11, 3H3, and 3H7, were obtained. Flow cytometry and immunocytochemistry results showed that the new monoclonal antibodies recognized CD44v6 on the cell surface. This novel panel of anti-CD44v6 antibodies has the potential for investigating the role of CD44v6 in cancer pathogenesis. PMID:25723282

  12. Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

    Jiang, Guoying; Yu, Christopher; Yadav, Daniela B; Hu, Zhilan; Amurao, Annamarie; Duenas, Eileen; Wong, Marc; Iverson, Mark; Zheng, Kai; Lam, Xanthe; Chen, Jia; Vega, Roxanne; Ulufatu, Sheila; Leddy, Cecilia; Davis, Helen; Shen, Amy; Wong, Pin Y; Harris, Reed; Wang, Y John; Li, Dongwei

    2016-07-01

    Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes. PMID:27262204

  13. Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

    ESPÍNDOLA Noeli M.

    2000-01-01

    Full Text Available We describe the production of the potential monoclonal antibodies (MoAbs using BALB/c mice immunized with vesicular fluid (VF-Tcra (T. crassiceps antigen. Immune sera presented anti-VF-Tcra (<20kD IgG and IgM antibodies with cross-reactivity with T. solium (Tso antigen (8-12, 14, and 18 kD. After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.

  14. Korean Red Ginseng Extract Enhances the Anticancer Effects of Imatinib Mesylate Through Abrogation p38 and STAT5 Activation in KBM-5 Cells.

    Jung, Sang Yoon; Kim, Chulwon; Kim, Wan-Seok; Lee, Seok-Geun; Lee, Jun-Hee; Shim, Bum Sang; Kim, Sung-Hoon; Ahn, Kyoo Seok; Ahn, Kwang Seok

    2015-07-01

    Although imatinib mesylate (IM) in the treatment of chronic myelogenous leukemia (CML) remains the best example of successful targeted therapy, majority of patients with CML suffer its toxicity profile and develop chemoresistance to existing therapeutic agents. Thus, there is a need to develop novel alternative therapies for the treatment of CML. Here, we investigated whether Korean red ginseng extract (KRGE) could suppress the proliferation and induce chemosensitization in human CML cells. Also, we used a human phospho-antibody array containing 46 antibodies against signaling molecules to examine a subset of phosphorylation events after treatment. Korean red ginseng extract broadly suppressed the proliferation of five different cell lines, but KRGE was found to be the most potent inducer of apoptosis against KBM-5 cells. It also abrogated the expression of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), survivin, inhibitors of apoptosis protein 1/2, COX-2 (Cyclooxygenase-2), cyclin D1, matrix metalloproteinase-9, and VEGF (Vascular endothelial growth factor), as well as upregulated the expression of pro-apoptotic gene products. Interestingly, KRGE also enhanced the cytotoxic and apoptotic effect of IM in KBM-5 cells. The combination treatment of KRGE and IM caused pronounced suppression of p38 and signal transducer and activator of transcription 5 phosphorylation and induced phosphorylation of p53 compared with the individual treatment. Our results demonstrate that KRGE can enhance the anticancer activity of IM and may have a substantial potential in the treatment of CML. PMID:25857479

  15. Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus

    One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA),Western blotting and fluorescence-activated cell sorter (FACS), mAb 3...

  16. Study on the preparation of antibody coated tubes for radioimmunoassay kit production

    The polystyrene tubes are coated with T3/ T4 antibodies by γ-globulin, second antibody and specific antibodies. They are immobilized on the solid at a suitably dilution and incubation for 24 h, pH 9.6. The variation of the binding capacity values (obtained for 10 consecutive preparations) was less than 10%. NSB <3%, Binding 30-50%. Using dried tubes coated either with anti-T3 or anti-T4 antibody according to the developed coating approach for the determination of total T3 and total T4 in human serum. The recovery of T3 was found to be between 85.5% and 104% while the recovery of T4 ranged between 90.9% and 119%. The cross-reactivity for T4 in the T3 assay was 0.22%. Both assays were sensitive, the detection limit of the RIA for total T3 assay was 0.15 ng/ml while the detection limit of the RIA for total T4 assay was 5 ng/ml. (author)

  17. Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

    Four murine monoclonal antibodies against rat platelets were produced by fusion of spleen cells from mice intravenously immunized with whole rat platelets. All four antibodies immunoprecipitated two major platelet membrane proteins with apparent molecular weights of 130,000 and 82,000 (nonreduced) and of 120,000 and 98,000 (reduced), which were structurally analogous to human glycoprotein (GP) IIb/IIIa, i.e. rat GPIIb/IIIa. Two of four antibodies, named P9 and P55, strongly inhibited adenosine diphosphate (ADP)-induced aggregation of washed rat platelets and caused approximately 50% inhibition of human fibrinogen binding to ADP-stimulated rat platelets, suggesting that rat GPIIb/IIIa serves as a fibrinogen receptor in ADP-induced aggregation. In contrast, two other antibodies, named P14 and P34, themselves caused aggregation of rat platelets in platelet-rich plasma (PRP) and the secretion of 14C-serotonin from 14C-serotonin-labeled PRP. These results indicate that rat GPIIb/IIIa plays an important role in platelet aggregation

  18. Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum.

    Shkoporov, A N; Khokhlova, E V; Savochkin, K A; Kafarskaia, L I; Efimov, B A

    2015-06-01

    Bifidobacteria constitute a significant part of healthy intestinal microbiota in adults and infants and present a promising platform for construction of genetically modified probiotic agents for treatment of gastrointestinal disorders. In this study, three strains of Bifidobacterium longum were constructed that express and secrete biologically active single-chain antibodies against human TNF-α and Clostridium difficile exotoxin A. Anti-TNF-α scFv antibody D2E7 was produced at the level of 25 μg L(-1) in broth culture and was mostly retained in the cytoplasm, while VHH-type antibodies A20.1 and A26.8 against C. difficile exotoxin A were produced at the levels of 0.3-1 mg L(-1) and secreted very efficiently. The biological activity of both antibody types was demonstrated in the mammalian cell-based assays. Expression of A20.1 and A26.8 was also observed in vivo after intragastric administration of transformed B. longum strains to (C57/BL6 × DBA/2)F1 mice. The obtained B. longum strains may serve as prototypes for construction of novel probiotic medications against inflammatory bowel disease and C. difficile-associated disease. PMID:25994292

  19. Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

    Rong LI; Dong-sheng XIONG; Xiao-feng SHAO; Jia LIU; Yuan-fu XU; Yuan-sheng XU; Han-zhi LIU; Zhen-ping ZHU; Chun-zheng YANG

    2004-01-01

    AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domainⅢ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain Ⅲ (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial ceils.RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. Ycom1D3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

  20. Interval between first dose and booster affected antibody production in cattle vaccinated against rabies

    A. Albas

    2006-01-01

    Full Text Available In this study, we compared the levels of neutralizing antibodies induced by inactivated rabies vaccine in cattle by using three alternative immunization procedures. Forty-five bovines (breed nelore were then organized in three groups (A, B and C, with 15 animals/group. Group A received only one vaccine dose at day zero and Group B received the first dose at day zero and then another dose at day 30 (early booster. Group C was also immunized with two doses; however, the booster was postponed until day 180 after the first dose (delayed booster. Blood samples were withdrawn at days zero (before the first dose and 30, 210, 390, and 540 after the beginning of immunization and the antibody titers were evaluated by mouse neutralization test. The protocol used to immunize Group C (booster at day 180 was clearly more efficient. In this group, antibody levels were higher and also remained higher for longer periods in comparison with the other two groups. These results show that booster timing significantly affected antibody levels. Therefore, programs addressed to control this disease in cattle should consider not only the use of a booster but also its administration time.

  1. Immune antibodies and helminth products promote CXCR2-dependent repair of parasite-induced injury

    Helminth parasites cause massive damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination. We observed that mice lacking antibodies (AID-/-) or activating Fc receptors (FcR'-/-) displayed impaired intestinal repair followi...

  2. Effects of Inula Britannica on the production of antibodies and cytokines and on T cell differentiation in C57BL/6 mice immunized by ovalbumin.

    Song, Qing-Hua; Kobayashi, Takao; Hong, Tie; Cyong, Jong-Chol

    2002-01-01

    In this study, we investigated the effects of Inula Britannica on the production of antibodies against ovalbumin, and the differentiation of T cells, in C57BL/6 mice. The oral administration of Inula Britannica suppressed IL-4 and IL-6 production in lymphocytes collected from an inguinal lymph node in the immunized leg. On the other hand, the intraperitoneal administration of Inula Britannica suppressed IgG1 production, the ratio of IFN-gamma+IL-4-/IFN-gamma-IL-4+ cells and cytokine production of IL-6. It was presumed that the effects of Inula Britannica on the production of antibodies were induced by regulation of the balance of Th1 and Th2. Further, IL-4 and IL-6 production by lymphocytes collected from an inguinal lymph node in the immunized leg were suppressed, and therefore production of antibodies was suppressed. PMID:12230018

  3. Xenohybridization of Epstein-Barr virus-transformed cells for the production of human monoclonal antibodies.

    Tiebout, R F; Stricker, E A; Oosterhof, F; van Heemstra, D J; Zeijlemaker, W P

    1985-12-01

    Transformation of human B lymphocytes, obtained from hyperimmune donors with Epstein-Barr virus, yields polyclonal cell populations in which a minority of cells produce IgG antibodies of predetermined specificity, whereas the majority of cells produce 'non-specific' immunoglobulin (mainly of the IgM class). Such lymphoblastoid cell lines can be easily propagated in high-density cultures. Because cloning at 1 cell per well is not possible, stabilization of lymphoblastoid cell lines by limiting dilution is not feasible and most newly established lines cease to produce specific antibody within a few weeks. Xenohybrids, resulting from fusion of Epstein-Barr virus-transformed cells with NS1 mouse plasmacytoma cells, can be cloned at 1 cell per well. Stable xenohybridoma subclones, producing antibody of the desired specificity, can be isolated after a series of limiting dilutions. In a model system, we have studied the efficiency of xenohybridization of human lymphoblastoid cells. Using this system, we have constructed IgG anti-tetanus-toxoid- and IgG anti-HBsAg-producing cell lines. Next, we investigated whether transformation with Epstein-Barr virus is essential in such a two-step procedure or whether a polyclonal stimulator, such as pokeweed mitogen, could also be used. It was found that antibody-producing xenohybrids can be obtained after stimulation with pokeweed mitogen. However, this latter system is subject to more variations and lacks the advantage of pre-selection of antibody-producing cells as compared to xenohybridization after transformation. PMID:3003887

  4. Stable, continuous large-scale production of human monoclonal HIV-1 antibody using a computer-controlled pilot plant.

    Unterluggauer, F; Doblhoff-Dier, O; Tauer, C; Jungbauer, A; Gaida, T; Reiter, M; Schmatz, C; Zach, N; Katinger, H

    1994-01-01

    A completely automated pilot plant used for fermentation has been employed with direct digital control (DDC) technology for monitoring and regulating growth of human cells. A human hybridoma cell line (3D6) producing anti-human immunodeficiency virus (HIV)-1 antibodies was used as a model for large-scale production (300-liter airlift fermentor) in continuous culture. Parameters controlled were pH, dissolved oxygen, temperature and the flow rate of four gases used in the process. A control strategy was implemented to achieve constant fluid velocity and mixing by maintaining the rate of gas flow at a constant level. Another advantage of this approach was that the total gas flow required for optimal fluid circulation was reduced from 1 volume gas/volume fermenter/hour (vvh) to 0.3 vvh. Use of a low flow rate eliminated the serious problems of foaming, which contributed significantly to cell destruction, shorter filter-life and other considerations. Dilution rate was optimized at laboratory scale for maximum productivity, which results in relatively low viability. At a dilution rate of 0.0076 h-1, a total cell density of 6-7 x 10(5) cells/ml with a viability of approximately 75% was maintained during long-term continuous cultivation. These growth conditions resulted in a product titer stabilized in the range of 35 micrograms IgG/ml. Batchwise purification was achieved with a recovery of more than 50% and a final purification of active monoclonal antibody representing about 99% product. Results from isoelectric focusing and Western blotting demonstrated batch-to-batch consistency of the purified human monoclonal antibody to HIV-1 during the continuous growth process.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8136128

  5. Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

    Andrabi Raiees

    2012-09-01

    Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

  6. Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp

    Tossapon WONGTANGPRASERT; Wirongrong NATAKUATHUNG; Umaporn PIMPITAK; Anumart BUAKEAW; Tanapat PALAGA; Kittinan KOMOLPIS; Nanthika KHONGCHAREONPORN

    2014-01-01

    A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cellfusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50%inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118%for an intra-assay and 96%-113%for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9%and 5.5%-14.9%, respectively.%本文题目:抗土霉素单克隆抗体的制备及其在虾中土霉素残留的检测应用Production of a monoclonal antibody against oxytetracycline and its application for oxy-tetracycline residue detection in shrimp研究目的:抗土霉素单克隆抗体的制备和特性分析,并用于间接竞争酶联免疫吸附测定法(icELISA)分析虾样品中土霉素的残留。创新要点:本研究建立分泌抗土霉素单克隆抗体的杂交瘤细胞株,筛选出对虾中土霉素残留检测具有较高灵敏度的单克隆抗体2-4F。研究方法:用细胞杂交技术使土霉素-牛血清白蛋白偶联物免疫的BALC/c雌性小鼠的脾细胞与骨髓瘤细胞融合,建立三株分泌抗土霉素单克隆抗体的杂交瘤细胞株(2-4F、7-3G和11-11A),并制备它们的单克隆抗体。通过 icELISA 法分析单克隆抗体对土霉素的半抑制质量浓度(IC50)和交叉反

  7. The use of monoclonal antibodies for the characterization and production of Mycobacterium leprae antigens

    J. Ivanyi

    1987-01-01

    Full Text Available Similar immunizations of mice and hybridoma technology were used by several investigators to raise monoclonal antibodies which identified a limited range of epitopes and antigenic molecules. Further studies would have the scope for revealing yet more novel structures. The existing MABs are agreed standard reagents, avaiable to investigators and valuable for several applications. At least six epitopes specific for M. leprae were defined in molecular terms. Monoclonal antibody based immunoassays proved to be invaluable for the screening of recombinant DNA clones and for the topographic study of individual epitopes. Purification of antigens using affinity chromatography requires further development of techniques whilst serology of leprosy is open for clinical and epidemiological evaluation.

  8. Anti-enrofloxacin Antibody Production by Using Enrofloxacin-screened HSA as an Immunogen

    LIU Chune; LIN Hong; CAO Limin; JIANG Jie

    2005-01-01

    A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating enrofloxacin (EF) to human serum albumin (HSA). The derived conjugate was characterized by UV spectrum and then used for immunization of BALB/C mice. In enzyme-linked immunosorbent assay (ELISA) and competitive inhibition ELISA experiments, the derived antiserum exhibited high antibody titer (greater than 1: 250 000) as well as varied cross-reactivity (from 97.8% to 161.7%) to three analogs of EF belonging to fluoroquinolones family. But over the concentration range studied, no significant cross-reactivity was observed to other group of antibiotics (chloramphenicol, oxytetracycline, sulphamethoxazole and nysfungin). It was confirmed that the synthesized immunogen was highly antigenic and elicited specific antibody responses in BALB/C mice against EF.

  9. [Antibody production in the blood of donors immunized with staphylococcal anatoxin].

    Dyzyk, H M; Shumeha, I S; Tarasenko, A O; Patoka, V V

    1998-01-01

    Blood serum content was studied of specific antistaphylococcal antibodies (staphylolysins) in 576 donors immunized with staphylococcal anatoxin with the purpose of obtaining an antistaphylococcal plasma and antistaphylococcal immunoglobulin to be used in clinical settings. 292 donors had been immunized and examined prior to 1986, 284--after 1986 (before 1994). Comparison of the immune responses in the above periods of time permitted finding out that 13.03% of immunized donors responded to the antigenic stimulus by such paradoxical reaction as disappearance of specific antibodies; the number of persons-active respondents has gotten reduced from 17.12% to 5.98% as has the number of individuals having the baseline level of staphylolysins (1-2 ME/ml). The changes were at their greatest in donors with group A (II) blood. PMID:9621632

  10. Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb

    ZHANG Qi; LI Tie-jun; ZHU Xiao-xia; XU Li-na; LIU Feng-quan; HU Bai-shi; JIANG Ying-hua; CAO Bin

    2007-01-01

    The hapten, 3-{[1-(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 1H-nuclear magnetic resonance spectrometry (1H-NMR). Then,HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimide/dicyclohexylcarbodimide (NHS/DCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAb01 reached 1.28 × 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAb01 with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.

  11. Production and characterisation of monoclonal antibodies specific for Escherichia coli O157:H7.

    Luciani, M; Armillotta, G; Magliulo, M; Portanti, O; Di Febo, T; Di Giannatale, E; Roda, A; Lelli, R

    2006-01-01

    Seven monoclonal antibodies (MAbs) specific for Escherichia coli O157:H7, one of the major causes of haemorrhagic colitis in humans, were produced by immunising Balb/c mice with the strain E. coli O157:H7. These monoclonal antibodies do not cross-react with other bacteria such as Salmonella enterica serovar Typhimurium, E. coli O14, E. coli JM109, S. enterica serovar Enteritidis, S. panama, S. saintpaul, S. derby, S. muenchen, S. bredeney, S. hadar, Yersinia enterocolitica, Proteus vulgaris, Shigella flexneri, Listeria ivanovii, L. monocytogenes 13M, L. innocua, Enterobacter cloacae, E. agglomerans, E. amnigenus, Citrobacter freundii, Escherichia fergussoni or Klebsiella pneumoniae. Of the seven MAbs obtained, MAb 8B8C3 was selected to prepare a high-sensitivity sandwich ELISA method specific for O157:H7. PMID:20429059

  12. Optimization of microchip-based electrophoresis for monoclonal antibody product quality analysis revealed needs for extra surfactants during denaturation.

    Cai, Hui; Song, Yuanli; Zhang, Jian; Shi, Ting; Fu, Ya; Li, Rong; Mussa, Nesredin; Li, Zheng Jian

    2016-02-20

    Microchip-based electrophoresis has gained increasing popularity in biopharmaceutical development and testing laboratories because of its automation and rapid analysis capabilities. One application of microchip-based electrophoresis is the assessment of size-based variants for product purity analysis. However, monoclonal antibodies analyzed by this technique sometimes exhibited different electrophoretic behaviors. In this study, when three IgG1 and five IgG4 were analyzed using microchip-based electrophoresis under reducing conditions, one of the IgG1s, denoted as mAb1, exhibited an atypical profile attributed to its specific heterogeneity resulting in separation of its heavy chain into two main species. During investigation of the atypical profile, several parameters that were critical to optimal resolution were evaluated, and the data pointed toward incomplete denaturation of mAb1 due to lack of sufficient surfactant in the vendor provided sample buffer (0.7% surfactant). Denaturation studies demonstrated that, although typical antibody profiles could be achieved at 0.7% surfactant for most antibodies analyzed, five out of eight antibodies were not fully denatured until the surfactant concentration reached 0.9% or higher, and mAb1 required a surfactant concentration of 1.3% for complete denaturation. Molecular modeling analysis revealed features in surface charge, hydrophobicity, and structure from mAb1 that led to its unique surfactant concentration-dependent electrophoretic behaviors observed. The optimized method was further evaluated for specificity, linearity, precision, and limit of quantitation for mAb1, and compared with that of conventional CE-SDS. PMID:26704629

  13. Expression Enhancement in Trastuzumab Therapeutic Monoclonal Antibody Production using Genomic Amplification with Methotrexate

    Akbarzadeh-Sharbaf, Soudabeh; Yakhchali, Bagher; Minuchehr, Zarrin; Shokrgozar, Mohammad Ali; Zeinali, Sirous

    2013-01-01

    Background Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. Methods According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were sele...

  14. Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus.

    Ste-Marie, L; Sénéchal, S; Boushira, M; Garzon, S.; Strykowski, H; Pedneault, L; de Repentigny, L

    1990-01-01

    Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with ma...

  15. Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.

    Hattori, S; Clanton, D J; Satoh, T.; Nakamura, S.; Kaziro, Y; Kawakita, M; Shih, T Y

    1987-01-01

    The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

  16. A trade-off between natural and acquired antibody production in a reptile: implications for long-term resistance to disease

    Franziska C. Sandmeier

    2012-08-01

    Vertebrate immune systems are understood to be complex and dynamic, with trade-offs among different physiological components (e.g., innate and adaptive immunity within individuals and among taxonomic lineages. Desert tortoises (Gopherus agassizii immunised with ovalbumin (OVA showed a clear trade-off between levels of natural antibodies (NAbs; innate immune function and the production of acquired antibodies (adaptive immune function. Once initiated, acquired antibody responses included a long-term elevation in antibodies persisting for more than one year. The occurrence of either (a high levels of NAbs or (b long-term elevations of acquired antibodies in individual tortoises suggests that long-term humoral resistance to pathogens may be especially important in this species, as well as in other vertebrates with slow metabolic rates, concomitantly slow primary adaptive immune responses, and long life-spans.

  17. Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle.

    Dye, John M; Wu, Hua; Hooper, Jay W; Khurana, Surender; Kuehne, Ana I; Coyle, Elizabeth M; Ortiz, Ramon A; Fuentes, Sandra; Herbert, Andrew S; Golding, Hana; Bakken, Russell A; Brannan, Jennifer M; Kwilas, Steve A; Sullivan, Eddie J; Luke, Thomas C; Smith, Gale; Glenn, Gregory; Li, Wenfang; Ye, Ling; Yang, Chinglai; Compans, Richard W; Tripp, Ralph A; Jiao, Jin-An

    2016-01-01

    Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities. PMID:27109916

  18. Thyroid Antibodies

    ... be limited. Home Visit Global Sites Search Help? Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  19. Long-term Continuous Production of Monoclonal Antibody by Hybridoma Cells Immobilized in a Fibrous-Bed Bioreactor

    Zhu, Hui; Yang, Shang-Tian

    2004-01-01

    The kinetics and long-term stability of continuous production of monoclonal antibody IgG2b by hybridoma HD-24 cells immobilized in a fibrous-bed bioreactor (FBB) were studied for a period of ∼8 months. The cells were immobilized in the fibrous bed by surface attachment of cells and entrapment of large cell clumps in the void space of the fibrous matrix. A high viable cell density of 1.01 × 108/ml was attained in the bioreactor, which was about 63 times higher than those in conventional T-flas...

  20. High Production of IL-18 by Dendritic Cells Induced by Sera from Patients with Primary Antibody Deficiency

    Maryam Nourizadeh

    2007-06-01

    Full Text Available Predominantly antibody deficiencies are a category of primary immunodeficiency diseases, whichconsist of several rare disorders such as common variable immunodeficiency (CVID and X-linked agammaglobulinemia (XLA. We evaluated the effects of CVID and XLA patients’ sera as a source of microenviromental factors on maturation and function of monocyte-derived DCs.Blood was collected from 10 CVID and 5 XLA patients before immunoglobulin replacementtherapy and also from 8 healthy volunteers in order to obtain necessary sera for this study. Monocyte derived DCs were generated from blood cells obtained from healthy volunteers in the presence of GM-CSF, IL-4 and 10% serum concentrations from cases and controls. Immature DCs were incubated with monocyte conditioned medium (MCM and TNF-α in order to generate mature DCs. Interleukin 18 (IL-18 production by CD40L-activated mature DCs was measured after 24 hours of culture in vitro.IL-18 production by DCs generated in the presence of CVID and XLA patients’ sera were6.75±2.59 and 7.08±1.75 ng/ml, respectively, which were significantly higher than normal serumconditioned DCs (3.55±0.68 ng/ml.These results suggest that the sera of patients with predominantly antibody deficiencies maycontain soluble factor(s that can induce a significant increase in IL-18 production by DCs.

  1. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human

  2. The Art of Making Antibodies.

    Headon, Denis R.

    1986-01-01

    Provides background information for teachers on the nature and production of antibodies. Points out that the production of monoclonal antibodies blends the malignant with the beneficial to create a medical tool of exciting potential. (JN)

  3. Identifying bottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in Chinese hamster ovary cells

    Mason, Megan; Sweeney, Bernadette; Cain, Katharine; Stephens, Paul; Sharfstein, Susan T.

    2012-01-01

    The increasing demand for antibody-based therapeutics has emphasized the need for technologies to improve recombinant antibody titers from mammalian cell lines. Moreover, as antibody therapeutics address an increasing spectrum of indications, interest has increased in antibody engineering to improve affinity and biological activity. However, the cellular mechanisms that dictate expression and the relationships between antibody sequence and expression level remain poorly understood. Fundamenta...

  4. PRODUCTION AND PURIFICATION OF IgY ANTIBODIES AS A NOVEL TOOL TO PURIFY THE NR1 SUBUNIT OF NMDA RECEPTO

    Edgar Antonio Reyes Montaño

    2011-12-01

    Full Text Available Producing polyclonal antibodies (IgY inchickens has advantages over those obtainedin other animal models, since theyhave been used as a tool for studyingdifferent proteins (NMDA glutamate receptorin our case, specifically the NR1subunit. We produced specific antibodiesagainst expression products by thealternative splicing of the gene encodingNMDA receptor NR1 subunit in adult ratbrain. Three peptides corresponding tothe splicing sites (N1, C1 and C2’ cassetteswere designed, synthesised and usedindividually as antigens in hens. Specificimmunoglobulins were purified fromyolks. The antibodies were then used forpurifying the NMDA receptor NR1 subunitusing affinity chromatography couplingthe three antibodies to the support.R

  5. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

    Pleckaityte Milda

    2011-12-01

    Full Text Available Abstract Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY, the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the sc

  6. Production and characterization of monoclonal antibodies to the edta extract of Leptospira interrogans, serovar icterohaemorrhagiae

    Lilian Terezinha de Queiroz Leite

    1996-10-01

    Full Text Available Monoclonal antibodies (MABs ivere produced against an etbylenediaminetetraacetate (EDTA extract of Leptospira interrogans serovar icterohaemorrhagiae being characterized by gel precipitation as IgM and IgG (IgGl and IgG2b. The EDTA extract was detected as several bands by silver staining in SDS-PAGE. In the Western blot the bands around 20 KDa reacted with a monoclonal antibody, 47B4D6, and was oxidized by periodate and was not digested by pronase, suggesting that the determinant is of carbohydrate nature, lmmunocytochemistry, using colloidal gold labeling, showed that an EDTA extract determinant recognized by monoclonal antibody 47B4D6, is localized under the outer envelope of serovar icterohaemorrhagiae. Hoe AIAB raised against the EDTA extract was not able to protect hamsters from lethal challenge with virulent homologous leptospires.Anticorpos monoclonais (AcM foram produzidos contra o extrato EDTA obtido de Leptospira interrogans, sorovar icterohaemorrhagiae. Pelo teste de precipitação foram caracterizados como IgM e IgG (IgGl e IgG2. A eletroforese em gel de poliacrilamida do extrato EDTA revelou diversas bandas quando corada pela prata. No "Western blot", as bandas em torno de 20 kDa reagiram com o AcM 47B4D6, foram oxidadas pelo periodato e não digeridas pela pronase, sugerindo que o determinante é de natureza carboidrato. O determinante reconhecido pelo AcM 47B4D6 estã localizado sob o envelope externo como revelado pela imunocitoquímica usando marcação com ouro coloidal. O AcM contra extrato EDTA do sorovar icterohaemorrahagiae não protegeu hamsters quando inoculados com lepstopira homóloga virulenta.

  7. Production and characterization of monoclonal antibodies to IgM of Pacific herring (Clupea pallasii)

    Purcell, Maureen K.; Bromage, Erin S.; Silva, Jessica; Hansen, John D.; Badil, Samantha M.; Woodson, James C.; Hershberger, Paul K.

    2012-01-01

    Pacific herring (Clupea pallasii) have a central role in the North Pacific ecosystem as a forage fish species and are natural reservoirs of several important finfish pathogens, including Viral hemorrhagic septicemia virus (VHSV). Here, we report the identification of the gene encoding the immunoglobulin mu (IgM) heavy chain, as well as the development and characterization of monoclonal antibodies (MAbs) that specifically react with Pacific herring IgM. Pacific herring immunoglobulin was purified and consisted of heavy and light chains of approximately 80 and 25 kDa. Three hybridoma clones were initially identified by ELISA as reactive with purified immunoglobulin but only one clone was able to detect an 80 kDa protein in Pacific and Atlantic herring (Clupea harengus) whole plasma by denaturing western blot. However, all three MAbs were able to precipitate an 80 kDa protein from Pacific herring and LCMS sequencing of peptide fragments derived from this protein matched the predicted amino acid sequence of the cloned, heavy chain gene. In addition, two of the MAbs stained cells within the putative lymphocyte gates for the spleen, anterior kidney and posterior kidney but were not reactive for myeloid/granulocyte gates, which is consistent with these MAbs reacting with surface IgM+ B-cells. To our knowledge, this is the first report of IgM-related gene sequences and anti-IgM monoclonal antibodies from any member of the family Clupeidae. The antibodies produced in this study are critical for achieving our long-term goal of conducting serological surveillance to assess pathogen exposure in natural populations of Pacific herring.

  8. Production and Characterization of a Peptide-based Monoclonal Antibody Against CD44 Variant 6

    ZAREI, Saeed; Bayat, Ali Ahmad; Hadavi, Reza; Mahmoudi, Ahmad R.; Tavangar, Banafsheh; VOJGANI, Yasaman; Jeddi-Tehrani, Mahmood; Amirghofran, Zahra

    2015-01-01

    The gene that codes for the CD44 family members consists of 20 exons, nine of which encode the standard form of the molecule. The other exons can be inserted in various combinations into the membrane proximal region of the extracellular domain of the protein, giving rise to variant isoforms (CD44v). CD44 variants, especially the CD44v6, have been reported to regulate tumor invasion, progression, and metastasis of carcinomas. Producing a high affinity monoclonal antibody against human CD44v6 p...

  9. Production and Characterization of Monoclonal Antibodies against Vegetative Cells of Bacillus cereus

    Charni, Nadine; Perissol, Claude; Le Petit, Jean; Rugani, Nathalie

    2000-01-01

    Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to dev...

  10. Interval between first dose and booster affected antibody production in cattle vaccinated against rabies

    A. Albas; O. L. Fontolan; P. E. Pardo; H. Bremer Neto; A. Sartori

    2006-01-01

    In this study, we compared the levels of neutralizing antibodies induced by inactivated rabies vaccine in cattle by using three alternative immunization procedures. Forty-five bovines (breed nelore) were then organized in three groups (A, B and C, with 15 animals/group). Group A received only one vaccine dose at day zero and Group B received the first dose at day zero and then another dose at day 30 (early booster). Group C was also immunized with two doses; however, the booster was postponed...