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Sample records for abrogates antibody production

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

    Science.gov (United States)

    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

    2016-01-01

    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

  2. A camelid anti-PrP antibody abrogates PrP replication in prion-permissive neuroblastoma cell lines.

    Directory of Open Access Journals (Sweden)

    Daryl Rhys Jones

    Full Text Available The development of antibodies effective in crossing the blood brain barrier (BBB, capable of accessing the cytosol of affected cells and with higher affinity for PrP(Sc would be of paramount importance in arresting disease progression in its late stage and treating individuals with prion diseases. Antibody-based therapy appears to be the most promising approach following the exciting report from White and colleagues, establishing the "proof-of-principle" for prion-immunotherapy. After passive transfer, anti-prion antibodies were shown to be very effective in curing peripheral but not central rodent prion disease, due to the fact that these anti-prion antibodies are relatively large molecules and cannot therefore cross the BBB. Here, we show that an anti-prion antibody derived from camel immunised with murine scrapie material adsorbed to immunomagnetic beads is able to prevent infection of susceptible N2a cells and cure chronically scrapie-infected neuroblastoma cultures. This antibody was also shown to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrP(C. In contrast, treatment with a conventional anti-prion antibody derived from mouse immunised with recombinant PrP protein was unable to prevent recurrence of PrP(Sc replication. Furthermore, our camelid antibody did not display any neurotoxic effects following treatment of susceptible N2a cells as evidenced by TUNEL staining. These findings demonstrate the potential use of anti-prion camelid antibodies for the treatment of prion and other related diseases via non-invasive means.

  3. Natural Mutations in Streptococcus agalactiae Resulting in Abrogation of β Antigen Production.

    Science.gov (United States)

    Vasilyeva, Anastasia; Santos Sanches, Ilda; Florindo, Carlos; Dmitriev, Alexander

    2015-01-01

    Streptococcus agalactiae genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of bac gene, coding for β antigen at transcriptional level. Inactivation of bgrR abrogated bac gene expression and increased virulence properties of S. agalactiae. In this study, a total of 140 strains were screened for the presence of bac gene, and the TCS bgrR and bgrS genes. A total of 53 strains carried the bac, bgrR and bgrS genes. Most of them (48 strains) expressed β antigen, while five strains did not express β antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of β antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of β antigen indicating the crucial regulatory role of TCS BgrRS. The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of S. agalactiae infection compared to parental strain A49V. In conclusion we showed that disruption of β antigen expression is associated with: i) insertion of ISSa4 upstream the bac gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the bac gene and a truncated form of β antigen; iii) single deletion (G) in position 439 of the bgrR gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in bgrR and bgrS genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of S. agalactiae gives a novel clue for understanding of S

  4. Natural Mutations in Streptococcus agalactiae Resulting in Abrogation of β Antigen Production

    Science.gov (United States)

    Vasilyeva, Anastasia; Santos Sanches, Ilda; Florindo, Carlos; Dmitriev, Alexander

    2015-01-01

    Streptococcus agalactiae genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of bac gene, coding for β antigen at transcriptional level. Inactivation of bgrR abrogated bac gene expression and increased virulence properties of S. agalactiae. In this study, a total of 140 strains were screened for the presence of bac gene, and the TCS bgrR and bgrS genes. A total of 53 strains carried the bac, bgrR and bgrS genes. Most of them (48 strains) expressed β antigen, while five strains did not express β antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of β antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of β antigen indicating the crucial regulatory role of TCS BgrRS. The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of S. agalactiae infection compared to parental strain A49V. In conclusion we showed that disruption of β antigen expression is associated with: i) insertion of ISSa4 upstream the bac gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the bac gene and a truncated form of β antigen; iii) single deletion (G) in position 439 of the bgrR gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in bgrR and bgrS genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of S. agalactiae gives a novel clue for understanding of S

  5. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the uni

  6. Production and Screening of Monoclonal Peptide Antibodies.

    Science.gov (United States)

    Trier, Nicole Hartwig; Mortensen, Anne; Schiolborg, Annette; Friis, Tina

    2015-01-01

    Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

  7. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    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 structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist...

  8. Enhanced Phagocytosis and Antibody Production by Tinospora ...

    African Journals Online (AJOL)

    SERVER

    2008-01-18

    Jan 18, 2008 ... antibody production through in vitro and in vivo studies. MATERIALS AND METHODS. Collection of plant .... and were fed orally to each animal for 14 days. After 14 days of .... Rubiadin: A new antioxidant from Rubia cardifolia.

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

    DEFF Research Database (Denmark)

    Ellebaek, Sofie; Pedersen, Susanne Brix; Grandal, Michael

    2016-01-01

    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 m...... 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...

  10. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

  11. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

    NARCIS (Netherlands)

    Joosten, V.; Lokman, C.; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2003-01-01

    In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and VHH) by these lower eukaryotes and the possible applications

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

    Energy Technology Data Exchange (ETDEWEB)

    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

    DEFF Research Database (Denmark)

    Ellebaek, Sofie; Pedersen, Susanne Brix; Grandal, Michael

    2016-01-01

    Abs is development of acquired resistance through mechanisms such as alterated 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....... 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......-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...

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

    Science.gov (United States)

    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.

  15. Production and purification of polyclonal antibody against bovine ...

    African Journals Online (AJOL)

    SERVER

    2007-06-18

    Jun 18, 2007 ... that ion-exchange chromatography could be an appropriate method for purification of IgG ... researches, polyclonal antibodies are routinely used as .... production and characterization of anti- human IgG monoclonal antibody ...

  16. Production and Purification of Polyclonal Antibodies.

    Science.gov (United States)

    Nakazawa, Masami; Mukumoto, Mari; Miyatake, Kazutaka

    2016-01-01

    Polyclonal antibodies consist of a mixture of antibodies produced by multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. Polyclonal antibodies raised against an antigen recognize multiple epitopes on a target molecule, which results in a signal amplification in indirect immunoassays including immune-electron microscopy. In this chapter, we present a basic procedure to generate polyclonal antibodies in rabbits.

  17. Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease.

    Science.gov (United States)

    Seid, Christopher A; Jones, Kathryn M; Pollet, Jeroen; Keegan, Brian; Hudspeth, Elissa; Hammond, Molly; Wei, Junfei; McAtee, C Patrick; Versteeg, Leroy; Gutierrez, Amanda; Liu, Zhuyun; Zhan, Bin; Respress, Jonathan L; Strych, Ulrich; Bottazzi, Maria Elena; Hotez, Peter J

    2017-03-04

    A therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.

  18. Inhibition of lanthanide nanocrystal-induced inflammasome activation in macrophages by a surface coating peptide through abrogation of ROS production and TRPM2-mediated Ca(2+) influx.

    Science.gov (United States)

    Yao, Han; Zhang, Yunjiao; Liu, Liu; Xu, Youcui; Liu, Xi; Lin, Jun; Zhou, Wei; Wei, Pengfei; Jin, Peipei; Wen, Long-Ping

    2016-11-01

    necessary for inflammasome activation, and this event was completely inhibited by RE-1 coating. We conclude from these studies that inhibition of ROS production, and the subsequent abrogation of TRPM2-mediated Ca(2+) influx, is the primary mechanism underlying RE-1's inhibitory effect on LNs-induced inflammasome activation. The ability of regulating the inflammatory response of nanocrystals through peptide surface coating may be of great value for in vivo applications of LNs and other engineered nanomaterials.

  19. Production of Monoclonal Antibody against Human Nestin.

    Science.gov (United States)

    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.

  20. Antibody Production in Plants and Green Algae.

    Science.gov (United States)

    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.

  1. Production and assay of forskolin antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ho, L.T.; Ho, R.J.

    1986-05-01

    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 /sup 3/H-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 /sup 3/H-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 IC/sub 50/ 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.

  2. 9 CFR 113.450 - General requirements for antibody products.

    Science.gov (United States)

    2010-01-01

    ... “monoclonal” shall be used. Example: “Duck Virus Hepatitis Antibody, Duck Origin.” (2) Bacterium-specific... “monoclonal” shall be used. Example: “Escherichia Coli Monoclonal Antibody, Murine Origin.” (3) Failure of... products. Retests shall be conducted as deemed necessary by the Administrator. (i) Before first use, horses...

  3. Polyclonal Antibody Production for Membrane Proteins via Genetic Immunization.

    Science.gov (United States)

    Hansen, Debra T; Robida, Mark D; Craciunescu, Felicia M; Loskutov, Andrey V; Dörner, Katerina; Rodenberry, John-Charles; Wang, Xiao; Olson, Tien L; Patel, Hetal; Fromme, Petra; Sykes, Kathryn F

    2016-02-24

    Antibodies are essential for structural determinations and functional studies of membrane proteins, but antibody generation is limited by the availability of properly-folded and purified antigen. We describe the first application of genetic immunization to a structurally diverse set of membrane proteins to show that immunization of mice with DNA alone produced antibodies against 71% (n = 17) of the bacterial and viral targets. Antibody production correlated with prior reports of target immunogenicity in host organisms, underscoring the efficiency of this DNA-gold micronanoplex approach. To generate each antigen for antibody characterization, we also developed a simple in vitro membrane protein expression and capture method. Antibody specificity was demonstrated upon identifying, for the first time, membrane-directed heterologous expression of the native sequences of the FopA and FTT1525 virulence determinants from the select agent Francisella tularensis SCHU S4. These approaches will accelerate future structural and functional investigations of therapeutically-relevant membrane proteins.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

    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....... Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated...

  6. Probiotics stimulate production of natural antibodies in chickens.

    Science.gov (United States)

    Haghighi, Hamid R; Gong, Jianhua; Gyles, Carlton L; Hayes, M Anthony; Zhou, Huaijun; Sanei, Babak; Chambers, James R; Sharif, Shayan

    2006-09-01

    Commensal bacteria in the intestine play an important role in the development of immune response. These bacteria interact with cells of the gut-associated lymphoid tissues (GALT). Among cells of the GALT, B-1 cells are of note. These cells are involved in the production of natural antibodies. In the present study, we determined whether manipulation of the intestinal microbiota by administration of probiotics, which we had previously shown to enhance specific systemic antibody response, could affect the development of natural antibodies in the intestines and sera of chickens. Our findings demonstrate that when 1-day-old chicks were treated with probiotics, serum and intestinal antibodies reactive to tetanus toxoid (TT) and Clostridium perfringens alpha-toxin in addition to intestinal immunoglobulin A (IgA) reactive to bovine serum albumin (BSA) were increased in unimmunized chickens. Moreover, IgG antibodies reactive to TT were increased in the intestines of probiotic-treated chickens compared to those of untreated controls. In serum, IgG and IgM reactive to TT and alpha-toxin were increased in probiotic-treated, unimmunized chickens compared to levels in untreated controls. However, no significant difference in serum levels of IgM or IgG response to BSA was observed. These results are suggestive of the induction of natural antibodies in probiotic-treated, unimmunized chickens. Elucidating the role of these antibodies in maintenance of the chicken immune system homeostasis and immune response to pathogens requires further investigation.

  7. Competency development in antibody production in cancer cell biology

    Energy Technology Data Exchange (ETDEWEB)

    Park, M.S.

    1998-12-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The main objective of this project was to develop a rapid recombinant antibody production technology. To achieve the objective, the authors employed (1) production of recombinant antigens that are important for cell cycle regulation and DNA repair, (2) immunization and specific selection of antibody-producing lymphocytes using the flow cytometry and magnetic bead capturing procedure, (3) construction of single chain antibody library, (4) development of recombinant vectors that target, express, and regulate the expression of intracellular antibodies, and (5) specific inhibition of tumor cell growth in tissue culture. The authors have accomplished (1) optimization of a selection procedure to isolate antigen-specific lymphocytes, (2) optimization of the construction of a single-chain antibody library, and (3) development of a new antibody expression vector for intracellular immunization. The future direction of this research is to continue to test the potential use of the intracellular immunization procedure as a tool to study functions of biological molecules and as an immuno-cancer therapy procedure to inhibit the growth of cancer cells.

  8. Cell culture processes for monoclonal antibody production.

    Science.gov (United States)

    Li, Feng; Vijayasankaran, Natarajan; Shen, Amy Yijuan; Kiss, Robert; Amanullah, Ashraf

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including 1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; 2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; 3) appropriate on-line and off-line sensors capable of providing information that enhances process knowledge; and 4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation that is compliant with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., engineering of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development.

  9. Cell culture processes for monoclonal antibody production

    Science.gov (United States)

    Li, Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; (2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; (3) appropriate on-line and off-line sensors capable of providing information that enhances process control; and (4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation and ensure compliance with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., generation of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development. PMID:20622510

  10. The effects of propolis on antibody production by laying hens.

    Science.gov (United States)

    Freitas, J A; Vanat, N; Pinheiro, J W; Balarin, M R S; Sforcin, J M; Venancio, E J

    2011-06-01

    Propolis is a honeybee product showing several biological properties that enhance the immune response, depending on the concentration and intake period. Because propolis possesses an immunomodulatory action on mammals, the objective of our study was to investigate the effects of propolis on the humoral immune response of laying hens by evaluating antibody production. Laying hens (ISA Brown) were divided into 5 groups with 7 birds each. Group 1 was a nonimmunized control, whereas birds in group 2 were immunized intravenously with SRBC, and those in groups 3, 4, and 5 were treated intraperitoneally with propolis (2, 10, and 50 mg/kg, respectively) on 3 consecutive days and then inoculated intravenously with SRBC. Hematological and serological analyses were carried out on d 0, 3, and 38. Natural and specific antibody levels were determined by hemagglutination with rabbit red blood cells and SRBC, respectively. Propolis-treated birds (50 mg/kg) showed a significant decline in heterophils and in the heterophil:lymphocyte ratio. After SRBC immunization, significant increases in levels of IgG were observed in groups 4 and 5. Furthermore, higher levels of natural antibodies were observed in propolis-treated laying hens. The administration of propolis to laying hens increased the production of IgG specific to SRBC and natural antibodies, and could be used to increase antigen-specific antibody responses to vaccines.

  11. Enhancement of antibody production to hepatitis B surface antigen by anti-idiotypic antibody.

    OpenAIRE

    Kakumu, S; Murase, K.; A Tsubouchi; Yoshioka, K.; Sakamoto, N.

    1986-01-01

    Studies were undertaken to determine whether anti-idiotypic antibody (anti-Id) against antibody to hepatitis B surface antigen (anti-HBs) could modulate in vitro anti-HBs production by human peripheral blood mononuclear cells stimulated with pokeweed mitogen. Peripheral blood mononuclear cells from patients positive for serum anti-HBs produced significantly increased amounts of anti-HBs by the addition of IgG fraction of anti-anti-HBs as well as purified HBsAg in a soluble form when compared ...

  12. Antibody production in early life supported by maternal lymphocyte factors.

    Science.gov (United States)

    Shimamura, Michio; Huang, Yi-Ying; Goji, Hiroshi

    2003-01-20

    To examine the influence of maternal lymphocyte factors on the immune responses in offspring in early life, antibody production in neonates born to either normal or lymphocyte-deficient mothers was analyzed. Recombination activating gene (Rag)-2(+/-) mouse neonates born to Rag-2(+/+), Rag-2(+/-)or Rag-2(-/-)mothers were injected with goat anti-mouse IgD antiserum, and IgE and IgG(1) production was evaluated. The levels of IgE and IgG(1) were higher in the pups born to Rag-2(+/+)and Rag-2(+/-) dams than to lymphocyte-deficient Rag-2(-/-) dams. The enhanced antibody production in the former compared with the latter neonates was also found following immunization with ovalbumin or TNP-Ficoll. Thus, the presence of maternal lymphocyte factors was suggested in neonates that augmented antigen-specific antibody production in both T cell-dependent and -independent pathways. A reduction in antibody production was observed in normal neonates when they were foster-nursed by Rag-2(-/-) mothers. Thus, the maternal lymphocyte factors enhancing the immune responses in newborns were shown to be present in breast-milk.

  13. Cell culture processes for monoclonal antibody production

    OpenAIRE

    LI Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert; Amanullah, Ashraf

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizin...

  14. Immunogenicity assessment of monoclonal antibody products: A simulated case study correlating antibody induction with clinical outcomes.

    Science.gov (United States)

    Knezevic, Ivana; Kang, Hye-Na; Thorpe, Robin

    2015-09-01

    Monoclonal antibodies are large molecules with complex structure and functions. They have a wide application for treatment of a broad range of chronic diseases and represent the largest class of biotherapeutic products. Given that biotherapeutic products may induce unwanted humoral and/or cellular immune responses in recipients, it is essential to investigate the immunogenicity of a product prior to licensure. The immune response is influenced by many factors and data generated in the pre-licensure studies are usually somewhat difficult for regulatory review. The knowledge and expertise required for this requires a thorough understanding of animal and human immunology as well as specific product characteristics, including mechanism of action, antibody assays and assessment of results in a given clinical context. The appropriate interpretation of immunogenicity data is of critical importance for defining the safety profile of a monoclonal antibody. Two case studies described in this paper were prepared to mimic a real situation in which regulators need to evaluate immunogenicity studies conducted by manufacturers of monoclonal antibody products. The specific objective of the case studies was to illustrate assessment of unwanted immunogenicity and the important factors that need to be considered in this context. Regulators and manufacturers who attended the World Health Organization (WHO) implementation workshop on Evaluation of Biotherapeutic Products, held in Seoul, Republic of Korea, in May 2014, participated in the case studies and provided valuable input. This article outlines the main aspects of immunogenicity discussed in these case studies and a summary of the lessons learned at this occasion. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. The in vitro biological activity of the HLA-DR-binding clinical IgG4 antibody 1D09C3 is a consequence of the disruption of cell aggregates and can be abrogated by Fab arm exchange.

    NARCIS (Netherlands)

    Hansen, K.; Ruttekolk, I.R.R.; Glauner, H.B.; Becker, F.; Brock, R.E.; Hannus, S.

    2009-01-01

    Antibodies of the IgG4 subclass, directed against cell surface antigens have received attention as therapeutic molecules due to their poor induction of the complement system. The MHC class II-directed IgG4 antibody 1D09C3 has been explored for the treatment of lymphomas. The mechanism-of-action is s

  16. Production and characterization of antibodies to advanced glycation products on proteins.

    Science.gov (United States)

    Nakayama, H; Taneda, S; Kuwajima, S; Aoki, S; Kuroda, Y; Misawa, K; Nakagawa, S

    1989-07-31

    Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.

  17. [Production and characteristics of monoclonal antibodies to the diphtheria toxin].

    Science.gov (United States)

    Valiakina, T I; Lakhtina, O E; Komaleva, R L; Simonova, M A; Samokhvalova, L V; Shoshina, N S; Kalinina, N A; Rubina, A Iu; Filippova, M A; Vertiev, Iu V; Grishin, E V

    2009-01-01

    Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

  18. Production and purification of polyclonal antibody against melatonin hormone

    Directory of Open Access Journals (Sweden)

    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.

  19. The Influence of Immunosuppressive Agents on the Risk of De Novo Donor-Specific HLA Antibody Production in Solid Organ Transplant Recipients.

    Science.gov (United States)

    OʼLeary, Jacqueline G; Samaniego, Millie; Barrio, Marta Crespo; Potena, Luciano; Zeevi, Adriana; Djamali, Arjang; Cozzi, Emanuele

    2016-01-01

    Production of de novo donor-specific antibodies (dnDSA) is a major risk factor for acute and chronic antibody-mediated rejection and graft loss after all solid organ transplantation. In this article, we review the data available on the risk of individual immunosuppressive agents and their ability to prevent dnDSA production. Induction therapy with rabbit antithymocyte globulin may achieve a short-term decrease in dnDSA production in moderately sensitized patients. Rituximab induction may be beneficial in sensitized patients, and in abrogating rebound antibody response in patients undergoing desensitization or treatment for antibody-mediated rejection. Use of bortezomib for induction therapy in at-risk patients is of interest, but the benefits are unproven. In maintenance regimens, nonadherent and previously sensitized patients are not suitable for aggressive weaning protocols, particularly early calcineurin inhibitor withdrawal without lymphocyte-depleting induction. Early conversion to mammalian target of rapamycin inhibitor monotherapy has been reported to increase the risk of dnDSA formation, but a combination of mammalian target of rapamycin inhibitor and reduced-exposure calcineurin inhibitor does not appear to alter the risk. Early steroid therapy withdrawal in standard-risk patients after induction has no known dnDSA penalty. The available data do not demonstrate a consistent effect of mycophenolic acid on dnDSA production. Risk minimization for dnDSA requires monitoring of adherence, appropriate risk stratification, risk-based immunosuppression intensity, and prospective DSA surveillance.

  20. Generation and applications of monoclonal antibodies for livestock production.

    Science.gov (United States)

    Van Der Lende, T

    1994-01-01

    Monoclonal antibodies (MCAs) have found widespread applications in livestock production. Although the generation of murine MCAs is at present a routine, the production of homologous MCAs, especially important for in vivo applications, is still hampered by the lack of efficient homologous fusion partners for immortalization of antibody producing lymphocytes of livestock species. At present, MCAs are used in immunodiagnostic tests e.g. to monitor livestock reproduction and quality of livestock products. In the future MCAs will also be used in immunosensors for real-time and on-site applications in the same areas. The commercial application of MCAs for the immunomodulation of (pharmacologically induced) physiological processes underlying important (re)production traits is at present limited to the use of anti-PMSG MCAs in PMSG-induced superovulation. However, many potentially interesting applications are under investigation (e.g. immunopotentiation of growth hormone to enhance growth; immunocytolysis of adipocytes to increase lean meat production; immunoneutralization of GnRH for immunocastration; immunoimitation of hormone activity with anti-idiotype antibodies). Attempts to use specific MCAs for the sexing of embryos have been disappointing, mainly because of the relatively low accuracy. In the future, MCAs against membrane proteins which are specific for X- or Y-chromosome bearing spermatozoa might be used for bulk separation of livestock sperm. In general, it is expected that engineered (homologous) recombinant MCAs will largely contribute to the development of a new generation of rapid immunodiagnostic tests and effective immunomodulation applications. They will further increase the use of MCAs in livestock production.

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

    Science.gov (United States)

    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

    2014-01-01

    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 strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  2. Antibody

    Science.gov (United States)

    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 ...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

    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...

  6. Specific Antibody Production by Blood B Cells is Retained in Late Stage Drug-naïve HIV-infected Africans

    Directory of Open Access Journals (Sweden)

    Lydie Béniguel

    2004-01-01

    Full Text Available Unseparated peripheral blood mononuclear cells (PBMCs obtained from drug-naïve African individuals living in a context of multi-infections and presenting with high viral load (VL, were cultured in vitro and tested for their ability to produce antibodies (Abs reacting with HIV-1 antigens. Within these PBMCs, circulating B cells were differentiated in vitro and produced IgG Abs against not only ENV, but also GAG and POL proteins. Under similar experimental conditions, HAART treated patients produced Abs to ENV proteins only. The in vitro antibody production by drug-naïve individuals' PBMCs depended on exogenous cytokines (IL-2 and IL-10 but neither on the re-stimulation of reactive cells in cultures by purified HIV-1-gp 160 antigen nor on the re-engagement of CD40 surface molecules. Further, it was not abrogated by the addition of various monoclonal Abs (mAbs to co-stimulatory molecules. This suggests that the in vitro antibody production by drug-naïve individuals' PBMCs resulted from the maturation of already envelope and core antigen-primed, differentiated B cells, presumably pre-plasma cells, which are not known to circulate at homeostasy. As in vitro produced Abs retained the capacity of binding antigen and forming complexes, this study provides pre-clinical support for functional humoral responses despite major HIV- and other tropical pathogen-induced B cell perturbations.

  7. Thyroid Antibodies

    Science.gov (United States)

    ... AACC products and services. Advertising & Sponsorship: Policy | Opportunities Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  8. Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products.

    Science.gov (United States)

    Koren, Eugen; Smith, Holly W; Shores, Elizabeth; Shankar, Gopi; Finco-Kent, Deborah; Rup, Bonita; Barrett, Yu-Chen; Devanarayan, Viswanath; Gorovits, Boris; Gupta, Shalini; Parish, Thomas; Quarmby, Valerie; Moxness, Michael; Swanson, Steven J; Taniguchi, Gary; Zuckerman, Linda A; Stebbins, Christopher C; Mire-Sluis, Anthony

    2008-04-20

    The appropriate evaluation of the immunogenicity of biopharmaceuticals is of major importance for their successful development and licensure. Antibodies elicited by these products in many cases cause no detectable clinical effects in humans. However, antibodies to some therapeutic proteins have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In addition, antibodies can affect drug efficacy. In non-clinical studies, anti-drug antibodies (ADA) can complicate interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, it is important to develop testing strategies that provide valid assessments of antibody responses in both non-clinical and clinical studies. This document provides recommendations for antibody testing strategies stemming from the experience of contributing authors. The recommendations are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry. The strategies proposed are also expected to contribute to better understanding of antibody responses and to further advance immunogenicity evaluation.

  9. Efficient antibody production in the methylotrophic yeast Ogataea minuta by overexpression of chaperones.

    Science.gov (United States)

    Suzuki, Takeshi; Baba, Satoshi; Ono, Minako; Nonaka, Koichi; Ichikawa, Kimihisa; Yabuta, Masayuki; Ito, Rie; Chiba, Yasunori

    2017-08-01

    A production system for a therapeutic monoclonal antibody was developed using the methylotrophic yeast Ogataea minuta IFO10746. The genetically engineered O. minuta secreted a detectable amount of anti-TRAIL receptor antibody into the culture supernatant, and the secreted antibody was purified by multiple column chromatography steps. In the purification process, both fully and partially assembled antibodies were detected and isolated. The fully assembled antibody from O. minuta showed almost the same biological activity as that derived from mammalian cells despite the distinct glycosylation profile, whereas the partially assembled antibody showed no cytotoxic activity. To increase the production of active antibody in O. minuta, we overexpressed selected chaperone proteins (included protein disulfide isomerase (OmPDI1), thiol oxidase (OmERO1), and immunoglobulin heavy chain binding protein (OmKAR2)) known to assist in the proper folding (in the endoplasmic reticulum) of proteins destined for secretion. Each of these chaperones enhanced antibody secretion, and together these three factors yielded 16-fold higher antibody accumulation while increasing the ratio of the fully assembled antibody compared to that from the parental strain. Supplementation of a rhodanine-3-acetic acid derivative (R3AD_1c), an inhibitor of O-mannosylation, further increased the secretion of the correctly assembled antibody. These results indicated that the co-overexpression of chaperones is an effective way to produce the correctly assembled antibody in O. minuta. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. Overcoming the susceptibility gap between maternal antibody disappearance and auto-antibody production.

    Science.gov (United States)

    Yosipovich, Roni; Aizenshtein, Elina; Shadmon, Roy; Krispel, Simcha; Shuster, Efrat; Pitcovski, Jacob

    2015-01-09

    In the first 10-14 days of a chick's life, protection is conferred by maternal antibodies. Further broiler protection is achieved by active vaccination. However, the high level of maternal antibodies interferes with the induction of an effective immune response by vaccination at a young age. As a result, there is a gap between the reduction in protective maternal antibodies and elevation of self-produced antibodies following active vaccination. The major aim of this study was to test an approach consisting of passive and active vaccination to overcome this gap and to provide continuous resistance to infectious viral diseases during the broiler's growth period. Newcastle disease virus (NDV), which is one of the world's most prevalent infectious diseases of poultry, was tested as a model. Following subcutaneous injection of 18 hemagglutination-inhibiting (HI) units of anti-NDV immunoglobulin Y per 1-day-old chick, protective log2 antibody titers above 4 could be detected to at least 17 days of age. The combination of passive immunization on day 1 of age with attenuated live vaccination on day 10 led to high protective titers throughout the entire growth period, up to 41 days of age. Moreover, the HI titers in the group of birds immunized with the combined vaccination were significantly more homogeneous than those in the group vaccinated only with live virus. Thus, full protection against NDV of all broilers in flock during their entire growth period was achieved by a vaccination regime that combines passive immunization and live vaccination.

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

    OpenAIRE

    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...

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

    OpenAIRE

    Haredy, AM; Nishizawa, A.; Honda, K.; T. Ohya; Ohtake, H; Omasa, T

    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...

  13. Production of Potent Fully Human Polyclonal Antibodies Against Zaire Ebola Virus in Transchromosomal Cattle

    Science.gov (United States)

    2016-07-01

    1 Production of potent fully human polyclonal antibodies against Zaire Ebola virus in transchromosomal cattle John M. Dye1, Hua Wu2, Jay...mail: jjiao@sabbiotherapeutics.com Keywords: Ebola virus, virus neutralization assay, human polyclonal antibodies, transchromosomal bovine...recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV)-Makona isolate. Serum collected from these hyperimmunized Tc

  14. Antibodies against Hepatitis A and Hepatitis B Virus in Intravenous Immunoglobulin Products.

    Science.gov (United States)

    Lee, Soyoung; Kim, Han Wool; Kim, Kyung Hyo

    2016-12-01

    The worldwide seroprevalence of hepatitis A virus (HAV) and hepatitis B virus (HBV) has changed over the last two decades, indicating a declining incidence of HAV and HBV infections. Therefore, vaccinations against HAV and HBV are recommended for unimmunized people before traveling to an endemic area. Unfortunately, primary antibody deficiency (PAD) patients can only obtain humoral immunity through intravenous immunoglobulin G (IVIG) replacement and not from vaccination because of a defect in antibody production. However, few studies have analyzed the titers of antibodies against HAV or HBV in IVIG products. In this study, the titers of anti-HAV and anti-HBs antibodies were measured in nineteen lots of IVIG products from five manufacturers from three countries (A, B from Korea; C, D from Japan; and E from the USA), and trough titers in plasma were estimated. Concentrations of anti-HAV antibody ranged from 1,888-8,927 mIU/mL and estimated trough titers exceeded the minimal protective value in all evaluated IVIG products. Concentrations of anti-HBs antibody ranged from 438-965 mIU/mL in products A and B and were 157, 123, and 1,945 mIU/mL in products C, D, and E, respectively. Estimated trough titers in products A, B, and E exceeded the minimal protective value but those in products C and D did not reach this threshold. These data demonstrated that available IVIG products generally provide sufficient antibodies against HAV and HBV to protect patients with PAD, although the trough concentrations of anti-HBs antibody in two IVIG products did not reach the minimum protective value.

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

    Science.gov (United States)

    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.

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

    National Research Council Canada - National Science Library

    Fomentini, Maíra; Haese, Douglas; Kill, João Luís; Sobreiro, Rodrigo Pereira; Puppo, Débora Del; Haddade, Ismail Ramalho; Lima, Anderson Lazarini; Saraiva, Alysson

    2016-01-01

    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...

  17. Production and potential use of monoclonal antibodies against polio viruses.

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); A.L. van Wezel; G. van Steenis (Bert); A.G. Hazendonk

    1982-01-01

    textabstractLymphocyte hybridomas secreting monoclonal antibodies against different strains of polio virus type 1, 2, or 3 have been produced. For this purpose Balb/C mice were immunized with purified and inactivated virus suspensions and their splenocytes were fused with P3X63Ag8 mouse myeloma cell

  18. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    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.

  19. Fed-batch CHO cell culture for lab-scale antibody production

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Ley, Daniel; Andersen, Mikael Rørdam

    2016-01-01

    Fed-batch culture is the most commonly used upstream process in industry today for recombinant monoclonal antibody production using Chinese hamster ovary cells. Developing and optimizing this process in the lab is crucial for establishing process knowledge, which enable rapid and predictable tech......-transfer to manufacturing scale. In this chapter, we will describe stepwise how to carry out fed-batch CHO cell culture for lab-scale antibody production....

  20. Process performance and product quality in an integrated continuous antibody production process.

    Science.gov (United States)

    Karst, Daniel J; Steinebach, Fabian; Soos, Miroslav; Morbidelli, Massimo

    2017-02-01

    Continuous manufacturing is currently being seriously considered in the biopharmaceutical industry as the possible new paradigm for producing therapeutic proteins, due to production cost and product quality related benefits. In this study, a monoclonal antibody producing CHO cell line was cultured in perfusion mode and connected to a continuous affinity capture step. The reliable and stable integration of the two systems was enabled by suitable control loops, regulating the continuous volumetric flow and adapting the operating conditions of the capture process. For the latter, an at-line HPLC measurement of the harvest concentration subsequent to the bioreactor was combined with a mechanistic model of the capture chromatographic unit. Thereby, optimal buffer consumption and productivity throughout the process was realized while always maintaining a yield above the target value of 99%. Stable operation was achieved at three consecutive viable cell density set points (20, 60, and 40 × 10(6) cells/mL), together with consistent product quality in terms of aggregates, fragments, charge isoforms, and N-linked glycosylation. In addition, different values for these product quality attributes such as N-linked glycosylation, charge variants, and aggregate content were measured at the different steady states. As expected, the amount of released DNA and HCP was significantly reduced by the capture step for all considered upstream operating conditions. This study is exemplary for the potential of enhancing product quality control and modulation by integrated continuous manufacturing. Biotechnol. Bioeng. 2017;114: 298-307. © 2016 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Economics of recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation.

    Science.gov (United States)

    Hammerschmidt, Nikolaus; Tscheliessnig, Anne; Sommer, Ralf; Helk, Bernhard; Jungbauer, Alois

    2014-06-01

    Standard industry processes for recombinant antibody production employ protein A affinity chromatography in combination with other chromatography steps and ultra-/diafiltration. This study compares a generic antibody production process with a recently developed purification process based on a series of selective precipitation steps. The new process makes two of the usual three chromatographic steps obsolete and can be performed in a continuous fashion. Cost of Goods (CoGs) analyses were done for: (i) a generic chromatography-based antibody standard purification; (ii) the continuous precipitation-based purification process coupled to a continuous perfusion production system; and (iii) a hybrid process, coupling the continuous purification process to an upstream batch process. The results of this economic analysis show that the precipitation-based process offers cost reductions at all stages of the life cycle of a therapeutic antibody, (i.e. clinical phase I, II and III, as well as full commercial production). The savings in clinical phase production are largely attributed to the fact that expensive chromatographic resins are omitted. These economic analyses will help to determine the strategies that are best suited for small-scale production in parallel fashion, which is of importance for antibody production in non-privileged countries and for personalized medicine.

  3. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

    Science.gov (United States)

    Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

    2015-12-23

    Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

  4. Large-scale production of monoclonal antibodies in suspension culture.

    Science.gov (United States)

    Backer, M P; Metzger, L S; Slaber, P L; Nevitt, K L; Boder, G B

    1988-10-01

    Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.

  5. Time course of antibodies against IgG and type II collagen in adjuvant arthritis. Role of mycobacteria administration in antibody production.

    Science.gov (United States)

    Franch, A; Cassany, S; Castellote, C; Castell, M

    1994-02-01

    The aim of this study was to elucidate, during the time course of adjuvant arthritis, the existence of antibodies directed to IgG (rheumatoid factor-like) and antibodies against type II collagen. In a second study, we also studied the relation between antibody production, arthritic process and mycobacteria administration. We have demonstrated the presence of antibodies to IgG and type II collagen by means of ELISA techniques. This reactivity appeared on day 7 post-induction, decreased later, and increased progressively from day 21 until last day studied (day 56 post-induction). We have also quantified antibodies against a soluble fraction of Mycobacterium butyricum, the inductor of the disease. Anti-mycobacteria antibodies appeared during the first seven days after induction, but from day 14, when systemic inflammation began, their levels suddenly increased. There is a positive correlation between anti-mycobacteria antibody levels and articular swelling. Anti-IgG and anti-collagen antibody production was not directly linked to arthritic process since these antibodies were synthesized when M. butyricum was administered intraperitoneally, which does not induce arthritis. Anti-mycobacteria antibody concentration was higher when arthritis induction by mycobacterial was successful than when it was unsuccessful.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2013-12-01

    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 cell line. Clone CHO-ATF4-16 did not show any change in growth rate compared with the parental cells or mock-transfected CHO-DP12-SF cells. The expression levels of mRNAs encoding both the antibody heavy and light chains in the CHO-ATF4-16 clone were analyzed. This analysis showed that ATF4 overexpression improved the total production and specific production rate of antibody without affecting the mRNA transcription level. These results indicate that ATF4 overexpression is a promising method for improving recombinant IgG production in CHO cells.

  8. REGULATION OF ANTI-SRBC ANTIBODY PRODUCTION BY OPIOIDS AND THEIR MECHANISMS

    Institute of Scientific and Technical Information of China (English)

    王慧琴; 林嘉友; 刘景生

    1995-01-01

    This study focused on the influences of opioids on the generation of antibody againse sheep erythrocyte in vitro.It was found that morphine,a-CAO,DADLE,MENK were able to inhibit the capacity of murine spleen cells to generate antibody and leukotriene C4 and conversely,dynorphin was able to stimulate the capacity of murine spleen cells to generate antibody and leukotriene C4. Morphine,a-CAO,MENK,DA-DLE,dynorphin decreased intracellular cAMP level,increased [Ca2+]i and calmodulin activity.The effects were completely blocked by naloxone,the specific opioid antagonist.Our results showed that opioids regulate the production of antibody in murine spleen cells,and alter intracellular cAMP,[Ca2+]i calmodulin activity,and leukotriene C4 production by way of binding to different receptor types.

  9. Monoclonal antibody disulfide reduction during manufacturing: Untangling process effects from product effects.

    Science.gov (United States)

    Hutterer, Katariina M; Hong, Robert W; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C

    2013-01-01

    Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.

  10. Nutritional abrogation of photoimmunosuppression: in vivo investigations.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    Science.gov (United States)

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

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

    Science.gov (United States)

    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).

  14. Production of monoclonal antibody for the detection of meat and bone meal in animal feed.

    Science.gov (United States)

    Kim, Shin-Hee; Huang, Tung-Shi; Seymour, Thomas A; Wei, Cheng-i; Kempf, Stephen C; Bridgman, C Roger; Clemens, Roger A; An, Haejung

    2004-12-15

    For the detection of prohibited meat and bone meal (MBM) in animal feed, monoclonal antibodies (MAbs) were raised against heat-stable h-caldesmon purified from bovine intestinal smooth muscle. The obtained hybridoma cells were screened against extracts of the bovine MBM and heat-treated smooth muscle, and MAb 5E12 was identified as having the best performance. Antibody 5E12 did not react with animal feed, milk product, plant proteins, and other ingredients used for commercial animal feed except for the gelatin. This antibody diluted to 100-fold was able to detect MBM mixed in animal feed at 0.05% in an ELISA, and it showed strong affinity toward bovine smooth muscle autoclaved at 130 degrees C. Therefore, this antibody can be used in the ELISA system for field testing of the presence of MBM in animal feed.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  16. Production and Characterization of Anti-Her2 Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    A.S. Tabatabaei-Panah

    2008-01-01

    Full Text Available Objective: Breast cancer is the most common cancer among women in the world.Early diagnosis of this cancer is a key element for its treatment. One of the approachesfor diagnosis of breast cancer is detection of its tumour-associated markers. Hence,Her2 has been the main focus of the researches in the field.Materials and Methods: For diagnosis of Her2 overexpression, monoclonalantibodies (mAb reacting against Her2 were produced in this study. For thispurpose, two peptides from extracellular domain of Her2 were selected and themAbs reacting against them were produced by hybrodoma technology. Reactivityof these antibodies were then evaluated in different immunological assays includingELISA, Immunoflurescence (IF, western blot (WB and immunoprecipitation (IP.Results: Total of 5 clones were produced from two separate fusions, and antibodyisotyping revealed that all clones were IgM. These mAbs showed appropriatereactivities in the following assays: ELISA, immunofluresence by staining of breastcancer cell line (SKBR3, WB and IP by detecting the 185 KD band of Her2.Conclusion: In conclusion, it seems that the mAbs are useful diagnostic tools fordetection of Her2 expression in patients with breast cancer.

  17. Exploring the antigenic response to multiplexed immunizations in a chicken model of antibody production

    DEFF Research Database (Denmark)

    Kousted, Tina Mostrup; Kalliokoski, Otto; Christensen, Sofie Kjellerup

    2017-01-01

    . With an upper limit of 1 mg protein material recommended for chicken immunizations, we found that the maximum number of immunogens that can be reliably used is most likely in the low double digits. The limiting factor for a response to an immunogen could not be related to the number of splenic plasma cells...... plasma cell involvement of less than 5%. Two breeds of egg-layers were compared with respect to antibody production in an initial experiment, but differences in antibody productivity were negligible. Although our findings support the use of multiplexed immunizations in the hen, we find that the number...

  18. Antidotes, antibody-mediated immunity and the future of pharmaceutical product development.

    Science.gov (United States)

    Caoili, Salvador Eugenio C

    2013-02-01

    If new scientific knowledge is to be more efficiently generated and applied toward the advancement of health, human safety must be more effectively addressed in the conduct of research. Given the present difficulties of accurately predicting biological outcomes of novel interventions in vivo, the imperative of human safety suggests the development of novel pharmaceutical products in tandem with their prospective antidotes in anticipation of possible adverse events, to render the risks of initial clinical trials more acceptable from a regulatory standpoint. Antibody-mediated immunity provides a generally applicable mechanistic basis for developing antidotes to both biologicals and small-molecule drugs (such that antibodies may serve as antidotes to pharmaceutical agents as a class including other antibodies) and also for the control and prevention of both infectious and noninfectious diseases via passive or active immunization. Accordingly, the development of prophylactic or therapeutic passive-immunization strategies using antipeptide antibodies is a plausible prelude to the development of corresponding active-immunization strategies using peptide-based vaccines. In line with this scheme, global proliferation of antibody- and vaccine-production technologies, especially those that obviate dependence on the cold chain for storage and transport of finished products, could provide geographically distributed breakout capability against emerging and future health challenges.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Immunisation - Choice of host, adjuvants and boosting schedules with emphasis on polyclonal antibody production.

    Science.gov (United States)

    Delahaut, Philippe

    2017-03-01

    Polyclonal antibodies are frequently used as immunodiagnostic tools in fundamental research. They are also used for routine diagnostic purposes in human and veterinary medicine and for quality control procedures in the food-processing industry. The antibody is a major component of the detection system. It binds with the molecule to be identified. This conjugate is subsequently revealed by means of binding the antibody with a radio-isotope, a fluorescent substance, an enzyme inducing a color change, or a biosensor based analytical system. Polyclonal antibodies are also used for treatment purposes in various pathologies. They might have immunomodulating or anti-inflammatory properties. Snake venom, rabies and tetanus antisera are examples of a therapeutic application; immunosuppressive antithymocyte serum used in order to avoid rejection in organ transplantation is another example from human medicine. These therapeutic aids need hyperimmunisation of animals. Since these are subject to a certain number of interventions such as injections and blood samplings, animal welfare prescriptions have to be taken into account. The optimisation of the immunisation protocol allows for reducing the numbers of animals used as well as reducing stress and pain while obtaining high quality antibodies. This article describes the critical steps in polyclonal antibody production with a particular focus on the choice of the animal species, the age of the subjects, the injection protocol and the sampling times.

  1. A Cassette Vector System for the Rapid Cloning and Production of Bispecific Tetravalent Antibodies

    Directory of Open Access Journals (Sweden)

    Stefanie Claudia Pohl

    2012-04-01

    Full Text Available Bivalent single chain (scFv-Fc antibodies have been used for years as recombinant alternatives of natural immunoglobulins. We have extended this approach to the scFv-Fc-scFv antibody format to obtain tetravalent antigen binding and the possibility to generate bispecific antibodies. We developed a mammalian expression vector system to construct tetravalent scFv-Fc-scFv antibodies with two NcoI+NotI compatible cloning sites flanking the Fc gene fragment. We demonstrated direct cloning from single chain antibody gene libraries and tested various scFv combinations. Transient production of scFv-Fc-scFv antibodies in human embryonic kidney (HEK 293T cells achieved volumetric yields of up to 10 mg/L. However, expression levels were strongly dependent on the carboxyterminal scFv and the scFv combination. All scFv-Fc-scFv antibodies exclusively formed disulfide-linked homodimers. Antigen binding studies revealed dual specificity for all scFv-Fc-scFv employing different scFv fragments. Comparison of C-reactive protein (CRP specific monovalent scFv LA13-IIE3, bivalent scFv-Fc and Fc-scFv LA13-IIE3, and tetravalent scFv-Fc-scFv (scFv LA13-IIE3 in combination with scFvs LA13-IIE3, TOB4-B11, or TOB5-D4 revealed an up to 500-fold increased antigen binding. This novel scFv-Fc-scFv antibody expression system allows simple and fast testing of various scFv combinations.

  2. Production and characterisation of a neutralising chimeric antibody against botulinum neurotoxin A.

    Directory of Open Access Journals (Sweden)

    Julie Prigent

    Full Text Available Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains. Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14 and insect cells (Sf9. After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.

  3. Effects of glutamine and asparagine on recombinant antibody production using CHO-GS cell lines.

    Science.gov (United States)

    Xu, Ping; Dai, Xiao-Ping; Graf, Erica; Martel, Richard; Russell, Reb

    2014-01-01

    A unique and nontraditional approach using glutamine and asparagine supplements for CHO-glutamine synthetase (GS) cell lines was studied. In our experiments, we found that a decrease in pH and an increase in cell death occurred in production phase of a GS cell line, leading to reduced antibody expression and lower antibody yields. The experimental results and the statistical analysis (ANOVA) indicated that additions of glutamine and asparagine in the basal and feed media were effective to buffer the cell culture pH, reduce lactate generation, maintain a higher cell viability profile, and improve antibody productivity. In bench-top bioreactors, glutamine and asparagine supplementation helped to prevent cell death, improve antibody yield, and reduce base usage. Glutamine is normally excluded from culture media for GS cell lines to prevent the bypass of selection pressure. In this study, however, the addition of glutamine did not affect cell population homogeneity, protein quality, or decrease antibody yield of two GS cell lines.

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

    NARCIS (Netherlands)

    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

  5. Improved production and function of llama heavy chain antibody fragments by molecular evolution

    NARCIS (Netherlands)

    Linden, van der R.H.; Geus, de B.; Frenken, G.J.; Peters, H.; Verrips, C.T.

    2000-01-01

    The aim of this study was to improve production level of llama heavy chain antibody fragments (V (HH)) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V (HH) fragments specific for the azo-dye reactive red-6. In

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

    NARCIS (Netherlands)

    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

  7. Development of mass spectrometry based techniques for the identification and determination of compositional variability in recombinant polyclonal antibody products.

    Science.gov (United States)

    Persson, Pia; Engström, Anders; Rasmussen, Lone K; Holmberg, Erland; Frandsen, Torben P

    2010-09-01

    Recombinant polyclonal antibodies are a new class of protein biologics, combining a defined number of target-specific antibodies, developed for therapeutic use across various indications. Development, manufacture, and release of recombinant polyclonal antibodies as well characterized biological products have required development of new chemistry, manufacturing, and control (CMC) technologies. Sym001 is a recombinant polyclonal antibody product containing 25 unique antibodies specific for the Rhesus D antigen. Sym001 drug substance is manufactured using a single batch technology, Sympress. Here, we describe the development of two novel mass spectrometry based methods that allows identification of individual antibodies in the Sym001 drug substance, through the determination of unique marker peptides or antibody light chains. The two methods provide an unambiguous identification of the 25 unique antibodies comprised in the Sym001 drug substance. Furthermore, the light chain liquid chromatography-mass spectrometry (LC-MS) method has been developed to allow the determination of the relative distribution of the 25 antibodies. The light chain LC-MS method has demonstrated linearity, specificity, precision, and accuracy, thus qualifying it for use in the quality control of recombinant polyclonal antibodies for human use. The development of such quantitative methods is central for the development and quality control of additional therapeutic recombinant polyclonal antibody products.

  8. Spleen vagal denervation inhibits the production of antibodies to circulating antigens.

    Directory of Open Access Journals (Sweden)

    Ruud M Buijs

    Full Text Available BACKGROUND: Recently the vagal output of the central nervous system has been shown to suppress the innate immune defense to pathogens. Here we investigated by anatomical and physiological techniques the communication of the brain with the spleen and provided evidence that the brain has the capacity to stimulate the production of antigen specific antibodies by its parasympathetic autonomic output. METHODOLOGY/PRINCIPAL FINDINGS: This conclusion was reached by successively demonstrating that: 1. The spleen receives not only sympathetic input but also parasympathetic input. 2. Intravenous trinitrophenyl-ovalbumin (TNP-OVA does not activate the brain and does not induce an immune response. 3. Intravenous TNP-OVA with an inducer of inflammation; lipopolysaccharide (LPS, activates the brain and induces TNP-specific IgM. 4. LPS activated neurons are in the same areas of the brain as those that provide parasympathetic autonomic information to the spleen, suggesting a feed back circuit between brain and immune system. Consequently we investigated the interaction of the brain with the spleen and observed that specific parasympathetic denervation but not sympathetic denervation of the spleen eliminates the LPS-induced antibody response to TNP-OVA. CONCLUSIONS/SIGNIFICANCE: These findings not only show that the brain can stimulate antibody production by its autonomic output, it also suggests that the power of LPS as adjuvant to stimulate antibody production may also depend on its capacity to activate the brain. The role of the autonomic nervous system in the stimulation of the adaptive immune response may explain why mood and sleep have an influence on antibody production.

  9. In vitro anti-HIV-1 antibody production in subjects in different stages of HIV-1 infection.

    Science.gov (United States)

    Rusconi, S; Riva, A; Meroni, L; Zehender, G; Cocchi, F; Scapellato, L; Galli, M

    1995-01-01

    We evaluated the in vitro antibody production from peripheral blood mononuclear cells (PBMC) against HIV-1 proteins in infected adults. Fifty-four HIV-1 infected patients (four recent seroconverters, 15 asymptomatics with a CD4 count higher than 500/microliters, 27 asymptomatics with a CD4 count between 200 and 500/microliters and eight symptomatic patients) were tested. PBMC were incubated in the presence or absence of 1% pokeweed mitogen (PWM) at 37 degrees C for 8 days. Western blot assay, p24 antigen ELISA and anti-p24 antibody ELISA were performed on serum and culture supernatants. Spontaneous production of anti-env antibody in culture supernatants was evidenced in all subjects. All the positive supernatants for anti-core antibodies (18/54) were derived from asymptomatic patients. PBMC from recent seroconverters and from symptomatic patients did not produce any anti-core antibody. Antibody production decreased after stimulation with PWM. The concentration of p24 antigen did not significantly increase in p24 positive supernatants following acidification (P = 0.1), suggesting that the inability to detect p24 antibody was not due to the anti-p24 antibody complexed to p24 antigen in culture supernatants. In vitro production of anti-p24 antibodies was significantly more frequent in asymptomatic subjects with high CD4+ cell counts (P = 0.02) and was absent in recent seroconverters. This last finding suggests that during the initial phases of the infection, anti-p24 antibody production may be restricted to cells residing in lymphoid organs. In addition, the lower percentage of anti-core antibody in people with low CD4+ cell counts is not merely a consequence of the binding of the antibody to an increased amount of antigen, but probably reflects an impaired production or a sequestration of producing cells in lymphoid tissue during the late stages of the infection. PMID:7554395

  10. Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies.

    Science.gov (United States)

    Stöger, E; Vaquero, C; Torres, E; Sack, M; Nicholson, L; Drossard, J; Williams, S; Keen, D; Perrin, Y; Christou, P; Fischer, R

    2000-03-01

    This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 microg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

  11. LUZ-Y, a Novel Platform for the Mammalian Cell Production of Full-length IgG-bispecific Antibodies*

    Science.gov (United States)

    Wranik, Bernd J.; Christensen, Erin L.; Schaefer, Gabriele; Jackman, Janet K.; Vendel, Andrew C.; Eaton, Dan

    2012-01-01

    The ability of bispecific antibodies to simultaneously bind two unique antigens has great clinical potential. However, most approaches utilized to generate bispecific antibodies yield antibody-like structures that diverge significantly from the structure of archetype human IgG, and those that do approach structural similarity to native antibodies are often challenging to engineer and manufacture. Here, we present a novel platform for the mammalian cell production of bispecific antibodies that differ from their parental mAbs by only a single point mutation per heavy chain. Central to this platform is the addition of a leucine zipper to the C terminus of the CH3 domain of the antibody that is sufficient to drive the heterodimeric assembly of antibody heavy chains and can be readily removed post-purification. Using this approach, we developed various antibody constructs including one-armed Abs, bispecific antibodies that utilize a common light chain, and bispecific antibodies that pair light chains to their cognate heavy chains via peptide tethers. We have applied this technology to various antibody pairings and will demonstrate the engineering, purification, and biological activity of these antibodies herein. PMID:23118228

  12. Development of novel monoclonal antibodies against starch and ulvan - Implications for antibody production against polysaccharides with limited immunogenicity

    DEFF Research Database (Denmark)

    Rydahl, Maja Gro; Kračun, Stjepan K.; Fangel, Jonatan U.

    2017-01-01

    Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody...

  13. Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies

    OpenAIRE

    Pörtner, Ralf; Lüdemann, Ines; Märkl, Herbert

    1997-01-01

    An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new “nutrient-split” feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a...

  14. [Salmonella typhi vaccination response study reveals defective antibody production selective IgA deficiency patient].

    Science.gov (United States)

    Pleguezuelo, Daniel E; Gianelli, Carla

    2015-01-01

    Selective IgA deficiency (SIgAD) is the most prevalent immunodeficiency worldwide, progressing to common variable immunodeficiency only in few reported cases. We report the case of a Spanish female aged 22 and diagnosed of selective IgA deficiency, a long history of bronchitis, several episodes of pneumonia, bilateral bronchiectasis, normal IgG, IgM, IgG subclasses, and detectable pre-vaccination IgG antibodies against tetanus toxoid and Streptococcus pneumoniae. She was evaluated in our clinic in order to rule out common variable immunodeficiency. We observed good antibody response to tetanus toxoid, absence of circulating switched memory B cells, decreased response to pneumococcal polysaccharide antigens and a lack of response to Salmonella typhi vaccine. Most SIgAD patients presents with upper respiratory tract infections or mild diarrhea. Those with lower tract infections, pneumonia or untreatable diarrhea should follow B-cell subpopulations' study and antibody response to vaccines. Absence of response to Salmonella typhi vaccine allowed us to expose the defective antibody production.

  15. [Production and characteristics of monoclonal antibodies against individual prekeratins in simple types of rat epithelium].

    Science.gov (United States)

    Troianovskiĭ, S M; Krutovskikh, V A; Bannikov, G A

    1986-06-01

    BALB/c mice were immunized with intermediate filaments (IF) from the rat colon mucosa, and their splenocytes were fused with myeloma cells to obtain hybridomas. Specific antibody production was assessed by indirect immunofluorescence on cultured rat hepatoma 27 containing prekeratins. The clones that stained IF in hepatoma and not in fibroblasts were judged positive. Clones E3 and E6 were shown to produce monoclonal antibodies against prekeratin with molecular mass of 40 kD (PK40), while clones E2 and E7 produced antibodies against prekeratin with molecular mass of 55 kD (PK55). This was established by immunoblotting with 125I-protein A in cell lysates from the colon, bladder, and hepatoma 27. Only PK55 was revealed in liver and salivary gland lysates. The above proteins were not detected in esophagus, fibroblast and skeletal muscle cell lysates. The monoclonal antibodies make it possible to study individual prekeratin expression in embryogenesis, differentiation and neoplastic transformation of simple epithelium.

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

    Science.gov (United States)

    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 and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma (clone: 2F9-C9) was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Results Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity (2.34×109 M -1) and the isotype was determined to be IgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. Conclusion This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic kit if other requirements of the kit are met. PMID:24285995

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Antithyroglobulin antibody

    Science.gov (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  19. Design and operation of a continuous integrated monoclonal antibody production process.

    Science.gov (United States)

    Steinebach, Fabian; Ulmer, Nicole; Wolf, Moritz; Decker, Lara; Schneider, Veronika; Wälchli, Ruben; Karst, Daniel; Souquet, Jonathan; Morbidelli, Massimo

    2017-07-10

    The realization of an end-to-end integrated continuous lab-scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter-based cell-retention, a continuous two column capture process, a virus inactivation step, a semi-continuous polishing step (twin-column MCSGP), and a batch-wise flow-through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity-yield trade-off of classical batch-wise bind-elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight-through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end-to-end integration. The steady-state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products.

    Science.gov (United States)

    Shankar, Gopi; Devanarayan, Viswanath; Amaravadi, Lakshmi; Barrett, Yu Chen; Bowsher, Ronald; Finco-Kent, Deborah; Fiscella, Michele; Gorovits, Boris; Kirschner, Susan; Moxness, Michael; Parish, Thomas; Quarmby, Valerie; Smith, Holly; Smith, Wendell; Zuckerman, Linda A; Koren, Eugen

    2008-12-15

    Most biological drug products elicit some level of anti-drug antibody (ADA) response. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. In humans, ADA often causes no detectable clinical effects, but in the instances of some therapeutic proteins these antibodies have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In nonclinical (preclinical) studies, ADA can affect drug exposure, complicating the interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess the immunogenic potential of biological drug molecules, and be able to correlate laboratory results with clinical events, it is important to develop reliable laboratory test methods that provide valid assessments of antibody responses in both nonclinical and clinical studies. For this, method validation is considered important, and is a necessary bioanalytical component of drug marketing authorization applications. Existing regulatory guidance documents dealing with the validation of methods address immunoassays in a limited manner, and in particular lack information on the validation of immunogenicity methods. Hence this article provides scientific recommendations for the validation of ADA immunoassays. Unique validation performance characteristics are addressed in addition to those provided in existing regulatory documents pertaining to bioanalyses. The authors recommend experimental and statistical approaches for the validation of immunoassay performance characteristics; these recommendations should be considered as examples of best practice and are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry.

  2. Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.

    Science.gov (United States)

    Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H

    2015-07-14

    Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice.

  3. Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging

    Directory of Open Access Journals (Sweden)

    Peter T. Sage

    2015-07-01

    Full Text Available Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR cells to stimulatory T follicular helper (TFH cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  5. A sulfanyl-PEG derivative of relaxin-like peptide utilizable for the conjugation with KLH and the antibody production.

    Science.gov (United States)

    Katayama, Hidekazu; Mita, Masatoshi

    2016-08-15

    A small peptide-keyhole limpet hemocyanin (KLH) conjugate is generally used as an antigen for producing specific antibodies. However, preparation of a disulfide-rich heterodimeric peptide-KLH conjugates is difficult. In this study, we developed a novel method for preparation of the conjugate, and applied it to the production of specific antibodies against the relaxin-like gonad-stimulating peptide (RGP) from the starfish. In this method, a sulfanyl group necessary for the conjugation with KLH was site-specifically introduced to the peptide after regioselective disulfide bond formation reactions. Using the conjugate, we could obtain specific antibodies with a high antibody titer. This method might also be useful for the production of antibodies against other heterodimeric peptides with disulfide cross-linkages, such as vertebrate relaxins.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Davoud Koolivand

    2016-10-01

    Full Text Available The genomic region of Grapevine fanleaf virus (GFLV encoding the movement protein (MP was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3 to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG concentrations (1, 1.5, and 2 mM each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

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

    Energy Technology Data Exchange (ETDEWEB)

    Amancio, Francisco F. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil)]|[Pernambuco Univ., Recife, PE (Brazil). Dept. de Biofisica e Radiobiologia; Andrade Junior, Heitor F. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina. Inst. de Medicina Tropical

    1997-12-01

    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 {sup 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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  10. {sup 90}Nb - a potential PET nuclide. Production and labeling of monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Radchenko, V.; Roesch, F. [Mainz Univ. (Germany). Inst. of Nuclear Chemistry; Hauser, H.; Eisenhut, M. [German Cancer Research Center, Heidelberg (Germany). Radiopharmaceutical Chemistry; Vugts, D.J.; Dongen, G.A.M.S. van [VU University Medical Center, Amsterdam (Netherlands). Dept. of Nuclear Medicine and PET Research; VU University Medical Center, Amsterdam (Netherlands). Dept. of Otolaryngology/Head and Neck Surgery

    2012-07-01

    Fast progressing immuno-PET gives reasons to develop new potential medium-long and long-lived radioisotopes. One of the promising candidates is {sup 90}Nb. It has a half-life of 14.6 h, which allows visualizing and quantifying processes with medium and slow kinetics, such as tumor accumulation of antibodies and antibodies fragments or polymers and other nanoparticles. {sup 90}Nb exhibits a high positron branching of 53% and an optimal energy of {beta}{sup +} emission of E{sub mean} = 0.35 MeV only. Consequently, efficient radionuclide production routes and Nb{sup V} labeling techniques are required. {sup 90}Nb was produced by the {sup 90}Zr(p,n){sup 90}Nb nuclear reaction on natural zirconium targets. No-carrier-added (n.c.a.) {sup 90}Nb was separated from the zirconium target via a multi-step separation procedure including extraction steps and ion-exchange chromatography. Protein labeling was exemplified using the bifunctional chelator desferrioxamine attached to the monoclonal antibody rituximab. Desferrioxamine was coupled to rituximab via two different routes, by the use of N-succinyl-desferrioxamine (N-suc-Df) and by means of the bifunctional derivative p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS), respectively. Following antibody modification, labeling with {sup 90}Nb was performed in HEPES buffer at pH 7 at room temperature. In vitro stability of the radiolabeled conjugates was tested in saline buffer at room temperature and in fetal calf serum (FCS) at 37 C. The selected production route led to a high yield of 145 {+-} 10 MBq/{mu}A h of {sup 90}Nb with high radioisotopic purity of > 97%. This yield may allow for large scale production of about 10 GBq {sup 90}Nb. The separation procedure resulted in 76-81% yield. The Zr/{sup 90}Nb decontamination factor reaches 10{sup 7}. Subsequent radiolabeling of the two different conjugates with {sup 90}Nb gave high yields; after one hour incubation at room temperature, more than 90% of {sup 90}Nb-Df-mAb was

  11. Produksi dan Karakterisasi Antibodi Monoklonal Anti-Cysticercus cellulosae (PRODUCTION AND CHRACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST CYSTICERCUS CELLULOSAE

    Directory of Open Access Journals (Sweden)

    Ida Bagus Ngurah Swacita

    2015-10-01

    Full Text Available The purpose of this study is to make a monoclonal antibody against- Cysticercus cellulosae and itscharacterization. Samples antigen prepared from T. solium larvae (C. cellulosae was then used to immunizeBalb/c. The immune response of mice assessed by ELISA test, then the lymphocytes of mice used for theproduction of monoclonal antibodies (MoAb. Origin lymphocytes of mice that produce antibodies againstC. cellulosae antigen, fused with myeloma cells (NS1. Results fusion of two cells produces hybrid cellscalled hybridomas; cells are then screened by ELISA test. Hybridoma cells that produce only MoAb, usedto produce large quantities in vitro. Characterization of MoAb against-C.cellulosae was tested by usingELISA and Western blotting. Mice were immunized with C.cellulosae antigen showed an immune responseproducing antibodies to C.cellulosae. Based on the results of fusion, produced a total of 51 hybridoma cellclones and after being screened, only three clones of hybridoma cells that produced MoAb against–C.cellulosae. MoAb produced, named after the hole where the growth of the ELISA micro plate, the BE6,BE7, and EE9. Characteristics of this MoAb capable of tracking cellulosae of fluid larvae and recognizeantigen protein bands with molecular weight 78kDa.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies.

    Science.gov (United States)

    Pörtner, R; Lüdemann, I; Märkl, H

    1997-01-01

    An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new "nutrient-split" feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l(-1) was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system. c(Glc) - glucose concentration, mmol l(-1); c(Gln) - glutamine concentration, mmol l(-1); c(Amm) - ammonia concentration, mmol l(-1); c(Lac) - lactate concentration, mmol l(-1); c(MAb) - MAb concentration, mg l(-1); D - dilution rate, d(-1); D(i) - dilution rate in the inner chamber of the membrane dialysis reactor, d(-1); D(0) - dilution rate in the outer chamber of the membrane dialysis reactor, d(-1); q*(FB,Glc) - volume specific glucose uptake rate related to the fixed bed volume, mmol l(FB) (-1) h(-1); q*(FB,Gln) - volume specific glutamine uptake rate related to the fixed bed volume, mmol l(FB) (-1) h(-1).

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  16. Extract of Gynostemma pentaphyllum enhanced the production of antibodies and cytokines in mice.

    Science.gov (United States)

    Huang, Wen-Chung; Kuo, Ming-Ling; Li, Ming-Liang; Yang, Rong-Chi; Liou, Chian-Jiun; Shen, Jiann-Jong

    2007-05-01

    Gynostemma pentaphyllum is a popular herbal tea in China and some Asian countries. The modulatory function of G. pentaphyllum total plant extracts on immune cells was evaluated in this study. The extract was intraperitoneally injected into mice for 5 consecutive days. The production of antibodies from B cells or cytokines from T cells was determined mainly with ELISA. After the treatment, serum IgM and IgG2a were significantly enhanced and showed dose-dependent effect. Moreover, serum IgA and IgG1 were also increased when received the extract at the doses of 0.05 or 0.50 g/kg/day. In addition to the serum levels, the injection of the extract enhanced the production of all antibodies from LPS-activated spleen cells. Furthermore, more cytokines were secreted from Con A-stimulated splenocytes of G. pentaphyllum-treated mice. Our results suggest that the extract of G. pentaphyllum might promote immune responses through the activation of T and B cells.

  17. Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products.

    Science.gov (United States)

    Gahoual, Rabah; Beck, Alain; Leize-Wagner, Emmanuelle; François, Yannis-Nicolas

    2016-10-01

    Out of all categories, monoclonal antibodies (mAbs), biosimilar, antibody-drug conjugates (ADCs) and Fc-fusion proteins attract the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide comprehensive in-depth characterization of these molecules. CE presents some obvious benefits as high resolution separation and miniaturized format to be widely applied to the analysis of biopharmaceuticals. CE is an effective method for the separation of proteins at different levels. capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) have been particularly relevant for the characterization of size and charge variants of intact and reduced mAbs, while CE-MS appears to be a promising analytical tool to assess the primary structure of mAbs and related products. This review will be dedicated to detail the current and state-of-the-art CE-based methods for the characterization of mAbs and related products.

  18. Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids.

    Science.gov (United States)

    Kumar, Jaideep; Chaudhury, Ashok; Bera, Bidhan C; Kumar, Ritesh; Kumar, Rajender; Tatu, Utpal; Yadav, Suresh Chandra

    2015-12-01

    The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.

  19. Model based adaptive control of a continuous capture process for monoclonal antibodies production.

    Science.gov (United States)

    Steinebach, Fabian; Angarita, Monica; Karst, Daniel J; Müller-Späth, Thomas; Morbidelli, Massimo

    2016-04-29

    A two-column capture process for continuous processing of cell-culture supernatant is presented. Similar to other multicolumn processes, this process uses sequential countercurrent loading of the target compound in order maximize resin utilization and productivity for a given product yield. The process was designed using a novel mechanistic model for affinity capture, which takes both specific adsorption as well as transport through the resin beads into account. Simulations as well as experimental results for the capture of an IgG antibody are discussed. The model was able to predict the process performance in terms of yield, productivity and capacity utilization. Compared to continuous capture with two columns operated batch wise in parallel, a 2.5-fold higher capacity utilization was obtained for the same productivity and yield. This results in an equal improvement in product concentration and reduction of buffer consumption. The developed model was used not only for the process design and optimization but also for its online control. In particular, the unit operating conditions are changed in order to maintain high product yield while optimizing the process performance in terms of capacity utilization and buffer consumption also in the presence of changing upstream conditions and resin aging.

  20. Production of polyclonal and monoclonal antibodies against the Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16.

    Science.gov (United States)

    Ben Hamadou-Charfi, Dorra; Sauer, Annette Juliane; Abdelkafi-Mesrati, Lobna; Jaoua, Samir; Stephan, Dietrich

    2015-03-01

    The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. However, the detection of Vip3Aa16 on Western blot showed in addition to the toxin two other strips (62 and 180 kDa) recognized by the anti-Vip3Aa16 polyclonal antibodies prepared at the Centre of Biotechnology of Sfax Tunisia. For that reason and in order to develop a technique for reliable quantification of the toxin, we have considered the production of polyclonal antibodies at the Julius Kühn Institute, Germany. These antibodies were the basis for the production of monoclonal antibodies directed against the protein produced by the Vip3Aa16 recombinant strain Escherichia coli BL21 (DE3). These monoclonal antibodies were tested by plate-trapped antigen (PTA) and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The selection of hybridoma supernatants gave us four positive clones producing monoclonal antibodies.

  1. Use of Taguchi's methods as a basis to optimize hybridoma cell line growth and antibody production in a spinner flask

    OpenAIRE

    Kallel, Héla; Zaïri, Hind; Rourou, Samia; Essafi, Makram; Barbouche, Ridha; Dellagi, Koussay; Fathallah, Dahmani M.

    2002-01-01

    Taguchi’s methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or β 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the α subunit CD11b. Anti β 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental desi...

  2. FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

    Science.gov (United States)

    Louie, Salina; Haley, Benjamin; Marshall, Brett; Heidersbach, Amy; Yim, Mandy; Brozynski, Martina; Tang, Danming; Lam, Cynthia; Petryniak, Bronislawa; Shaw, David; Shim, Jeongsup; Miller, Aaron; Lowe, John B; Snedecor, Brad; Misaghi, Shahram

    2017-03-01

    During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. [Effects of Celosia argentea and Cucurbita moschata extracts on anti-DNP IgE antibody production in mice].

    Science.gov (United States)

    Imaoka, K; Ushijima, H; Inouye, S; Takahashi, T; Kojima, Y

    1994-05-01

    We have already reported that the Perilla frutescens extract (PFE) suppressed anti-DNA IgE antibody production in mice. In this study, we prepared extracts of Celosia argentea L. (CAE) and Cucurbita moschata Duch (CME), which are Chinese herbal medicines like Perilla frutescens, and examined the effects on anti-DNP antibody responses in mice. To examine the effects of CAE & CME on primary antibody responses, CAE & CME were intraperitoneally injected the day before primary immunization of DNP-ovalbumin. Anti-DNP antibody production was markedly suppressed. Then, we examined the effects on secondary antibody responses. CEA & CME were injected only the day before secondary immunization. Anti-DNP IgE production was markedly suppressed, but IgG responses were not affected. It was also found that mitogenic activity occurred in CAE & CME dose dependently in vitro. These effects of CAE & CME were superior to that of PFE. These results suggest that CAE & CME may be more useful than PFE for the suppression of IgE antibody in certain allergic disorders.

  4. Identification and characterization of host cell protein product-associated impurities in monoclonal antibody bioprocessing.

    Science.gov (United States)

    Levy, Nicholas E; Valente, Kristin N; Choe, Leila H; Lee, Kelvin H; Lenhoff, Abraham M

    2014-05-01

    Downstream processing of monoclonal antibodies (mAbs) has evolved to allow the specific process for a new product to be developed largely by empirical specialization of a platform process that enables removal of impurities of different kinds. A more complete characterization of impurities and the product itself would provide insights into the rational design of efficient downstream processes. This work identifies and characterizes host cell protein (HCP) product-associated impurities, that is, HCP species carried through the downstream processes via direct interactions with the mAb. Interactions between HCPs and mAbs are characterized using cross-interaction chromatography under solution conditions typical of those used in downstream processing. The interacting species are then identified by two-dimensional gel electrophoresis and mass spectrometry. This methodology has been applied to identify product-associated impurities in one particular purification step, namely protein A affinity chromatography, for four therapeutic mAbs as well as the Fab and Fc domains of one of these mAbs. The results show both the differences in HCP-mAb interactions among different mAbs, and the relative importance of product association compared to co-elution in protein A affinity chromatography. © 2013 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Ajda Biček

    2004-01-01

    Full Text Available 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 turkey, pheasant and peafowl. Chicken IgY light chain specific mAb 3E10 revealed the presence of common epitopes on immunoglobulins of turkey, pheasant and sparrow. Monoclonal antibody clone 1F5/3G2 was used to prepare horseradish peroxidase (HRP conjugate and immunoadsorbent column. Conjugated mAbs were demonstrated to be excellent secondary antibodies for diagnostics of certain infections in different avian species. Since they do not react with mammalian immunoglobulins using our mAbs as secondary antibodies in human serodiagnostics would minimize background staining that appears when using mouse detection system. In dot immunobinding assay (DIBA and immunoblot assay they recognized specific IgY antibodies against Mycoplasma synoviae, Mycoplasma gallisepticum and Newcastle disease virus in sera of infected or vaccinated birds. Immunoadsorption as a method for removal of IgY from samples in which Mycoplasma synoviae specific IgY was predominant immunoglobulin class enabled more exact demonstration of specific IgA and IgM antibodies. Herein we are presenting effective mAbs useful in diagnostics of avian and mammalian infections as well as in final steps of detection and purification of chicken antibodies and their subunits produced in vivo or in vitro as polyclonal, monoclonal or recombinant antibodies.

  6. Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens.

    Science.gov (United States)

    Geier, Christoph B; Piller, Alexander; Linder, Angela; Sauerwein, Kai M T; Eibl, Martha M; Wolf, Hermann M

    2015-01-01

    Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency.

  7. Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens.

    Directory of Open Access Journals (Sweden)

    Christoph B Geier

    Full Text Available Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency.

  8. Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies.

    Science.gov (United States)

    Hu, Zhonglan; Chen, Zhenyu; Huang, Nongyu; Teng, Xiu; Zhang, Jun; Wang, Zhen; Wei, Xiaoqiong; Qin, Ke; Liu, Xiao; Wu, Xueping; Tang, Huan; Zhu, Xiaofeng; Cui, Kaijun; Li, Jiong

    2015-03-01

    Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl β-D-1-thio galactoside (IPTG) at 37°C for 4h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 8,9-Epoxyeicosatrienoic acid inhibits antibody production of B lymphocytes in mice.

    Directory of Open Access Journals (Sweden)

    Yanxiang Gao

    Full Text Available Epoxyeicosatrienoic acids (EETs, synthesized from arachidonic acid by cytochrome P450 epoxygenases, are converted to dihydroxyeicosatrienoic acids by soluble epoxide hydrolase. EETs exert anti-inflammatory effects. However, the effect of EETs on humoral immunity is poorly understood. The present study is to investigate the potential role of EETs on B cell function and mechanisms. We examined the role of EETs on antibody production of splenic B cells from C57BL/6 and apolipoprotein E-deficient (ApoE-/- mice by means of ELISA. Of the 4 EET regioisomers, 8,9-EET decreased basal and activation-induced B cell antibody secretion. As well, 8,9-EET significantly inhibited B-cell proliferation and survival, plasma cell differentiation and class-switch recombination. Western blot analysis revealed that lipopolysaccharide-induced nuclear translocation of NF-κB could be attenuated by 8,9-EET. Furthermore, germinal center formation was impaired by 8,9-EET in mice in vivo. 8,9-EET may inhibit B-cell function in vitro and in vivo, which suggests a new therapeutic strategy for diseases with excess B cell activation.

  12. Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection.

    Science.gov (United States)

    Kapoor, Reetika; Mandal, Bikash; Paul, Prabir Kumar; Chigurupati, Phaneendra; Jain, Rakesh Kumar

    2014-02-01

    Cocktail of polyclonal antibodies (PAb) were produced that will help in multiple virus detection and overcome the limitation of individual virus purification, protein expression and purification as well as immunization in multiple rabbits. A dual fusion construct was developed using conserved coat protein (CP) sequences of Cucumber mosaic virus (CMV) and Papaya ringspot virus (PRSV) in an expression vector, pET-28a(+). The fusion protein (∼40kDa) was expressed in Escherichia coli and purified. Likewise, a triple fusion construct was developed by fusing conserved CP sequences of CMV and PRSV with conserved nucleocapsid protein (N) sequence of Groundnut bud necrosis virus (GBNV) and expressed as a fusion protein (∼50kDa) in pET-28a(+). PAb made separately to each of these three viruses recognized the double and triple fusion proteins in Western blot indicating retention of desired epitopes for binding with target antibodies. The fusion proteins (∼40kDa and ∼50kDa) were used to produce cocktail of PAb by immunizing rabbits, which simultaneously detected natural infection of CMV and PRSV or CMV, PRSV and GBNV in Cucurbitaceous, Solanaceous and other hosts in DAC-ELISA. This is the first report on production of a cocktail of PAb to recombinant fusion protein of two or three distinct viruses.

  13. Determination of Aspergillus pathogens in agricultural products by a specific nanobody-polyclonal antibody sandwich ELISA.

    Science.gov (United States)

    Wang, Ting; Li, Peiwu; Zhang, Qi; Zhang, Wen; Zhang, Zhaowei; Wang, Tong; He, Ting

    2017-06-28

    Aspergillus and its poisonous mycotoxins are distributed worldwide throughout the environment and are of particular interest in agriculture and food safety. In order to develop a specific method for rapid detection of Aspergillus flavus to forecast diseases and control aflatoxins, a nanobody, PO8-VHH, highly reactive to A. flavus was isolated from an immunized alpaca nanobody library by phage display. The nanobody was verified to bind to the components of extracellular and intracellular antigen from both A. flavus and A. parasiticus. To construct a sandwich format immunoassay, polyclonal antibodies against Aspergillus were raised with rabbits. Finally, a highly selective nanobody-polyclonal antibody sandwich enzyme-linked immunosorbent assay was optimized and developed. The results revealed that the detection limits of the two fungi were as low as 1 μg mL(-1), and that it is able to detect fungal concentrations below to 2 μg mg(-1) of peanut and maize grains in both artificially and naturally contaminated samples. Therefore, we here provided a rapid and simple method for monitoring Aspergillus spp. contamination in agricultural products.

  14. A fully defined, fed-batch, recombinant NS0 culture process for monoclonal antibody production.

    Science.gov (United States)

    Hermes, Paul A; Castro, Chris D

    2010-01-01

    To manufacture a glycoprotein, mammalian cells expressing the desired protein are often grown in fed-batch mode. Feeding an undefined, nonanimal hydrolysate helps the cells receive sufficient nutrition, but makes systems difficult to optimize. Even different lots of the same hydrolysate may have significant variability; furthermore, individual components may actually be detrimental to the cells. Switching to fully defined feeds could eliminate these issues. For monoclonal antibody (mAb) production by fed-batch NS0 cells, this article describes the replacement of a hydrolysate-based feed with a fully defined, animal-component-free feed system. The defined feed initially had 67 components, but additional experiments allowed a reduction to 25 components. The mAb titer is approximately 20% higher than in the undefined system, and the feed volume is circa 20% lower. The two systems generated antibodies with similar glycosylation profiles. Other benefits of the defined feed system include lower raw material costs, the ability to optimize key nutrient concentrations, greater confidence in raw material quality, and the elimination of potential, hydrolysate-associated endotoxin issues.

  15. Entrapment of animal cells for the production of biomolecules such as monoclonal antibodies.

    Science.gov (United States)

    Scheirer, W; Nilsson, K; Merten, O W; Katinger, H W; Mosbach, K

    1983-01-01

    An important problem in the production of monoclonal antibodies is the large-scale cultivation of hybridoma cells in vitro. Fragility of cells and suboptimal in vitro cultivation methods have led to poor results in larger scale production up to now. To lower the mechanical stress on the cells we tried to entrap the cells into microspheres made of polymer material. In addition to other materials, agarose as embedding medium was investigated and results with hybridoma and other, non anchorage-dependent cell lines are given. The conclusion of the results is that encapsulation of living cells is possible and entrapped cells remain viable and continue to produce the desired substance for at least several weeks. The substances are secreted through the polymer matrix. Handling of microspheres is shown to be easy and simple fermentation apparatus may be used for the production on a reliable technical scale. Some problems remain unsolved, such as the determination of viable cell count within the microspheres and cultivation in columns which seems to be the simplest form of continuous production process.

  16. Use of Taguchi's methods as a basis to optimize hybridoma cell line growth and antibody production in a spinner flask.

    Science.gov (United States)

    Kallel, Héla; Zaïri, Hind; Rourou, Samia; Essafi, Makram; Barbouche, Ridha; Dellagi, Koussay; Fathallah, Dahmani M

    2002-05-01

    Taguchi's methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or beta 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the alpha subunit CD11b. Anti beta 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi's methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways.

  17. PLACEBO-DRIVEN CLINICAL TRIALS OF TRANSFER POINT GLUCAN #300 IN CHILDREN WITH CHRONIC RESPIRATORY PROBLEMS: ANTIBODY PRODUCTION

    Directory of Open Access Journals (Sweden)

    Vetvicka Vaclav

    2013-01-01

    Full Text Available The role of glucan in stimulation of immune reactions has been well-established. In this report, we focused on the antibody production in glucan-supplemented children with chronic respiratory problems. We measured the levels of salivary IgA, IgM and IgG in 40 children aged 8-12 years and evaluated the effects of 100 mg d-1 oral dose of glucan. We found a significant increase in production of all tested antibodies in the glucan-stimulated group, but a decrease of antibody production in the control group. A thirty-day oral application of yeast-based natural immunomodulator β-glucan strongly stimulated the mucosal immunity of children with chronic respiratory problems.

  18. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Influence of long-term treatment with tetracycline and niacinamide on antibody production in dogs with discoid lupus erythematosus.

    Science.gov (United States)

    Mueller, Ralf S; Fieseler, Kathryn V; Bettenay, Sonya V; Rosychuk, Rodney A W

    2002-04-01

    To evaluate the effect of long-term treatment with tetracycline and niacinamide on antibody production in dogs by measuring postvaccinal serum concentrations of antibodies against canine parvovirus and canine distemper virus. 10 dogs receiving long-term treatment with tetracycline and niacinamide (treatment group) and 10 healthy dogs (control group). The treatment group included 9 dogs with discoid lupus erythematosus and 1 dog with pemphigus foliaceus on long-term treatment (> 12 months) with tetracycline and niacinamide. The control group included 10 healthy dogs with no clinical signs of disease and no administered medications for the past 3 months. Blood samples were obtained from all dogs by jugular venipuncture. Serum antibody titers against canine parvovirus and canine distemper virus antigens were measured, using hemaglutination inhibition and serum neutralization, respectively, and compared between groups. A significant difference in antibody titers between treatment- and control-group dogs was not found. All dogs had protective antibody titers against canine distemper virus, and 8 of 10 dogs from each group had protective titers against canine parvovirus infection. These results provide evidence that long-term treatment with tetracycline and niacinamide does not interfere with routine vaccinations and thus does not seem to influence antibody production in dogs.

  1. Are we ready to abrogate compulsory vaccinations for children?

    Science.gov (United States)

    Martinelli, Domenico; Tafuri, Silvio; Fortunato, Francesca; Cozza, Vanessa; Germinario, Cinzia A; Prato, Rosa

    2015-01-01

    In Italy, vaccination against diphtheria, tetanus, polio and hepatitis B is compulsory for infants countrywide, except in Veneto region where since 2007 Health Authorities have experimented the suspension of mandatory vaccination. In light of the recent discussion on the potential abrogation in other regions, we explored the opinion of family pediatricians who play a crucial role in promoting immunization programmes in Italy. In November 2009, we interviewed by phone the family pediatricians working in Puglia region using a standardised, ad hoc and piloted questionnaire. Of the 596 contacted, 502 (84.2%) completed the questionnaire (54% female, median age = 52 y). Among the respondents, 72 (14.3%) would agree on the hypothesis of abrogation. This judgment was associated with having a good opinion on the level of awareness of the importance of vaccinations in the general public (OR = 6.6; 95% CI: 3.6-12.1) and having the perception of adequate organization of Vaccination Services in supporting the abrogation (OR = 3.6; 95% CI: 1.7-5.9). Family pediatricians appeared really sceptical about the abrogation of compulsory vaccination that could be hypothesized only increasing public awareness, communication skills and capability of Vaccination Services personnel in offering vaccinations.

  2. Interference of infectious bursal disease virus on antibody production against Newcastle disease and infectious bronchitis virus

    Directory of Open Access Journals (Sweden)

    WM Cardoso

    2006-09-01

    Full Text Available This work has the objective of verifying the interference of infectious bursal disease virus in the antibody production against Newcastle disease virus and infectious bronchitis virus. The experiment was carried out with 640 day-old-chicks from a 42 weeks old hen flock. The birds were separated into eight experimental groups (n=80/group and were submitted to different combinations of vaccinations, with live vaccines, to Newcastle disease, avian infectious bronchitis, and infectious bursal disease with diverse combinations of days of vaccination. We verified that the utilization of polyvalent vaccinal programs have a different efficacy comparing to monovalent vaccinations when Newcastle disease, infectious bronchitis, and infectious bursal disease vaccinations are applied. This way, the use of vaccinations to infectious bursal disease in polyvalent vaccinal programs is desirable due to improvement of NDV response with the presence of IBV by the probable reduction of interference of IBV under NDV.

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

    Science.gov (United States)

    de Marco, Ario

    2015-09-02

    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 the different options to help the researchers interested in expressing recombinant antibodies in their choice. Rather than the compilation of an exhaustive list of the recent publications in the field, this review intendeds to analyze the development of the most innovative or fast-growing strategies. These have been illustrated with some significant examples and, when possible, compared with the existing alternatives. Space has also been given to those solutions that might represent interesting opportunities or that investigate critical aspects of the production optimization but for which the available data as yet do not allow for a definitive judgment. The take-home message is that there is a clear process of progressive diversification concerning the antibody expression platforms and an effort to yield directly application-adapted immune-reagents rather than generic naked antibodies that need further in vitro modification steps before becoming usable.

  4. Elevated PC responsive B cells and anti-PC antibody production in transgenic mice harboring anti-PC immunoglobulin genes.

    Science.gov (United States)

    Pinkert, C A; Manz, J; Linton, P J; Klinman, N R; Storb, U

    1989-12-01

    The rearrangement of heavy and light chain immunoglobulin genes is necessary for the production of functional antibody molecules. The myeloma MOPC 167 produces specific antibodies to the antigen phosphorylcholine (PC), which is present on bacterial surfaces, fungi and other environmental contaminants. Rearranged heavy and light chain immunoglobulin genes cloned from MOPC 167 were microinjected into mouse eggs. Within the resulting transgenic mice, expression of the transgenes were limited to lymphoid tissues. Transgenic mice produced elevated levels of anti-PC antibodies constitutively, at 16 days of age, when normal non-transgenic mice were not fully immunocompetent. A triggering antigenic stimulus was not necessary to evoke anti-PC immunoglobulin production. Additionally, the frequency of PC-responsive B cells in these transgenic mice was further increased upon specific immunization.

  5. Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.

    Science.gov (United States)

    Choudhary, Nandlal; Roy, Avijit; Leon, M G; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2017-05-01

    The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

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

    Science.gov (United States)

    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.

  7. Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein.

    Science.gov (United States)

    Ding, Kai; Han, Lei; Zong, Huifang; Chen, Junsheng; Zhang, Baohong; Zhu, Jianwei

    2017-03-01

    Demonstration of reproducibility and consistency of process and product quality is one of the most crucial issues in using transient gene expression (TGE) technology for biopharmaceutical development. In this study, we challenged the production consistency of TGE by expressing nine batches of recombinant IgG antibody in human embryonic kidney 293 cells to evaluate reproducibility including viable cell density, viability, apoptotic status, and antibody yield in cell culture supernatant. Product quality including isoelectric point, binding affinity, secondary structure, and thermal stability was assessed as well. In addition, major glycan forms of antibody from different batches of production were compared to demonstrate glycosylation consistency. Glycan compositions of the antibody harvested at different time periods were also measured to illustrate N-glycan distribution over the culture time. From the results, it has been demonstrated that different TGE batches are reproducible from lot to lot in overall cell growth, product yield, and product qualities including isoelectric point, binding affinity, secondary structure, and thermal stability. Furthermore, major N-glycan compositions are consistent among different TGE batches and conserved during cell culture time.

  8. Antibodies Against Sporothrix schenckii Enhance TNF-α Production and Killing by Macrophages.

    Science.gov (United States)

    Franco, D de Lima; Nascimento, R C; Ferreira, K S; Almeida, S R

    2012-02-01

    Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti-gp70. Additionally, we show an increase in the levels of pro-inflammatory cytokines such as TNF-α and IL-1β. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF-α production and fungus killing by macrophages in experimental sporotrichosis.

  9. Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer.

    Science.gov (United States)

    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 A; Siddiqui, Javed; Wei, John T; Chinnaiyan, Arul M; Qian, Wei-Jun; 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. Studies of TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies suitable for quantitative studies. 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 provided confident detection of 6 unique ERG peptides in both TMPRSS2-ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2-ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2-ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2-ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing "signature" peptides for detection of ERG over-expression resulting from TMPRSS2-ERG gene fusion. The PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products in prostate cancer.

  10. RELATIONSHIP BETWEEN CYTOMEGALOVIRUS INFECTION AND THE PRODUCTION OF AN TICARDIOLIPIN ANTIBODY IN RENAL TRANSPLANT RECIPIENTS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the relationship bet wee n cytomegalovirus (CMV) infection and the production of anticardiolipin antibody (ACA) in renal transplant recipients.Methods Polymerase c hain reaction (PCR) was used qualitat ively for detection of CMV-DNA in 146 renal transplant recipients.Meanwhile,enz yme-linked immunosorbent assay (ELISA) was used for detection of ACA-IgG in bl ood serum samples from these recipients and 32 healthy individuals. Results The ACA positive rate was 17.1% among the 146 ren al transplant recipients,and that of the control group was 6.3%.There was no sig nificant difference.However,the ACA positive rate of the renal transplant recipi ents infected with CMV was 31.2%.It was clearly higher than that of those with n o infection of CMV and that of the control group (P<0.005). Con clusion The production of ACA was closely related to CMV infection.It m ight be one of the factors of chronic angiopathy of the transpl anted kidney due to CMV infection.

  11. Evaluation of bovine coronavirus antibody levels, virus shedding, and respiratory disease incidence throughout the beef cattle production cycle

    Science.gov (United States)

    Objective- Determine how levels of serum antibody to bovine coronavirus (BCV) are related to virus shedding patterns and respiratory disease incidence in beef calves at various production stages. Animals- 890 crossbred beef calves from four separately managed herds at the U.S. Meat Animal Research C...

  12. Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

    Directory of Open Access Journals (Sweden)

    Shephard Enid G

    2011-10-01

    Full Text Available Abstract Background HIV-1 Gag virus like particles (VLPs used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates

  13. Production and immunoanalytical application of 32 monoclonal antibodies against metacestode somatic antigens of Echinococcus multilocularis.

    Science.gov (United States)

    Wang, Xin; Lu, Rui; Liu, Qiao-Feng; Chen, Jian-Ping; Deng, Qiang; Zhang, Ya-Lou; Zhang, Bing-Hua; Xu, Jia-Nan; Sun, Lei; Niu, Qin-Wang; Liang, Quan-Zeng

    2010-06-01

    Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles.

  14. Guardians at the gate: patent protection of therapeutic monoclonal antibodies through product life cycle management--Part 3.

    Science.gov (United States)

    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 these characteristics are positioned in a marketplace that is competition-free for as long as possible. In an era of mAbs with billion dollar markets, the loss of even a single day of sales could cost companies millions of dollars in lost revenue.

  15. Radioimmunoassay for zearalenone and zearalanol in human serum: production, properties, and use of porcine antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Thouvenot, D.; Morfin, R.F.

    1983-01-01

    To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6'-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The procine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  17. THE PRODUCTION OF KIDNEY ANTIBODIES BY INJECTION OF HOMOLOGOUS KIDNEY PLUS BACTERIAL TOXINS

    Science.gov (United States)

    Schwentker, Francis F.; Comploier, Frank C.

    1939-01-01

    Rabbits injected with emulsions of homologous kidney to which staphylcoccus or streptococcus toxins had been added produced complement fixing antibodies which reacted with both rabbit kidney and brain. By absorption tests it was demonstrated that the sera contained at least two antibodies, one specific for kidney, the other non-specific. Similar kidney antibodies were found in the blood of a majority of patients with scarlet fever but in only a few normal persons. The possibility that a similar or related antibody may be concerned in the etiology of scarlatinal nephritis is discussed. PMID:19870903

  18. Species-Specific Chromosome Engineering Greatly Improves Fully Human Polyclonal Antibody Production Profile in Cattle.

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsushita

    Full Text Available Large-scale production of fully human IgG (hIgG or human polyclonal antibodies (hpAbs by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.

  19. Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production.

    Science.gov (United States)

    Strauss, Daniel M; Cano, Tony; Cai, Nick; Delucchi, Heather; Plancarte, Magdalena; Coleman, Daniel; Blank, Gregory S; Chen, Qi; Yang, Bin

    2010-01-01

    The quality-by-design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design-of-experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus-like particles from CHO-cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained.

  20. Involvement of pentraxin-3 in anti-neutrophil cytoplasmic antibody production induced by aluminum salt adjuvant.

    Science.gov (United States)

    Nagai, Kei; Aratani, Yasuaki; Shibuya, Akira; Yamagata, Kunihiro

    2017-01-01

    Pentraxin 3 (PTX3) is a multifunctional soluble factor. PTX3 can be involved in the regulation of vasculitis and is expressed in the cytoplasm of neutrophils. As anti-neutrophil cytoplasmic antibody (ANCA) is recognised as a cause of vasculitis, we aimed to discover the role of PTX3 in ANCA production in vivo. To this end, we used aluminum salt (alum), which induces neutrophil extracellular traps, as an adjuvant for producing anti-myeloperoxidase-ANCA (MPO-ANCA). Specifically, we intraperitoneally injected alum and recombinant MPO (rMPO) into MPO-deficient mice and then measured the concentration of anti-MPO IgG in their blood. To show the involvement of extracellular PTX3 in this model, we assessed PTX3 protein content and host double-stranded DNA levels in the mice's peritoneal fluid after alum injection. In addition, we simultaneously administered recombinant PTX3, rMPO and alum to MPO-deficient mice to assess the function of PTX3 in producing anti-MPO IgG in vivo. Anti-MPO IgG was produced by the alum + rMPO immunisation model in MPO-deficient but not wildtype mice. Injection of alum induced extracellular PTX3 as well as double-stranded DNA and dead cells in MPO-deficient mice. Simultaneous injection of recombinant PTX3 with rMPO and alum attenuated the production of anti-MPO IgG in MPO-deficient mice. Our current findings provide evidence that PTX3 attenuates the production of murine MPO-ANCA.

  1. Post-transplant donor-specific antibody production and graft outcome in kidney transplantation: results of sixteen-year monitoring by flow cytometry.

    Science.gov (United States)

    Piazza, Antonina; Poggi, Elvira; Ozzella, Giuseppina; Borrelli, Laura; Scornajenghi, Alessandra; Iaria, Giuseppe; Tisone, Giuseppe; Adorno, Domenico

    2006-01-01

    Our data show that monitoring by sensitive flow cytometric techniques of the de novo production of anti-HLA antibodies in patients receiving kidney transplantation is a useful and noninvasive tool to identify the onset of an immune response towards the graft before any clinical manifestation of antibody-mediated graft injury. Consequently prospective posttransplant monitoring of anti-HLA donor-directed antibodies may offer the chance to realize an effective clinical intervention in order to prevent graft dysfunction and to prolong graft survival. The long follow-up period of the study allowed us to demonstrate a very low graft survival rate in patients who developed donor-specific HLA antibodies in comparison with patients who did not have antibodies, thus confirming the "humoral theory of transplantation". The posttransplant production of anti-HLA antibodies can predict not only graft failure but also chronic dysfunction of the graft. Moreover, our findings suggest that graft survival is influenced by the epitope- and locus-specificity of anti-HLA donor-directed antibodies. The interval between antibody appearance and loss of graft function was short in some patients but reached several years in others. Moreover, some patients showed consistent production of antibodies for many years and an uneventful clinical status. These findings suggest a mechanism of graft "accommodation" or the production of "harmless" antibodies. Immunosuppressive drug combinations able to inhibit T and B cell activation are useful tools to prevent the humoral immune response against graft and consequently to prolong graft survival.

  2. Production of anti-fullerene C60 polyclonal antibodies and study of their interaction with a conjugated form of fullerene

    Science.gov (United States)

    Hendrickson, O. D.; Fedyunina, N. S.; Martianov, A. A.; Zherdev, A. V.; Dzantiev, B. B.

    2011-09-01

    The aim of this study was to produce anti-fullerene C60 antibodies for the development of detection systems for fullerene C60 derivatives. To produce anti-fullerene C60 antibodies, conjugates of the fullerene C60 carboxylic derivative with thyroglobulin, soybean trypsin inhibitor, and bovine serum albumin were synthesized by carbodiimide activation and characterized. Immunization of rabbits by the conjugates led to the production of polyclonal anti-fullerene antibodies. The specificity of the immune response to fullerene was investigated. Indirect competitive immunoenzyme assay was developed for the determination of conjugated fullerene with detection limits of 0.04 ng/mL (calculated for coupled C60) and 0.4 ng/mL (accordingly to total fullerene-protein concentration).

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

    DEFF Research Database (Denmark)

    Nøstdal, Alexander; Wandall, Hans H

    2013-01-01

    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...

  4. Linking the seroresponse to infection to within-host heterogeneity in antibody production

    NARCIS (Netherlands)

    Teunis, P. F M; van Eijkeren, J. C H; de Graaf, W.F.; Marinović, A. Bonačić; Kretzschmar, M. E E

    2016-01-01

    A recently published model for the serum antibody response to infection appeared well suited for use in statistical analyses of longitudinal serological data. The published model assumed exponential decay with fixed rates for pathogen and serum antibody kinetics, ignoring any within-host

  5. Properties, production and applications of camelid single-domain antibody fragments

    NARCIS (Netherlands)

    Harmsen, M.M.; Haard, de H.J.

    2007-01-01

    Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms a

  6. The Synthesis of N-Morphine Hapten and Production of Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Those antibodies elicited by different tether site for attachment to carrier protein have different specificity. Herein we reported that a monoclonal antibody against morphine with high specificity and affinity was successfully produced by using different linkers to couple to different carrier proteins.

  7. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variab

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

    Directory of Open Access Journals (Sweden)

    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.

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

    NARCIS (Netherlands)

    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

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

    Institute of Scientific and Technical Information of China (English)

    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.

  11. Antibody-mediated activation of FGFR1 induces FGF23 production and hypophosphatemia.

    Directory of Open Access Journals (Sweden)

    Ai-Luen Wu

    Full Text Available The phosphaturic hormone Fibroblast Growth Factor 23 (FGF23 controls phosphate homeostasis by regulating renal expression of sodium-dependent phosphate co-transporters and cytochrome P450 enzymes involved in vitamin D catabolism. Multiple FGF Receptors (FGFRs can act as receptors for FGF23 when bound by the co-receptor Klotho expressed in the renal tubular epithelium. FGFRs also regulate skeletal FGF23 secretion; ectopic FGFR activation is implicated in genetic conditions associated with FGF23 overproduction and hypophosphatemia. The identity of FGFRs that mediate the activity of FGF23 or that regulate skeletal FGF23 secretion remains ill defined. Here we report that pharmacological activation of FGFR1 with monoclonal anti-FGFR1 antibodies (R1MAb in adult mice is sufficient to cause an elevation in serum FGF23 and mild hypophosphatemia. In cultured rat calvariae osteoblasts, R1MAb induces FGF23 mRNA expression and FGF23 protein secretion into the culture medium. In a cultured kidney epithelial cell line, R1MAb acts as a functional FGF23 mimetic and activates the FGF23 program. siRNA-mediated Fgfr1 knockdown induced the opposite effects. Taken together, our work reveals the central role of FGFR1 in the regulation of FGF23 production and signal transduction, and has implications in the pathogenesis of FGF23-related hypophosphatemic disorders.

  12. Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein.

    Science.gov (United States)

    Wangman, Pradit; Senapin, Saengchan; Chaivisuthangkura, Parin; Longyant, Siwaporn; Rukpratanporn, Sombat; Sithigorngul, Paisarn

    2012-03-20

    The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol µl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.

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

    Science.gov (United States)

    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.

  14. In vitro-induced antibody production in chronic hepatitis C virus infection

    Directory of Open Access Journals (Sweden)

    Oliveira Jr. E.B.

    2003-01-01

    Full Text Available The objectives of the present study were to assess the in vitro-induced anti-hepatitis C virus (HCV antibody production (IVIAP in relation to the clinical, biochemical, virologic and histologic variables of patients with HCV infection. The study included 57 patients (60% males with HCV infection (anti-HCV and HCV-RNA positive. Alanine aminotransferase (ALT was elevated in 89% of the patients. Mean viral load was 542,241 copies/ml and histology of the liver showed chronic hepatitis in 27/52 (52% and cirrhosis in 11/52 (21% patients. IVIAP levels were determined by immunoenzymatic assay at median absorbance of 0.781 at 450 nm. IVIAP was negative in 14% of the patients. When groups with IVIAP levels above and below the median were compared, high IVIAP levels were associated with the male sex, elevated ALT levels and more advanced disease stage. After logistic regression analysis, advanced histologic damage to the liver remained as the only independent variable associated with elevated IVIAP levels. Using a receiver operator characteristic curve, the best cut-off level for IVIAP was established (= 1.540, with 71% sensitivity and 94% specificity for the detection of more advanced disease stages (grades 3 and 4. These findings are consistent with the participation of immunological mechanisms in the genesis of the hepatic lesions induced by HCV and indicate that the IVIAP test may be useful as a noninvasive marker of liver damage either alone or in combination with other markers.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Structural comparison of two anti-CD20 monoclonal antibody drug products using middle-down mass spectrometry.

    Science.gov (United States)

    Wang, Bo; Gucinski, Ashley C; Keire, David A; Buhse, Lucinda F; Boyne, Michael T

    2013-05-21

    Liquid chromatography-mass spectrometry (LC-MS) is an information rich analytical tool that can provide fast, robust and sensitive characterization of protein therapeutics for quality assurance and structural comparison. Herein, structural characterization of two anti-CD20 monoclonal antibodies obtained from two different sources was performed using a middle-down LC-MS strategy to determine if they can be analytically differentiated. Through the use of a specific enzymatic digestion method using IdeS with subsequent LC-MS analysis, we show that the anti-CD20 monoclonal antibody that has been approved by the FDA can be partially characterized and differentiated analytically from an Indian sourced product that lacks FDA approval. In comparison to the FDA-approved product, differential modifications to both the N- and C-termini result in increased charge heterogeneity for the Indian product. In addition, significant differences in the intensities of the observed glycoforms between the two antibodies were detected. While this study assesses only one lot of each of a FDA approved drug product and the Indian sourced drug product, the observed differences may represent process specific fingerprints that could be useful for surveillance purposes.

  17. Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Haustraete Jurgen

    2009-08-01

    Full Text Available Abstract Background Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. Results Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. Conclusion Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression

  18. Long-term antibody synthesis in vitro. VI. Anti-allotype sera as probes of clonal products in affinity maturation.

    Science.gov (United States)

    Conway de Macario, E; Macario, A J; Tosi, R M; Celada, F; Landucci-Tosi, S

    1978-08-01

    A new experimental system is described for measuring the allotypic product of rabbit B cells during long-lasting in vitro antibody responses. The immunoenzymatic assays described allow determination of several parameters mapping in different regions of the same molecule, which can be measured and combined to yield a multidimensional picture of the time-course dynamics of antibody synthesis. The rabbit immune system responding to Escherichia coli beta-D-galactosidase was sample and disassembled by (a) culturing lymph node microfragments and (b) sorting out from among all anti-enzyme antibodies only those activating a mutant enzyme, AMEF, which bore the b4 or b9 allotype. A considerable simplification of the response was achieved in the microcultures as documented by cultures of heterozygous cells which produced only one allotype and by the fact that each culture showed a distinctive pattern when antibody titre, association constant, heterogeneity index, L-chain type, and k-chain allotype were considered together. This array of patterns was not an artifact but the result of disassembling a representative sample of the rabbit immune system into small components, since the b4/b9 ratio obtained by averaging the results of all cultures from a heterozygous rabbit lymph node was the same as the serum ratio. Despite the Poisson distribution of the responder microcultures, none of them was monoclonal; i.e. no antibodies homogeneous by all parameters tested were observed, This finidng supports the notion that in normal lymphoid tissue in its native tridimensional arrangement, one T cell can trigger several B cells clustered in one antibody-forming unit. This natural arrangement would ensure the monospecificity of the cluster (dictated by the T cell) while allowing for variation in affinity (depending upon the array of B cells in the unit). Accordingly our findings would results from the fact that as the size of the microfragments was reduced, the cells diluted out first were

  19. Mechanistic modeling of ion-exchange process chromatography of charge variants of monoclonal antibody products.

    Science.gov (United States)

    Kumar, Vijesh; Leweke, Samuel; von Lieres, Eric; Rathore, Anurag S

    2015-12-24

    Ion-exchange chromatography (IEX) is universally accepted as the optimal method for achieving process scale separation of charge variants of a monoclonal antibody (mAb) therapeutic. These variants are closely related to the product and a baseline separation is rarely achieved. The general practice is to fractionate the eluate from the IEX column, analyze the fractions and then pool the desired fractions to obtain the targeted composition of variants. This is, however, a very cumbersome and time consuming exercise. A mechanistic model that is capable of simulating the peak profile will be a much more elegant and effective way to make a decision on the pooling strategy. This paper proposes a mechanistic model, based on the general rate model, to predict elution peak profile for separation of the main product from its variants. The proposed approach uses inverse fit of process scale chromatogram for estimation of model parameters using the initial values that are obtained from theoretical correlations. The packed bed column has been modeled along with the chromatographic system consisting of the mixer, tubing and detectors as a series of dispersed plug flow and continuous stirred tank reactors. The model uses loading ranges starting at 25% to a maximum of 70% of the loading capacity and hence is applicable to process scale separations. Langmuir model has been extended to include the effects of salt concentration and temperature on the model parameters. The extended Langmuir model that has been proposed uses one less parameter than the SMA model and this results in a significant ease of estimating the model parameters from inverse fitting. The proposed model has been validated with experimental data and has been shown to successfully predict peak profile for a range of load capacities (15-28mg/mL), gradient lengths (10-30CV), bed heights (6-20cm), and for three different resins with good accuracy (as measured by estimation of residuals). The model has been also

  20. Synthesis and Characterization of Hapten-Protein Conjugates for Antibody Production against Cyanogenic Glycosides.

    Science.gov (United States)

    Bolarinwa, Islamiyat Folashade

    2015-07-01

    Consumption of cyanogenic plants can cause serious health problems for humans. The ability to detect and quantify cyanogenic glycosides, capable of generating cyanide, could contribute to prevention of cyanide poisoning from the consumption of improperly processed cyanogenic plants. Hapten-protein conjugates were synthesized with amygdalin and linamarin by using a novel approach. Polyclonal antibodies were generated by immunizing four New Zealand White rabbits with synthesized amygdalin-bovine serum albumin and linamarin-bovine serum albumin immunogen. This is the first time an antibody was produced against linamarin. Antibody titer curves were obtained from all the four rabbits by using a noncompetitive enzyme-linked immunosorbent assay. High antibody titer was obtained at dilutions greater than 1:50,000 from both immunogens. This new method is an important step forward in preventing ingestion of toxic cyanogenic glycosides.

  1. Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy

    OpenAIRE

    Maciej Żaczek; Marzanna Łusiak-Szelachowska; Ewa Jończyk-Matysiak; Beata Weber-Dąbrowska; Ryszard Międzybrodzki; Barbara Owczarek; Agnieszka Kopciuch; Wojciech Fortuna; Paweł Rogóż; Andrzej Górski

    2016-01-01

    In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties towards applied phages (K rate). Among 20 examined patients receiving...

  2. Production and Application of Monoclonal Antibodies Against Methamphetamine (MA) and Morphine (MP)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Three monoclonal antibodies (MAbs) against metharnphetamine (BD1, BD2, BD10) and four MAbs against morphine (MP6A8, MP6D9, MP7D6, MP8D9) are obtained by using MA-conjugated bovine serum albumin (BSA) and MP-conjugated BSA as immunogens, respectively. The MAbs against MA belonged to the IgG1 subclass with λ chains. The association constants (Ka) of the antibodies are higher than 107 L/mol (RIA). The MAbs against MP also belonged to the IgG1 subclass with λ chains. Ka of these antibodies is higher thanThree monoclonal antibodies (MAbs) against metharnphetamine (BD1, BD2, BD10) and four MAbs against morphine (MP6A8, MP6D9, MP7D6, MP8D9) are obtained by using MA-conjugated bovine serum albumin (BSA) and MP-conjugated BSA as immunogens, respectively. The MAbs against MA belonged to the IgG1 subclass with λ chains. The association constants (Ka) of the antibodies are higher than 107 L/mol (RIA). The MAbs against MP also belonged to the IgG1 subclass with λ chains. Ka of these antibodies is higher than 108

  3. Peptide-based Polyclonal Antibody Production against P110 Protein of Mycoplasma genitalium

    Science.gov (United States)

    Zarei, Omid; Irajian, Gholam Reza; Zarnani, Amir Hassan; Chamani-Tabriz, Leili; Emami, Shaghayegh; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah

    2011-01-01

    Mycoplasma genitalium (M.genitalium) is a sexually transmitted pathogen. Detection of this microorganism in clinical specimens by culture is rather difficult and time consuming. The aim of this study was to produce polyclonal antibody against a synthetic peptide from P110 protein of M.genitalium in order to develop a diagnostic tool for detection of this microorganism in clinical specimens. A synthetic peptide from P110 protein was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of a white New Zealand rabbit. The produced antibody was purified by affinity chromatography and its specific interaction with immunizing peptide was determined by ELISA. Immunoreactivity of the antibody was also tested by Western blotting in bacterial cell lysate prepared from M.genitalium G-37. To confirm its application as a diagnostic tool, indirect immunofluorescent staining method was performed on M.genitalium-infected PBMC using anti-P110 as the primary antibody. The results showed that produced antibody has excellent reactivity with immunizing peptide and also detected a single band of 110 kDa corresponding to P110 protein. M.genitalium-infected PBMC showed a bright fluorescent signal in IF staining. This antibody might be used as a tool in diagnostic applications. PMID:23408484

  4. Production and Purification of Monoclonal Antibody Against Tumor Marker of TPA

    Directory of Open Access Journals (Sweden)

    Seyyed Amir Abbas Ghodrat

    2016-05-01

    Full Text Available Considering the invasive nature of cancer cells, one of the most important and best indicator of them is the markers inside them. One of the most important markers that observed in some types of cancer cells in various parts of the body is the Cytokeratin. Tissue plasminogen activator antigen (TPA is a Cytokeratin composed of molecules with various molecular weights. The level of TPA serum as associated with cellular growth level and tumorization of cells. In this research, the hybrid of spleen cells in BALB/c female mouse with myeloma cells was conducted with a ratio of 10:1. The resulting monoclonal antibodies were confirmed by SDS-PAGE and western blot. Protein G chromatography was utilized to purify monoclonal antibodies. The results for determining isotypes showed IgM and IgG classes. The titer of the antibody obtained from various clones was capable of identifying Cytokeratin antigen with a dilution of 1/10000. The resulting antibodies were finally confirmed by western blot and all the 5 resulting monoclonal antibodies were capable of identifying a 48 kDa protein. The results indicate that with the help of TPA marker and the monoclonal antibodies produced against them, this marker can be recognized quickly with great accuracy in suspicious cases of cancer. Thus, appropriate measures will be taken to prevent and fight off its probable side effects. This factor can be further used to build a diagonal kit with high sensitivity.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler.

    Science.gov (United States)

    Choo, Chiou-Yu; Tian, Yuan; Kim, Wan-Seop; Blatter, Erich; Conary, Jon; Brady, Ciaran P

    2007-01-01

    A perfusion system is described for the production of a human monoclonal antibody in non-secreting murine myeloma (NS0) cells that was previously shown to be difficult to produce at high levels using fed-batch culture. The perfusion system was based on the use of a commercially available cell settler as the separation device to separate the cells from the culture. Separation efficiency of the cell settler was above 98%. Based on the growth and glucose consumption rates, fresh media was added to the culture and the turnover rate for the bioreactor was set at a maximum of 1.5 times the bioreactor volume per day. The perfusion process resulted in twice the maximum viable cell densities and up to three times the total protein production in a 53-day run period when compared to the fed-batch process. In addition, charge heterogeneity of the antibody as measured by ion exchange chromatography was lower for material purified from the perfusion runs compared to fed-batch. Perfusion mode of culture using a commercially available gravity settler is therefore a viable alternative to fed-batch mode for high-level production of this monoclonal antibody in NS0 cells.

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

    Science.gov (United States)

    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.

  8. Benchmarking B-cell epitope prediction with quantitative dose-response data on antipeptide antibodies: towards novel pharmaceutical product development.

    Science.gov (United States)

    Caoili, Salvador Eugenio C

    2014-01-01

    B-cell epitope prediction can enable novel pharmaceutical product development. However, a mechanistically framed consensus has yet to emerge on benchmarking such prediction, thus presenting an opportunity to establish standards of practice that circumvent epistemic inconsistencies of casting the epitope prediction task as a binary-classification problem. As an alternative to conventional dichotomous qualitative benchmark data, quantitative dose-response data on antibody-mediated biological effects are more meaningful from an information-theoretic perspective in the sense that such effects may be expressed as probabilities (e.g., of functional inhibition by antibody) for which the Shannon information entropy (SIE) can be evaluated as a measure of informativeness. Accordingly, half-maximal biological effects (e.g., at median inhibitory concentrations of antibody) correspond to maximally informative data while undetectable and maximal biological effects correspond to minimally informative data. This applies to benchmarking B-cell epitope prediction for the design of peptide-based immunogens that elicit antipeptide antibodies with functionally relevant cross-reactivity. Presently, the Immune Epitope Database (IEDB) contains relatively few quantitative dose-response data on such cross-reactivity. Only a small fraction of these IEDB data is maximally informative, and many more of them are minimally informative (i.e., with zero SIE). Nevertheless, the numerous qualitative data in IEDB suggest how to overcome the paucity of informative benchmark data.

  9. Carbamoylation abrogates the antioxidant potential of hydrogen sulfide.

    Science.gov (United States)

    Praschberger, Monika; Hermann, Marcela; Laggner, Christian; Jirovetz, Leopold; Exner, Markus; Kapiotis, Stylianos; Gmeiner, Bernhard M K; Laggner, Hilde

    2013-11-01

    Hydrogen sulfide (H2S) has been identified as the third gasotransmitter. Beside its role as signaling molecule in the cardiovascular and nervous system the antioxidant and cyto-protective properties of H2S have gained much attention. In the present study we show that cyanate, an uremic toxin which is found in abundant concentration in sera of patients suffering from chronic kidney disease (CKD), can abrogate the antioxidant and cytoprotective activity of H2S via S-carbamoylation reaction, a reaction that previously has only been shown to have a physiological effect on cysteine groups, but not on H2S. Carbamoylation strongly inhibited the free radical scavenging (ABTS(+·) and alkylperoxyl ROO(·)) properties of H2S. The extent of intracellular ROS formation induced by ROO(·) was diminished by H2S whereas carbamoylation counteracted the protective effect. Reagent HOCl was rapidly inactivated by H2S in contrast to the carbamoylated compound. Protein modification by HOCl was inhibited by H2S but carbamoylation significantly reduced the effect. Thus, S-carbamoylation of low molecular weight thiols by abrogating their antioxidant potential may contribute to the higher oxidative stress observed in CKD.

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

    Science.gov (United States)

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

    1990-06-01

    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 cervical smears. The antibody reacts strongly and consistently with biopsy specimens containing HPV-16 or HPV-33, but very weak reactions were occasionally observed with biopsy specimens or smears containing HPV-6 or HPV-11. The potential advantages of using a vaccinia recombinant are (i) the target protein is synthesised in a eukoryotic cell so that its "processing" and location are normal; (ii) cells infected with vaccinia recombinants can be subjected to various fixing procedures similar to those used for routine clinical material. This greatly increases the probability that an identified antibody will be useful in a clinical setting.

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

    Directory of Open Access Journals (Sweden)

    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

  12. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay

    OpenAIRE

    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya

    2015-01-01

    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-ba...

  13. Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings

    Science.gov (United States)

    Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.

    1988-01-01

    A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Production and characterization of monoclonal antibodies to wall-localized peroxidases from corn seedlings

    Science.gov (United States)

    Kim, S. H.; Terry, M. E.; Hoops, P.; Dauwalder, M.; Roux, S. J.

    1988-01-01

    A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

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

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Pedersen, Susanne Brix; Frøkiær, Hanne

    2004-01-01

    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...

  17. Tumor microenvironment contributes to Epstein-Barr virus anti-nuclear antigen-1 antibody production in nasopharyngeal carcinoma.

    Science.gov (United States)

    Ai, Ping; Li, Zhiping; Jiang, Yong; Song, Changping; Zhang, Lin; Hu, Huaizhong; Wang, Tao

    2017-08-01

    Nuclear antigen-1 (NA1) protein of Epstein-Barr virus (EBV) is expressed in EBV-infected cells in the microenvironment of cancer. Since immune cells infiltrate abundantly in nasopharyngeal carcinoma (NPC) tumor tissues, we hypothesized that the local tumor microenvironment may perform an important role in the production of antibodies directed at NA1. Furthermore, we hypothesized that anti-NA1 antibody originating in the local microenvironment could be secreted into the saliva of patients with NPC. In the present study, 20 healthy controls and 39 patients with NPC treated with intensity-modulated radiation therapy were recruited for the study. Saliva and serum samples were collected from the NPC patients, and nasopharyngeal tissue samples from the patients with NPC. The titers of anti-NA1 antibody [immunoglobulin A (IgA)] were determined by ELISA. Expression of NA1, human leukocyte antigen-antigen D related (HLA-DR), cluster of differentiation (CD)80, CD86, CD3, CD4, CD19 and IgA was detected by immunohistochemical staining on paraffin-embedded nasopharyngeal tissue sections. Anti-NA1 antibodies were detected in the serum and saliva samples of the patients with NPC. In infiltrating cells, expression of HLA-DR, CD80, CD86, CD3, CD4, CD19 and IgA was detected, indicating that dendritic cells, T lymphocytes and B lymphocytes were all present in the local tumor tissues. Furthermore, expression of EBNA1 protein was detected on the membrane of the NPC tumor cells. Therefore, the NPC tumor microenvironment has the potential to initiate a humoral response to EBNA1 by producing IgA antibodies.

  18. Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor.

    Science.gov (United States)

    Sotiriadis, A; Keshavarz, T; Keshavarz-Moore, E

    2001-01-01

    A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1). A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1). A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1). Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment.

  19. Rapid Transient Production of a Monoclonal Antibody Neutralizing the Porcine Epidemic Diarrhea Virus (PEDV) in Nicotiana benthamiana and Lactuca sativa.

    Science.gov (United States)

    Rattanapisit, Kaewta; Srijangwad, Anchalee; Chuanasa, Taksina; Sukrong, Suchada; Tantituvanont, Angkana; Mason, Hugh S; Nilubol, Dachrit; Phoolcharoen, Waranyoo

    2017-06-02

    Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro. These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection. Georg Thieme Verlag KG Stuttgart · New York.

  20. Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair.

    Directory of Open Access Journals (Sweden)

    Julia Esser-von Bieren

    2015-03-01

    Full Text Available Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/- or activating Fc receptors (Fcrg-/- displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb, whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3 by macrophages (MΦ and myofibroblasts (MF within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

  1. Immune antibodies and helminth products drive CXCR2-dependent macrophage-myofibroblast crosstalk to promote intestinal repair.

    Science.gov (United States)

    Esser-von Bieren, Julia; Volpe, Beatrice; Sutherland, Duncan B; Bürgi, Jérôme; Verbeek, J Sjef; Marsland, Benjamin J; Urban, Joseph F; Harris, Nicola L

    2015-03-01

    Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

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

    Science.gov (United States)

    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. Published by Elsevier Inc.

  3. Mixing monoclonal antibody formulations using bottom-mounted mixers: impact of mechanism and design on drug product quality.

    Science.gov (United States)

    Gikanga, Benson; Chen, Yufei; Stauch, Oliver B; Maa, Yuh-Fun

    2015-01-01

    Using bottom-mounted mixers, particularly those that are magnetically driven, is becoming increasingly common during the mixing process in pharmaceutical and biotechnology manufacturing because of their associated low risk of contamination, ease of use, and ability to accommodate low minimum mixing volumes. Despite these benefits, the impact of bottom-mounted mixers on biologic drug product is not yet fully understood and is scarcely reported. This study evaluated four bottom-mounted mixers to assess their impact on monoclonal antibody formulations. Changes in product quality (size variants, particles, and turbidity) and impact on process performance (sterile filtration) were evaluated after mixing. The results suggested that mixers that are designed to function with no contact between the impeller and the drive unit are the most favorable and gentle to monoclonal antibody molecules. Designs with contact or a narrow clearance tended to shear and grind the protein and resulted in high particle count in the liquid, which would subsequently foul a filter membrane during sterile filtration using a 0.22 μm pore size filter. Despite particle formation, increases in turbidity of the protein solution and protein aggregation/fragmentation were not detected. Further particle analysis indicated particles in the range of 0.2-2 μm are responsible for filter fouling. A small-scale screening model was developed using two types of magnetic stir bars mimicking the presence or absence of contact between the impeller and drive unit in the bottom-mounted mixers. The model is capable of differentiating the sensitivity of monoclonal antibody formulations to bottom-mounted mixers with a small sample size. This study fills an important gap in understanding a critical bioprocess unit operation. Mixing is an important unit operation in drug product manufacturing for compounding (dilution, pooling, homogenization, etc.). The current trend in adopting disposable bottom-mounted mixers has

  4. Phenotyping and auto-antibody production by liver-infiltrating B cells in primary sclerosing cholangitis and primary biliary cholangitis.

    Science.gov (United States)

    Chung, Brian K; Guevel, Bardia T; Reynolds, Gary M; Gupta Udatha, D B R K; Henriksen, Eva Kristine Klemsdal; Stamataki, Zania; Hirschfield, Gideon M; Karlsen, Tom Hemming; Liaskou, Evaggelia

    2017-02-01

    Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are immune-mediated biliary diseases that demonstrate prominent and restricted genetic association with human leukocyte antigen (HLA) alleles. In PBC, anti-mitochondrial antibodies (AMA) are specific and used as diagnostic biomarkers. PSC-relevant auto-antibodies remain controversial despite a distinct HLA association that mirrors archetypical auto-antigen driven disorders. Herein, we compared antibody-secreting B cells (ASCs) in PSC and PBC liver explants to determine if liver-infiltrating ASCs represent an opportune and novel source of disease-relevant auto-antibodies. Using enzymatic digestion and mechanical disruption, liver mononuclear cells (LIMCs) were isolated from fresh PSC and PBC explants and plasmablast (CD19+CD27+CD38(hi)CD138-) and plasma cell (CD19+CD27+CD38(hi)CD138+) ASCs were enumerated by flow cytometry. We observed 45-fold fewer plasma cells in PSC explants (n = 9) compared to PBC samples (n = 5, p < 0.01) and 10-fold fewer IgA-, IgG- and IgM-positive ASCs (p < 0.05). Liver-infiltrating ASCs from PSC and PBC explants were functional and produced similar concentrations of IgA, IgG and IgM following 2 weeks of culture. Antibody production by PBC ASCs (n = 3) was disease-specific as AMA to pyruvate dehydrogenase complex E2 subunit (PDC-E2) was detected by immunostaining, immunoblotting and ELISA. Antibody profiling of PSC supernatants (n = 9) using full-length recombinant human protein arrays (Cambridge Protein Arrays) revealed reactivities to nucleolar protein 3 (5/9) and hematopoietic cell-specific Lyn substrate 1 (3/9). Array analysis of PBC supernatants (n = 3) detected reactivities to PDC-E2 and hexokinase 1 (3/3). In conclusion, we detected unique frequencies of liver-infiltrating ASCs in PSC and PBC and in so doing, highlight a feasible approach for understanding disease-relevant antibodies in PSC. Copyright © 2016. Published by Elsevier Ltd.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, H.; Tamura, S.; Sudo, T.; Suzuki, T. (Kirin Brewery Co., Ltd., Gunma (Japan))

    1990-09-15

    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.

  7. Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures.

    Science.gov (United States)

    Lonoce, Chiara; Salem, Reda; Marusic, Carla; Jutras, Philippe V; Scaloni, Andrea; Salzano, Anna Maria; Lucretti, Sergio; Steinkellner, Herta; Benvenuto, Eugenio; Donini, Marcello

    2016-09-01

    Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco-engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2-3 mg/L). N-glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant-typical complex structures for N. tabacum-derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT-derived counterpart. Both antibody glyco-formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co-infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor-targeting mAb H10 with a human-compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of 'next generation' human therapeutic antibodies.

  8. Intravenous immunoglobulin prevents murine antibody-mediated acute lung injury at the level of neutrophil reactive oxygen species (ROS production.

    Directory of Open Access Journals (Sweden)

    John W Semple

    Full Text Available Transfusion-related acute lung injury (TRALI is a leading cause of transfusion-associated mortality that can occur with any type of transfusion and is thought to be primarily due to donor antibodies activating pulmonary neutrophils in recipients. Recently, a large prospective case controlled clinical study of cardiac surgery patients demonstrated that despite implementation of male donors, a high incidence of TRALI still occurred and suggested a need for additional interventions in susceptible patient populations. To examine if intravenous immunoglobulin (IVIg may be effective, a murine model of antibody-mediated acute lung injury that approximates human TRALI was examined. When BALB/c mice were injected with the anti-major histocompatibility complex class I antibody 34-1-2s, mild shock (reduced rectal temperature and respiratory distress (dyspnea were observed and pre-treatment of the mice with 2 g/kg IVIg completely prevented these symptoms. To determine IVIg's usefulness to affect severe lung damage, SCID mice, previously shown to be hypersensitive to 34-1-2s were used. SCID mice treated with 34-1-2s underwent severe shock, lung damage (increased wet/dry ratios and 40% mortality within 2 hours. Treatment with 2 g/kg IVIg 18 hours before 34-1-2s administration completely protected the mice from all adverse events. Treatment with IVIg after symptoms began also reduced lung damage and mortality. While the prophylactic IVIg administration did not affect 34-1-2s-induced pulmonary neutrophil accumulation, bone marrow-derived neutrophils from the IVIg-treated mice displayed no spontaneous ROS production nor could they be stimulated in vitro with fMLP or 34-1-2s. These results suggest that IVIg prevents murine antibody-mediated acute lung injury at the level of neutrophil ROS production and thus, alleviating tissue damage.

  9. PRODUCTION OF POLYCLONAL ANTIBODY TO THE COAT PROTEIN OF CITRUS TRISTEZA VIRUS IN CHICKEN EGGS

    Directory of Open Access Journals (Sweden)

    Nurhadi Nurhadi

    2016-10-01

    Full Text Available Citrus tristeza virus (CTV is one of the most destructive diseases in many citrus growing areas of Indonesia. Effective strategies for controlling CTV depend on diagnostic procedure namely enzyme-linked immunosorbent assay (ELISA. Study aimed to purify the CTV antigen and produced its polyclonal antibody. Virion of the severe CTV isolate designated UPM/ T-002 was concentrated by polyethylene glycol (PEG precipitation combined with low speed centrifugation. Semipurified antigen was further purified by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE. The specific coat protein (CP band of CTV with molecular weight of 25 kD was excised and eluted using elution buffer containing 0.25 M Tris-HCl pH 6.8 + 0.1% SDS, then used as antigen for injection into 6-month-old female of White Leghorn chicken. Results, showed than the specific polyclonal antibody raised against the 25-kDa CP had a titer of approximately 104, gave low background reaction with healthy plant sap and reacted specifically with CTV isolates. The reaction was equally strong for a severe, a moderate, a mild, and a symptomless isolate, suggesting a broad reaction range of this antibody toward different CTV isolates. Optimal virus titer can be obtained since virus loss during purification could be minimized and the highly purified antigen as an immunogen could be obtained by cutting out the CP band from SDS-PAGE gels. Large amount of highly titer of CTV antibody can be produced in chicken egg. The simplicity of the procedure makes it economically acceptable and technically adoptable because the antibody can be produced in basic laboratory.

  10. Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains.

    Science.gov (United States)

    Balabashin, Dmitriy; Kovalenko, Elena; Toporova, Viktoria; Aliev, Teimur; Panina, Anna; Svirshchevskaya, Elena; Dolgikh, Dmitry; Kirpichnikov, Mikhail

    2015-10-01

    Tumor necrosis factor-α (TNF-α) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-α recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

  11. Cardiomyocyte Antihypertrophic Effect of Adipose Tissue Conditioned Medium from Rats and Its Abrogation by Obesity is Mediated by the Leptin to Adiponectin Ratio.

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    Suresh C Bairwa

    Full Text Available White adipocytes are known to function as endocrine organs by secreting a plethora of bioactive adipokines which can regulate cardiac function including the development of hypertrophy. We determined whether adipose tissue conditioned medium (ATCM generated from the epididymal regions of normal rats can affect the hypertrophic response of cultured rat ventricular myocytes to endothelin-1 (ET-1 administration. Myocytes were treated with ET-1 (10 nM for 24 hours in the absence or presence of increasing ATCM concentrations. ATCM supressed the hypertrophic response to ET-1 in a concentration-dependent manner, an effect enhanced by the leptin receptor antagonist and attenuated by an antibody against the adiponectin AdipoR1 receptor. Antihypertrophic effects were also observed with ATCM generated from perirenal-derived adipose tissue. However, this effect was absent in ATCM from adipose tissue harvested from corpulent JCR:LA-cp rats. Detailed analyses of adipokine content in ATCM from normal and corpulent rats revealed no differences in the majority of products assayed, although a significant increase in leptin concentrations concomitant with decreased adiponectin levels was observed, resulting in a 11 fold increase in the leptin to adiponectin ratio in ATCM from JCR:LA-cp. The antihypertrophic effect of ATCM was associated with increased phosphorylation of AMP-activated protein kinase (AMPK, an effect abrogated by the AdipoR1 antibody. Moreover, the antihypertrophic effect of ATCM was mimicked by an AMPK activator. There was no effect of ET-1 on mitogen-activated protein kinase (MAPK activities 24 hour after its addition either in the presence or absence of ATCM. Our study suggests that adipose tissue from healthy subjects exerts antihypertrophic effects via an adiponectin-dependent pathway which is impaired in obesity, most likely due to adipocyte remodelling resulting in enhanced leptin and reduced adiponectin levels.

  12. Cardiomyocyte Antihypertrophic Effect of Adipose Tissue Conditioned Medium from Rats and Its Abrogation by Obesity is Mediated by the Leptin to Adiponectin Ratio.

    Science.gov (United States)

    Bairwa, Suresh C; Rajapurohitam, Venkatesh; Gan, Xiaohong Tracey; Mangat, Rabban; Proctor, Spencer D; Karmazyn, Morris

    2016-01-01

    White adipocytes are known to function as endocrine organs by secreting a plethora of bioactive adipokines which can regulate cardiac function including the development of hypertrophy. We determined whether adipose tissue conditioned medium (ATCM) generated from the epididymal regions of normal rats can affect the hypertrophic response of cultured rat ventricular myocytes to endothelin-1 (ET-1) administration. Myocytes were treated with ET-1 (10 nM) for 24 hours in the absence or presence of increasing ATCM concentrations. ATCM supressed the hypertrophic response to ET-1 in a concentration-dependent manner, an effect enhanced by the leptin receptor antagonist and attenuated by an antibody against the adiponectin AdipoR1 receptor. Antihypertrophic effects were also observed with ATCM generated from perirenal-derived adipose tissue. However, this effect was absent in ATCM from adipose tissue harvested from corpulent JCR:LA-cp rats. Detailed analyses of adipokine content in ATCM from normal and corpulent rats revealed no differences in the majority of products assayed, although a significant increase in leptin concentrations concomitant with decreased adiponectin levels was observed, resulting in a 11 fold increase in the leptin to adiponectin ratio in ATCM from JCR:LA-cp. The antihypertrophic effect of ATCM was associated with increased phosphorylation of AMP-activated protein kinase (AMPK), an effect abrogated by the AdipoR1 antibody. Moreover, the antihypertrophic effect of ATCM was mimicked by an AMPK activator. There was no effect of ET-1 on mitogen-activated protein kinase (MAPK) activities 24 hour after its addition either in the presence or absence of ATCM. Our study suggests that adipose tissue from healthy subjects exerts antihypertrophic effects via an adiponectin-dependent pathway which is impaired in obesity, most likely due to adipocyte remodelling resulting in enhanced leptin and reduced adiponectin levels.

  13. Terasaki-ELISA for murine IgE-antibodies.I.Quality of the detecting antibody: production and specificity testing of antisera specific for IgE

    NARCIS (Netherlands)

    Savelkoul, H.F.J.; Soeting, P.W.C.; Radl, J.; Linde-Preesman, van der A.A.

    1989-01-01

    In order to develop an ELISA for the quantitative determination of murine IgE, antisera specific for murine IgE were prepared in the goat and rabbit. As immunogen, monoclonal IgE antibody mixtures of several allotypically different hybridomas were used. Before use, these antibodies were purified emp

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

    Institute of Scientific and Technical Information of China (English)

    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

  15. Production of anti-double-stranded DNA antibodies in activated lymphocyte derived DNA induced lupus model was dependent on CD4+ T cells.

    Science.gov (United States)

    Wen, Z; Xu, L; Xu, W; Xiong, S

    2012-04-01

    Our previous study demonstrated that activated lymphocyte derived DNA (ALD-DNA) could function as an autoantigen to induce production of anti-double-stranded DNA (anti-dsDNA) antibodies in syngeneic BALB/c mice. Here we carefully evaluated the potential role of T cells in the induction of anti-dsDNA antibody. We demonstrated that ALD-DNA could effectively induce production of anti-dsDNA antibodies in vivo and in vitro. In contrast, ALD-DNA could not induce the generation of anti-dsDNA antibodies in nude mice. We further showed that in vivo depletion of CD3(+) T cells blocked the induction of anti-dsDNA antibodies in BALB/c mice. Notably, we demonstrated that CD4(+) but not CD8(+) T cells conferred ALD-DNA to induce anti-dsDNA antibodies. Finally, we demonstrated that adoptive transfer of CD4(+) T cells could rescue ALD-DNA induced anti-dsDNA antibodies in nude mice. Our results suggested that T helper cells were required for ALD-DNA to induce anti-dsDNA antibodies. These findings could further our understanding about the immunogenic properties of DNA and throw new light on SLE pathogenesis.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  19. Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization

    DEFF Research Database (Denmark)

    Pedersen, Thomas Skamris; Tian, Xinsheng; Thorolfsson, Matthias

    2016-01-01

    PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody...... aggregation. METHODS: We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography......H IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were...

  20. Production and Characterisation of Anti-Cardiac Troponin-I Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Kh. H. Haider

    1994-01-01

    Full Text Available Cardiac troponin-I (cTn-I was isolated from bovine left ventricular tissue and used as immunogen. Sixteen murine hybridoma lines were produced with two of them. I D 12 and 5F4, showing a high specificity for cTn-I; both of these monoclonal antibodies (McAbs were isotyped as IgG I with kappa - light chains. The specificity of the McAbs for cTn-1 was confirmed by ELISA, western blotting and by the ability of the antibodies to block actomyosin ATPase inhibition by cTn-I. The McAbs may be useful for human ill vivo imaging of myocardial infarcts and other pathological conditions related to cardiac myocyte damage.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  2. Effect of operating conditions in production of diagnostic Salmonella Enteritidis O-antigen-specific monoclonal antibody in different bioreactor systems.

    Science.gov (United States)

    Ayyildiz-Tamis, Duygu; Nalbantsoy, Ayse; Elibol, Murat; Deliloglu-Gurhan, Saime Ismet

    2014-01-01

    In this study, different cultivation systems such as roller bottles (RB), 5-L stirred-tank bioreactor (STR), and disposable bioreactors were used to cultivate hybridoma for lab-scale production of Salmonella Enteritidis O-antigen-specific monoclonal antibody (MAb). Hybridoma cell line was cultivated in either serum-containing or serum-free medium (SFM) culture conditions. In STR, MAb production scaled up to 4 L, and production capabilities of the cells were also evaluated in different featured production systems. Moreover, the growth parameters of the cells in all production systems such as glucose consumption, lactate and ammonia production, and also MAb productivities were determined. Collected supernatants from the reactors were concentrated by a cross-flow filtration system. In conclusion, cells were not adapted to SFM in RB and STR. Therefore, less MAb titer in both STR and RB systems with SFM was observed compared to the cultures containing fetal bovine serum-supplemented medium. A higher MAb titer was gained in the membrane-aerated system compared to those in STR and RB. Although the highest MAb titer was obtained in the static membrane bioreactor system, the highest productivity was obtained in STR operated in semicontinuous mode with overlay aeration.

  3. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

    Science.gov (United States)

    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya

    2015-06-10

    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

  4. Production and characterization of monoclonal antibody specific to recombinant dengue multi-epitope protein.

    Science.gov (United States)

    Abhyankar, Ajay Vinayak; Bhargava, Rakesh; Jana, Asha Mukul; Sahni, Ajay Kumar; Rao, P V Lakshmana

    2008-06-01

    Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.

  5. Identification of two quantitative trait loci involved in antibody production on mouse chromosome 8.

    Science.gov (United States)

    Puel, A; Mevel, J C; Bouthillier, Y; Decreusefond, C; Fridman, W H; Feingold, N; Mouton, D

    1998-03-01

    Several quantitative trait loci (QTLs) contributing to the extreme phenotypes of the selected high (H) and low (L) antibody-responder lines of mice were mapped on distinct chromosomes. Successive backcrosses were bred to reduce the length of the QTL-bearing segment detected on chromosome 8 and to produce congenic lines to test gene effect independently of the other QTLs. An increase in antibody responses was repeatedly found to be associated with inheritance of the H-line allele at two markers separated by 30 cM on that chromosome. In the successive backcrosses, background and unlinked involved genes of H-line origin were progressively eliminated; however, unexpected within-progeny variations persisted in the third and even fourth backcross. Nevertheless, the presence of two QTLs within the considered interval was definitely demonstrated in distinct progenies of the fourth backcross which separately inherited one of the two gene-marker H-line alleles. The previously identified chromosome 8 segment therefore contains at least two QTLs involved in antibody responsiveness.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. The role of evolutionarily conserved germ-line DH sequence in B-1 cell development and natural antibody production.

    Science.gov (United States)

    Vale, Andre M; Nobrega, Alberto; Schroeder, Harry W

    2015-12-01

    Because of N addition and variation in the site of VDJ joining, the third complementarity-determining region of the heavy chain (CDR-H3) is the most diverse component of the initial immunoglobulin antigen-binding site repertoire. A large component of the peritoneal cavity B-1 cell component is the product of fetal and perinatal B cell production. The CDR-H3 repertoire is thus depleted of N addition, which increases dependency on germ-line sequence. Cross-species comparisons have shown that DH gene sequence demonstrates conservation of amino acid preferences by reading frame. Preference for reading frame 1, which is enriched for tyrosine and glycine, is created both by rearrangement patterns and by pre-BCR and BCR selection. In previous studies, we have assessed the role of conserved DH sequence by examining peritoneal cavity B-1 cell numbers and antibody production in BALB/c mice with altered DH loci. Here, we review our finding that changes in the constraints normally imposed by germ-line-encoded amino acids within the CDR-H3 repertoire profoundly affect B-1 cell development, especially B-1a cells, and thus natural antibody immunity. Our studies suggest that both natural and somatic selection operate to create a restricted B-1 cell CDR-H3 repertoire.

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

    Directory of Open Access Journals (Sweden)

    Kiefer Hans

    2009-06-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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

  9. Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette

    2004-01-01

    This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...... advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal...

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

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    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.

  11. Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments.

    Science.gov (United States)

    Kim, Sung Hyun; Lee, Gyun Min

    2009-06-01

    An efficient method of formulating serum-free medium (SFM) for production of therapeutic antibodies by recombinant CHO (rCHO) cells was developed using two rCHO cell lines producing a therapeutic antibody. In this method, ten kinds of SFM were prepared by supplementing the basal SFM with statistically designed mixtures (total 5 g L(-1)) of three non-animal-derived hydrolysates: yeastolate, soy hydrolysate, and wheat gluten hydrolysate. When the two rCHO cell lines were cultivated, the mixtures of soy hydrolysate and wheat gluten hydrolysate showed a positive effect on cell growth. On the other hand, the mixtures including a high portion of yeastolate significantly enhanced specific antibody productivity. To reconstitute the mixture ratios of the three hydrolysates for high growth and antibody production, the effect of each medium was analyzed by the statistical program Design-Expert(R). The resulting medium gave a 1.9-3.3-fold increase in the maximum antibody concentration, compared to the basal SFM. Taken together, the supplementation of hydrolysates to the basal SFM with the help of statistical analysis is an efficient means of developing SFM for therapeutic antibody production by rCHO cells.

  12. Synovial fluid mononuclear cells provide an environment for long-term survival of antibody-secreting cells and promote the spontaneous production of anti-citrullinated protein antibodies.

    Science.gov (United States)

    Kerkman, Priscilla F; Kempers, Ayla C; van der Voort, Ellen I H; van Oosterhout, Maikel; Huizinga, Tom W J; Toes, René E M; Scherer, Hans U

    2016-12-01

    In rheumatoid arthritis (RA), observations point to a crucial role for (autoreactive) B cells in disease pathogenesis. Here, we studied whether cells from the synovial environment impact on the longevity of autoreactive B cell responses against citrullinated antigens. Synovial fluid mononuclear cells and peripheral blood mononuclear cells (SFMC/PBMC) were obtained from patients with established RA and assessed for the presence of B cell subpopulations. Cells spontaneously secreting anti-citrullinated protein antibodies (ACPA-IgG) directly ex vivo were detected by antigen-specific Enzyme-Linked ImmunoSpot (ELISpot) assay. SFMC and PBMC were cultured to assess the degree of spontaneous ACPA-IgG secretion. Cells surviving for several weeks were characterised by carboxyfluorescein succinimidyl ester (CFSE) labelling and Ki-67 staining. Cells spontaneously secreting ACPA-IgG were readily detectable in peripheral blood and synovial fluid (SF) of patients with ACPA-positive RA. SFMC showed an up to 200-fold increase in ex vivo ACPA-IgG secretion compared with PBMC despite lower numbers of B cells in SFMC. ELISpot confirmed the presence of spontaneously ACPA-IgG-secreting cells, accounting for up to 50% (median 12%) of all IgG-secreting cells in SF. ACPA-IgG secretion was remarkably stable in SFMC cultures, maintained upon depletion of the CD20(+) B cell compartment and detectable for several months. CFSE labelling and Ki-67 staining confirmed the long-term survival of non-dividing plasma cells (PCs). This study demonstrates a high frequency of differentiated, spontaneously ACPA-IgG-secreting cells in SF. These cells are supported by SFMC for prolonged survival and autoantibody secretion, demonstrating that the synovial compartment is equipped to function as inflammatory niche for PC survival. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  13. Evolution of anti-Trypanosoma cruzi antibody production in patients with chronic Chagas disease: Correlation between antibody titers and development of cardiac disease severity

    Science.gov (United States)

    Georg, Ingebourg; Hasslocher-Moreno, Alejandro Marcel; Xavier, Sergio Salles; de Holanda, Marcelo Teixeira; Bonecini-Almeida, Maria da Gloria

    2017-01-01

    Chagas disease is one of the most important endemic infections in Latin America affecting around 6–7 million people. About 30–50% of patients develop the cardiac form of the disease, which can lead to severe cardiac dysfunction and death. In this scenario, the identification of immunological markers of disease progression would be a valuable tool for early treatment and reduction of death rates. In this observational study, the production of anti-Trypanosoma cruzi antibodies through a retrospective longitudinal follow-up in chronic Chagas disease patients´ cohort and its correlation with disease progression and heart commitment was evaluated. Strong inverse correlation (ρ = -0.6375, p = 0.0005) between anti-T. cruzi IgG1 titers and left ventricular ejection fraction (LVEF) in chronic Chagas cardiomyopathy (CCC) patients were observed after disease progression. Elevated levels of anti-T. cruzi IgG3 titers were detected in all T. cruzi-infected patients, indicating a lack of correlation of this IgG isotype with disease progression. Furthermore, low levels of anti-T. cruzi IgG2, IgG4, and IgA were detected in all patients through the follow-up. Although without statistical significance anti-T. cruzi IgE tends to be more reactive in patients with the indeterminate form (IND) of the disease (p = 0.0637). As this study was conducted in patients with many years of chronic disease no anti-T. cruzi IgM was detected. Taken together, these results indicate that the levels of anti-T. cruzi IgG1 could be considered to seek for promising biomarkers to predict the severity of chronic Chagas disease cardiomyopathy. PMID:28723905

  14. The modulatory influence of some Echinacea-based remedies on antibody production and cellular immunity in mice

    Science.gov (United States)

    Sokolnicka, Irena; Skopińska-Różewska, Ewa; Skopiński, Piotr

    2016-01-01

    Echinacea purpurea-containing remedies are herbal medicines used in respiratory tract infections and several inflammatory conditions as enhancers of non-specific and modulators of specific cellular immunity. They also exert anti-inflammatory, anti-viral, and anti-microbial activity. The aim of the present study was to compare the in vivo influence of orally administered three Echinacea purpurea-based remedies (IMMUNAL drops, ECHINACEA FORTE drops, IMMUNAL FORTE tablets) on some parameters of cellular and humoral immunity in mice. Results Feeding mice for seven days with IMMUNAL drops resulted in enhanced anti-SRBC antibody production and modulatory effect on proliferative response to PHA of their splenic lymphocytes. No stimulatory effect was observed on splenocytes chemokinesis. Mice fed with ECHINACEA FORTE drops presented enhanced response to PHA of their splenocytes. However, contrary to the previous group, no enhancement of antibody production was observed. In this group, lymphocyte-induced immunological angiogenesis (LIA) and chemokinesis (spontaneous migration – SM) of spleen lymphocytes was diminished after feeding mice with both doses (LIA) or with a higher dose (SM) of remedy. Lymphocyte-induced immunological angiogenesis activity of splenocytes collected from animals fed with prophylactic and therapeutic IMMUNAL FORTE tablet doses did not differ from the controls. PMID:27095917

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    Pleckaityte, Milda; Zvirbliene, Aurelija; Sezaite, Indre; Gedvilaite, Alma

    2011-12-15

    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. 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. Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and

  17. Modeling of cell culture damage and recovery leads to increased antibody and biomass productivity in CHO cell cultures.

    Science.gov (United States)

    Naderi, Saeideh; Nikdel, Ali; Meshram, Mukesh; McConkey, Brendan; Ingalls, Brian; Budman, Hector; Scharer, Jeno

    2014-09-01

    The development of an efficient and productive cell-culture process requires a deep understanding of intracellular mechanisms and extracellular conditions for optimal product synthesis. Mathematical modeling provides an effective strategy to predict, control, and optimize cell performance under a range of culture conditions. In this study, a mathematical model is proposed for the investigation of cell damage of a Chinese hamster ovary cell culture secreting recombinant anti-RhD monoclonal antibody (mAb). Irreversible cell damage was found to be correlated with a reduction in pH. This irreversible damage to cellular function is described mathematically by a Tessier-based model, in which the actively growing fraction of cells is dependent on an intracellular metabolic product acting as a growth inhibitor. To further verify the model, an offline model-based optimization of mAb production in the cell culture was carried out, with the goal of minimizing cell damage and thereby enhancing productivity through intermittent refreshment of the culture medium. An experimental implementation of this model-based strategy resulted in a doubling of the yield as compared to the batch operation and the resulting biomass and productivity profiles agreed with the model predictions.

  18. Curcumin and folic acid abrogated methotrexate induced vascular endothelial dysfunction.

    Science.gov (United States)

    Sankrityayan, Himanshu; Majumdar, Anuradha S

    2016-01-01

    Methotrexate, an antifolate drug widely used in rheumatoid arthritis, psoriasis, and cancer, is known to cause vascular endothelial dysfunction by causing hyperhomocysteinemia, direct injury to endothelium or by increasing the oxidative stress (raising levels of 7,8-dihydrobiopterin). Curcumin is a naturally occurring polyphenol with strong antioxidant and anti-inflammatory action and therapeutic spectra similar to that of methotrexate. This study was performed to evaluate the effects of curcumin on methotrexate induced vascular endothelial dysfunction and also compare its effect with that produced by folic acid (0.072 μg·g(-1)·day(-1), p.o., 2 weeks) per se and in combination. Male Wistar rats were exposed to methotrexate (0.35 mg·kg(-1)·day(-1), i.p.) for 2 weeks to induce endothelial dysfunction. Methotrexate exposure led to shedding of endothelium, decreased vascular reactivity, increased oxidative stress, decreased serum nitrite levels, and increase in aortic collagen deposition. Curcumin (200 mg·kg(-1)·day(-1) and 400 mg·kg(-1)·day(-1), p.o.) for 4 weeks prevented the increase in oxidative stress, decrease in serum nitrite, aortic collagen deposition, and also vascular reactivity. The effects were comparable with those produced by folic acid therapy. The study shows that curcumin, when concomitantly administered with methotrexate, abrogated its vascular side effects by preventing an increase in oxidative stress and abating any reduction in physiological nitric oxide levels.

  19. Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis.

    Science.gov (United States)

    Nakajima, Mie; Kodama, Michi; Yanase, Haruko; Iwanaga, Toshihiko; Mulenga, Albert; Ohashi, Kazuhiko; Onuma, Misao

    2003-08-14

    There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.

  20. Monoclonal Antibodies Recognising Sialyl-Tn: Production and Application to Immunochemistry

    Directory of Open Access Journals (Sweden)

    Peter L. Devine

    1994-01-01

    Full Text Available In order to develop reagents that can detect the exposed sialyl-Tn antigen (NeuAcα2,6GaINAcα 1-O-Ser/Thr on tumour-associated mucins, we have prepared monoclonal antibodies (mabs 3C2 and 301, both IgM against ovine submaxillary mucin (OSM; >98% of glycans as sialyl-Tn. These mabs showed strong reactivity with OSM and bovine submaxillary mucin (BSM; 50% of glycans as sialyl-Tn but did not react with desialylated OSM or BSM. Sialic acid at I mg/ml did not significantly inhibit mab binding to OSM, suggesting that the linkage to GalNAc may be important for mab binding. 3C2 and 3D I also showed similar reactivity to sialyl-Tn reactive mab Bn.3, and detected Bn.3 capturedOSM in a sandwich ELISA. In Western blotting of mucus from a patient with a mucinous ovarian tumour, the mabs reacted with high molecular weight (>200 kDa species. In immunohistochemistry, these mabs showed strong reactivity with most cancers of the colon, lung, and stomach, and also some tumours of the ovary and breast. There was only limited reactivity in normal tissue from these sites. The antibodies should be useful reagents for the detection of the sialyl-Tn antigen in human cancers.

  1. Transgenic Production of an Anti HIV Antibody in the Barley Endosperm.

    Directory of Open Access Journals (Sweden)

    Goetz Hensel

    Full Text Available Barley is an attractive vehicle for producing recombinant protein, since it is a readily transformable diploid crop species in which doubled haploids can be routinely generated. High amounts of protein are naturally accumulated in the grain, but optimal endosperm-specific promoters have yet to be perfected. Here, the oat GLOBULIN1 promoter was combined with the legumin B4 (LeB4 signal peptide and the endoplasmic reticulum (ER retention signal (SEKDEL. Transgenic barley grain accumulated up to 1.2 g/kg dry weight of recombinant protein (GFP, deposited in small roundish compartments assumed to be ER-derived protein bodies. The molecular farming potential of the system was tested by generating doubled haploid transgenic lines engineered to synthesize the anti-HIV-1 monoclonal antibody 2G12 with up to 160 μg recombinant protein per g grain. The recombinant protein was deposited at the periphery of protein bodies in the form of a mixture of various N-glycans (notably those lacking terminal N-acetylglucosamine residues, consistent with their vacuolar localization. Inspection of protein-A purified antibodies using surface plasmon resonance spectroscopy showed that their equilibrium and kinetic rate constants were comparable to those associated with recombinant 2G12 synthesized in Chinese hamster ovary cells.

  2. Expression of porcine sapovirus VP1 gene and VP1 specific monoclonal antibody production.

    Science.gov (United States)

    Ryu, Min-Sang; Jung, Eun-Hye; Cho, Kyoung-Oh; Kang, Shien-Young

    2012-06-01

    Sapovirus (SaV) is an agent of human and porcine gastroenteritis and a member of the family Caliciviridae. SaV has been classified based on VP1 full gene nucleotide sequences into five genogroups (GI-GV), among which GIII is known to infect pigs. The VP1 folds into two major domains designated S and P for the shell and protruding domain, respectively. The P domain is divided into two subdomains, P1 and P2. In this study, the VP1 full gene and the S, P, and P2 regions of the VP1 gene of porcine SaV were expressed using a baculovirus expression system. Expressed proteins in the recombinant virus were confirmed by polymerase chain reaction, indirect fluorescence antibody (IFA) testing, and Western blot analysis. Four hybridomas secreting VP1-specific monoclonal antibodies (MAbs) against porcine sapovirus were generated. Four MAbs were characterized according to their IFA and Western blot analysis results. All of the hybridomas produced in this study secreted MAbs binding to S domain of VP1 protein specifically. The MAbs produced in this study can be used as specific diagnostic reagents for detecting porcine SaV.

  3. Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

    Institute of Scientific and Technical Information of China (English)

    LIU Xian-jin; YAN Chun-rong; LIU Yuan; YU Xiang-yang; ZHANG Cun-zheng

    2008-01-01

    Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method (AEM) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide method (EDC). The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL-1 and an I50 value of 0.182 ug mL-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 ug mL-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semi-quantitation determination of the above former five organophosphorus pesticides.

  4. Production of monoclonal antibodies for Avian Metapneumovirus (SHS-BR-121 isolated in Brazil

    Directory of Open Access Journals (Sweden)

    LT Coswig

    2007-12-01

    Full Text Available Avian Metapneumovirus (aMPV, also called Turkey Rhinotracheitis Virus (TRTV, is an upper respiratory tract infection of turkeys, chickens and other avian species. Five monoclonal antibodies (MAbs were created against the Brazilian isolate (SHS-BR-121 of aMPV, MAbs 1A5B8; 1C1C4; 2C2E9 and 2A4C3 of IgG1 and MAb 1C1F8 of IgG2a. Four Mabs (1A5B8; 1C1C4; 2C2E9 and 2A4C3 showed neutralizing activity and three (1A5B8; 1C1C4 and 2A4C3 inhibited cellular fusion in vitro. These MAbs were used to investigate antigenic relationship among three strains (SHS-BR-121, STG 854/88 and TRT 1439/91 of aMPV subtypes A and B using cross-neutralization test. The results confirm that the monoclonal antibodies described can be used as a valuable tool in the epizootiological and serological studies, and also for the specific diagnosis of the subtypes in the infection for Avian Metapneumovirus.

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

    Directory of Open Access Journals (Sweden)

    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

  6. Silence of MCL-1 upstream signaling by shRNA abrogates multiple myeloma growth.

    Science.gov (United States)

    Wang, Mengchang; Wu, Di; Liu, Pengbo; Deng, Jiusheng

    2014-01-01

    Multiple myeloma (MM) is an incurable B-cell cancer with accumulated clonal abnormal plasma cells in bone marrow of patients. MCL-1 (myeloid cell leukemia sequence 1) protein is an anti-apoptotic molecule in MM cells and regulated by pro-inflammatory cytokine IL-6 and downstream signaling molecules STAT3, PI3K and MAPK. The purpose of this study is to investigate the effect of STAT3, PI3K and MAPK gene silence on MCL-1 expression in human MM cells and the consequence of cell survival. Lentivirus small hairpin RNA (shRNA) interference techniques were utilized to knock down STAT3, PI3K or MAPK genes. Gene and protein expression was quantified by quantitative real-time PCR and Western Blot. MM cell apoptosis was examined by annexin-V FITC/propidium iodide staining. Efficient silence of STAT3, PI3K, MAPK1 or MAPK2 gene robustly abrogated IL-6 enhanced MCL-1 expression and suppressed MM cell growth. Silencing STAT3 gene inhibited PI3K expression, silencing PI3K markedly abrogated STAT3 and MAPK production. Inhibition of MAPK2 gene by shMAPK2 suppressed STAT3, PI3K and MAPK1 expression in the cells. Silencing of STAT3, PI3K and MAPK2 together completely blocked MCL-1 expression in MM cells. There is a syngeneic effect among the three independent STAT3, PI3K and MAPK2 survival-signaling pathways related to MCL-1 expression in MM cells. shRNAs silencing of STAT3, PI3K and MAPK2 together could provide an effective strategy to treat MM.

  7. Production of monoclonal antibodies against the outer cell wall of Clostridium tyrobutyricum.

    Science.gov (United States)

    Talbot, F; Robreau, G; Gueguen, F; Malcoste, R

    1994-02-01

    Several hybridoma cell lines producing murine monoclonal antibodies (mAbs) directed to the Clostridium tyrobutyricum outer cell wall have been established and characterized. Whole bacteria, crude extract of cell wall, and polysaccharide fraction of crude extract have been used as immunogens. The immunizations were performed either in vivo or in vitro after priming in vivo. Amongst the clones obtained, six hybridoma cell lines were selected. Four mAbs recognized only the immunizing strain (ATCC 25755), while two mAbs recognized all the C. tyrobutyricum tested strains. Three mAbs were IgM, one IgG3, and two IgG1 isotypes. The antigens (proteins or polysaccharides) recognized by these mAbs have been characterized by Western Blot. These mAbs could be used for an early detection of C. tyrobutyricum in milk.

  8. Production and characterization of highly specific monoclonal antibodies to D-glutamic acid.

    Science.gov (United States)

    Sakamoto, Seiichi; Matsuura, Yurino; Yonenaga, Yayoi; Tsuneura, Yumi; Aso, Mariko; Kurose, Hitoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2014-12-01

    Most of the functions of D-amino acids (D-AA) remain unclear because of little analytic methods for specific detection/determination. In this study, a highly specific monoclonal antibody to D-glutamic acid (D-Glu-MAb) was produced using a hybridoma method. Characterization of D-Glu-MAb by indirect enzyme-linked immunosorbent assay (ELISA) revealed that it has high selectivity against D-Glu-glutaraldehyde (GA) conjugates, while no cross-reaction was observed when 38 other kinds of AA-GA conjugates were used. Moreover, subsequent indirect competitive ELISA disclosed that an epitope of D-Glu-MAb is a D-Glu-GA molecule in the conjugates, suggesting that D-Glu-MAb could be a useful tool to investigate the functional analysis of D-Glu in immunostaining.

  9. Production and characterisation of monoclonal antibodies to phytoene synthase of Lycopersicon esculentum.

    Science.gov (United States)

    Fraser, P D; Misawa, N; Sandmann, G; Johnson, J; Schuch, W; Bramley, P M

    1998-10-01

    Monoclonal antibodies have been prepared against the tomato (Lycopersicon esculentum Mill.) fruit ripening-enhanced phytoene synthase (PSY1). The antigen was prepared as a beta-galactosidase fusion protein by cloning a 1.13 kb fragment of Psy1 cDNA into pUR291, followed by transformation of E. coli. The fusion protein, induced by IPTG, was purified by preparative SDS-PAGE and used to elicit an immune response. The cell lines were screened for cross-reactivity against beta-galactosidase-phytoene synthase fusion protein in E. coli extracts using western blotting and ELISA detection procedures. Positive clones were further screened for their ability to cross-react with the mature phytoene synthase protein on western blots as well as their ability to inhibit enzyme activity. Eleven monoclonal lines were obtained. Nine of these, all of the IgM isotype, exhibited strong responses to phytoene synthase of ripe tomato fruit on western blots, but did not inhibit enzyme activity effectively. The other two lines (IgG/la 2 isotypes) inhibited phytoene synthase activity in ripe tomato stroma, but produced a poor response to the protein on western blots. The monoclonals identified a ripe fruit phytoene synthase of 38 kDa, exclusively located in the chromoplast. In contrast, antibodies were unable to detect microbial phytoene synthases, nor phytoene synthase of maize leaf, tomato chloroplast or mango fruit extracts, either on western blots or from inhibition of phytoene synthase activity. However, they did cross-react with a 44 kDa protein from carrot leaf stroma and with three different proteins (44, 41, and 37 kDa) in carrot root. Cross-reactivity was also found with a 37 kDa protein from pumpkin fruit stroma.

  10. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  11. Production of a human single-chain variable fragment antibody against esophageal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ming-Yan Xu; Xiao-Hu Xu; Geng-Zhen Chen; Xiao-Ling Deng; Jonathan Li; Xiao-Jun Yu; Mei-Zhen Chen

    2004-01-01

    AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFv antibody against esophageal cancer.METHODS: Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed. esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coli HB2151 which was transfected with the positive phage clone, then purified by affinity chromatography.Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.RESULTS: The size of scFv gene library was approximately 9×106 clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells, 25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Eca109 cells, but not to HeLa and NHEEC cells. Variable heavy (VH) gene from one of the positive clones was shown to be derived from the γ chain subgroup Ⅳ of immunoglobulin, and variable light (VL) gene from the κchain subgroup I of immunoglobulin.CONCLUSION: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.

  12. Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

    Directory of Open Access Journals (Sweden)

    L.C. Kreutz

    2000-12-01

    Full Text Available Three Brazilian isolates of bovine viral diarrhea virus (BVDV, antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs. Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11, were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid and 1:100 (hybridoma culture supernatant in IFA and immunoperoxidase (IPX staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

  13. Plant production of anti-β-glucan antibodies for immunotherapy of fungal infections in humans.

    Science.gov (United States)

    Capodicasa, Cristina; Chiani, Paola; Bromuro, Carla; De Bernardis, Flavia; Catellani, Marcello; Palma, Angelina S; Liu, Yan; Feizi, Ten; Cassone, Antonio; Benvenuto, Eugenio; Torosantucci, Antonella

    2011-09-01

    There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

  14. Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

    Science.gov (United States)

    Pybus, Leon P; Dean, Greg; West, Nathan R; Smith, Andrew; Daramola, Olalekan; Field, Ray; Wilkinson, Stephen J; James, David C

    2014-02-01

    Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of

  15. Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer.

    Science.gov (United States)

    Horenstein, A L; Poiesi, C; DeMonte, L; Camagna, M; Mariani, M; Albertini, A; Malavasi, F

    1993-01-01

    An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.

  16. Epithelial inactivation of Yy1 abrogates lung branching morphogenesis.

    Science.gov (United States)

    Boucherat, Olivier; Landry-Truchon, Kim; Bérubé-Simard, Félix-Antoine; Houde, Nicolas; Beuret, Laurent; Lezmi, Guillaume; Foulkes, William D; Delacourt, Christophe; Charron, Jean; Jeannotte, Lucie

    2015-09-01

    Yin Yang 1 (YY1) is a multifunctional zinc-finger-containing transcription factor that plays crucial roles in numerous biological processes by selectively activating or repressing transcription, depending upon promoter contextual differences and specific protein interactions. In mice, Yy1 null mutants die early in gestation whereas Yy1 hypomorphs die at birth from lung defects. We studied how the epithelial-specific inactivation of Yy1 impacts on lung development. The Yy1 mutation in lung epithelium resulted in neonatal death due to respiratory failure. It impaired tracheal cartilage formation, altered cell differentiation, abrogated lung branching and caused airway dilation similar to that seen in human congenital cystic lung diseases. The cystic lung phenotype in Yy1 mutants can be partly explained by the reduced expression of Shh, a transcriptional target of YY1, in lung endoderm, and the subsequent derepression of mesenchymal Fgf10 expression. Accordingly, SHH supplementation partially rescued the lung phenotype in vitro. Analysis of human lung tissues revealed decreased YY1 expression in children with pleuropulmonary blastoma (PPB), a rare pediatric lung tumor arising during fetal development and associated with DICER1 mutations. No evidence for a potential genetic interplay between murine Dicer and Yy1 genes during lung morphogenesis was observed. However, the cystic lung phenotype resulting from the epithelial inactivation of Dicer function mimics the Yy1 lung malformations with similar changes in Shh and Fgf10 expression. Together, our data demonstrate the crucial requirement for YY1 in lung morphogenesis and identify Yy1 mutant mice as a potential model for studying the genetic basis of PPB.

  17. A bioavailable cathepsin S nitrile inhibitor abrogates tumor development.

    Science.gov (United States)

    Wilkinson, Richard D A; Young, Andrew; Burden, Roberta E; Williams, Rich; Scott, Christopher J

    2016-04-21

    Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models. Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors. We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis. In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.

  18. Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv against human ICAM-1

    Directory of Open Access Journals (Sweden)

    H. Sun

    2014-07-01

    Full Text Available Intercellular adhesion molecule-1 (ICAM-1 is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

  19. Production, characterization and utility of a panel of monoclonal antibodies for the detection of toluene diisocyanate haptenated proteins.

    Science.gov (United States)

    Ruwona, Tinashe B; Johnson, Victor J; Hettick, Justin M; Schmechel, Detlef; Beezhold, Donald; Wang, Wei; Simoyi, Reuben H; Siegel, Paul D

    2011-10-28

    Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals used in the manufacture of polyurethane products and are the most commonly reported cause of occupational asthma. Mechanistic disease studies and development of biomonitoring and research tools, such as monoclonal antibodies (mAbs) have been hampered by dNCOs' ability to self-polymerize and to cross-link biomolecules. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs), with potential use in immunoassays for exposure and biomarker assessments, were produced and reactivities characterized against mono- and diisocyanate and dithioisocyanate protein conjugates. In general, TDI reactive mAbs displayed stronger recognition of isocyanate haptenated proteins when the NCO was in the ortho position relative to the tolyl group, and were capable of discriminating between isocyanate and isothiocyanate conjugates and between aromatic and aliphatic dNCOs. Preliminary studies using TDI vapor exposed cells suggest potential utility of these mAbs for both research and biomonitoring. Published by Elsevier B.V.

  20. ANTIBODY POLYCLONAL PRODUCTION ON RABBIT ANTI-OVINE PREGNANCY-ASSOCIATED GLYCOPROTEIN (Rabbit anti-ovPAG

    Directory of Open Access Journals (Sweden)

    E.T. Setiatin

    2014-10-01

    Full Text Available The aim of the study was to produce polyclonal antibody (rabbit anti-ovPAG which could detectPAG in the urine of pregnant ewes. Twelve rabbits were immunized against ovPG DEAE-TrisHCl (DT,DEAE-NaCl 20mM (DN2, DEAE-NaCl 40mM (DN4, DEAE-NaCl 80mM (DN8, DEAE-NaCl160mM (DN16, DEAE-NaCl 320mM (DN32 and DEAE-NaCl 1M (DN1 and NaCl 0.9 % as aplacebo. The 0.5 ml of isolate (purified from ovine cotyledon was emulsified in equal volume withcomplete and incomplete Freud’s adjuvant. The mixture of each isolate and adjuvant was injected atmutiple sites along the dorsal area of rabbits by subcutaneous route. Blood were collected from marginalear vein, starting before first injection (baseline and every 14 days. Rabbit anti-ovPAG were measuredusing Modified ELISA Technique. By using Western Blot Technique, DN32 showed the best immuneresponse among others and also could differenciate ovPAG in the urine of pregnant ewes It could beconcluded that ovPAG DN32 is a specific source of rabbit anti-ovPAG production. Protein of ovPAG atmolecular weight 31 kDa is a pregnancy protein marker of garut sheep and could be developed as amajor protein for producing antibodi.

  1. Does bone marrow-derived mesenchymal stem cell transfusion prevent antisperm antibody production after traumatic testis rupture?

    Science.gov (United States)

    Aghamir, Seyyed Mohammad Kazem; Salavati, Alborz; Yousefie, Reza; Tootian, Zahra; Ghazaleh, Noushin; Jamali, Mostafa; Azimi, Pourya

    2014-07-01

    To determine whether transfusion of mesenchymal stem cells (MSCs) could prevent humoral immune response and autoimmunization against sperms after traumatic testis rupture. Immunomodulatory properties of MSCs have been evaluated by a prospective cohort on 50 adult BALB/c mice. In each interventional arms of study, controlled testis rupture and surgical repair were exerted. In addition to tissue repair, single dose of 5×10(5) MSCs labeled by green fluorescent protein was delivered intravenously to 20 cases (cell therapy group). After euthanizing, seroconversion of antisperm antibody (ASA) was compared between 2 interventional groups as response of humoral immune system. Lung and testis tissues were examined for green fluorescent protein-positive cells to assess whether presence of stem cells is correlated with seroconversion rates. Six cases had been lost during the study. Fourteen of 16 mice in cell therapy control group formed ASA (87.5%) but 6 of 18 mice (33.3%) in cell therapy group were immunized and formed ASA (P=.002). Transplanted cells were traced in lungs of 55% (n=10) of cell therapy group and none were found in trauma site. Small volume of mice blood was our main limitation to trace seroconversion or quantitative measurement of ASA in each case. In this in vivo model of autoimmune infertility, bone marrow-derived MSC transfusion showed immunosuppressive effects on antibody production. Considering immunomodulatory properties of MSCs even in allogeneic settings, novel clinical application should be investigated further. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Large Scale Production and Characterization of Anti-Human IgG Monoclonal Antibody in Peritoneum of Balb/c MICE

    Directory of Open Access Journals (Sweden)

    B. Baradaran

    2005-01-01

    Full Text Available Monoclonal antibodies are key reagents that are used in biomedical researches, diagnosis of immunodeficiency diseases such as IgG subclasses deficiency and treatment of diseases like infections and cancers .For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human IgG were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After ten days, approximately 3 ml ascitic fluid was harvested from the peritoneum of each mouse. Ascitic fluid was assayed for the titer of monoclonal antibody in reaction with human IgG and its cross reactivity in reaction with IgM & IgA. The titer of mAb was 100,000 and didn't show cross reactivity with IgM & IgA. Immunobloting was done for confirming the ELISA method. In immunobloting, only one sharp band in the heavy chain position of IgG was developed. The subclass of antibody was IgG1 and its light chain was kappa. Ascitic fluid was purified by ion exchange chromatography and the purified monoclonal antibody was conjugated with HRP. The conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of all other infectious diseases.

  3. Peptide-based antibodies against glutathione-binding domains suppress superoxide production mediated by mitochondrial complex I.

    Science.gov (United States)

    Chen, Jingfeng; Chen, Chwen-Lih; Rawale, Sharad; Chen, Chun-An; Zweier, Jay L; Kaumaya, Pravin T P; Chen, Yeong-Renn

    2010-01-29

    Complex I (NQR) is a critical site of superoxide (O2-*) production and the major host of redox protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51- and 75-kDa subunits are known to be reversibly S-glutathionylated. Although several glutathionylated domains from NQR 51 and 75 kDa have been identified, their roles in the regulatory functions remain to be explored. To gain further insights into protein S-glutathionylation of complex I, we used two peptides of S-glutathionylated domain ((200)GAGAYICGEETALIESIEGK(219) of 51-kDa protein and (361)VDSDTLCTEEVFPTAGAGTDLR(382) of 75-kDa protein) as chimeric epitopes incorporating a "promiscuous" T-cell epitope to generate two polyclonal antibodies, AbGSCA206 and AbGSCB367. Binding of AbGSCA206 and AbGSCB367 inhibited NQR-mediated O2-* generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain ((21)SGDTTAPKKTSFGSLKDFDR(40) of 51-kDa peptide and (100)WNILTNSEKTKKAREGVMEFL(120) of 75-kDa peptide) were synthesized as chimeric epitopes to generate two polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated generation to a significant level. However, binding of Ab75 inhibited NQR-mediated O2-*generation by 35%. None of AbGSCA206, AbGSCB367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the glutathione-binding domain decreased electron leakage from the hydrophilic domain of NQR. When heart tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathionylation was enhanced in post-ischemic myocardium at the NQR 51-kDa subunit, but not at the 75-kDa subunit, indicating that the 51-kDa subunit of flavin subcomplex is more sensitive to oxidative stress resulting from myocardial infarction.

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

    National Research Council Canada - National Science Library

    Sainsbury, Frank; Sack, Markus; Stadlmann, Johannes; Quendler, Heribert; Fischer, Rainer; Lomonossoff, George P

    2010-01-01

    .... 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...

  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

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders

    2015-01-01

    Background: Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradica...

  6. Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae.

    NARCIS (Netherlands)

    Gorlani, A.; Hulsik, D.L.; Adams, H.; Vriend, G.; Hermans, P.; Verrips, T.

    2012-01-01

    Variable domains of llama heavy-chain antibodies (VHH) are becoming a potent tool for a wide range of biotechnological and medical applications. Because of structural features typical of their single-domain nature, they are relatively easy to produce in lower eukaryotes, but it is not uncommon that

  7. Production and Characterization of Monoclonal Antibodies against Human Nuclear Protein FAM76B.

    Directory of Open Access Journals (Sweden)

    Xiaojing Zheng

    Full Text Available Human FAM76B (hFAM76B is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s of FAM76B, murine monoclonal antibodies (MAbs against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s of FAM76B.

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

    Science.gov (United States)

    Wongtangprasert, Tossapon; Natakuathung, Wirongrong; Pimpitak, Umaporn; Buakeaw, Anumart; Palaga, Tanapat; Komolpis, Kittinan; Khongchareonporn, Nanthika

    2014-02-01

    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 cell fusion 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.

  9. Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

    Directory of Open Access Journals (Sweden)

    Yukihiro Shoyama

    2011-06-01

    Full Text Available To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs were prepared. Ginsenosides were developed on thin layer chromatography (TLC plates which were covered by a polyvinylidene difluoride (PVDF membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

  10. Monoclonal Antibody Production and Immunolocalization of a Salinity Stress-Related Protein in Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    Jackson MARCONDES; Ana Beatriz GARCIA

    2011-01-01

    Among various physiological responses to salt stress,the synthesis of a lectin-related protein of 14.5 kDa was observed in rice plants (Oryza sativa L.) under the treatment of 170 mmol/L NaCl.In order to better understand the role of the SALT protein in the physiological processes involving salinity,it was immunolocalized in mesophilic cells of leaf sheath and blade of a rice variety IAC-4440 following monoclonal antibodies produced by hybridome culture technique.This variety turned out to be an excellent model for that purpose,since it accumulates SALT protein even in absence of salt treatment and it has been classified as moderately sensitive to salinity and a superior grain producer.This feature was relevant for this work since it allowed the use of plants without the deleterious effects caused by salinity.Immunocytochemistry assays revealed that the SALT protein is located in the stroma of chloroplasts under non-stressing condition.Since the chloroplast is the main target affected by salinity and considering that the SALT protein does not present any apparent signal peptide for organelle localization,its lectin-like activity seems to play an important role in the establishment of stable complexes,either to other proteins or to oligosaccharides that are translocated to the chloroplast.

  11. Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures.

    Science.gov (United States)

    Serrato, J Antonio; Palomares, Laura A; Meneses-Acosta, Angélica; Ramírez, Octavio T

    2004-10-20

    It is known that heterogeneous conditions exist in large-scale animal cell cultures. However, little is known about how heterogeneities affect cells, productivities, and product quality. To study the effect of non-constant dissolved oxygen tension (DOT), hybridomas were subjected to sinusoidal DOT oscillations in a one-compartment scale-down simulator. Oscillations were forced by manipulating the inlet oxygen partial pressure through a feedback control algorithm in a 220-mL bioreactor maintained at a constant agitation. Such temporal DOT oscillations simulate spatial DOT gradients that can occur in large scales. Different oscillation periods, in the range of 800 to 12,800 s (axis of 7% (air saturation) and amplitude of 7%), were tested and compared to constant DOT (10%) control cultures. Oscillating DOT decreased maximum cell concentrations, cell growth rates, and viability indexes. Cultures at oscillating DOT had an increased glycolytic metabolism that was evidenced by a decrease in yield of cells on glucose and an increase in lactate yield. DOT gradients, even several orders of magnitude higher than those expected under practical large-scale conditions, did not significantly affect the maximum concentration of an IgG(1) monoclonal antibody (MAb). The glycosylation profile of the MAb produced at a constant DOT of 10% was similar to that reported in the literature. However, MAb produced under oscillating culture conditions had a higher amount of triantennary and sialylated glycans, which can interfere with effector functions of the antibody. It was shown that transient excursions of hybridomas to limiting DOT, as occurs in deficiently mixed large-scale bioreactors, is important to culture performance as the oscillation period, and thus the time cells spent at low DOT, affected cell growth, metabolism, and the glycosylation pattern of MAb. Such results underline the importance of monitoring protein characteristics for the development of large-scale processes.

  12. Effects of immunosuppression on avian coccidiosis: cyclosporin A but not hormonal bursectomy abrogates host protective immunity.

    Science.gov (United States)

    Lillehoj, H S

    1987-01-01

    The effects of cyclosporin A (CsA) treatment and hormonal bursectomy on Eimeria tenella infection of chickens were investigated to evaluate the role of humoral antibody and cell-mediated immunity (CMI) in the host protective immunity to an intestinal protozoan disease, coccidiosis. Hormonal bursectomy had no significant effect on the host response to E. tenella. CsA treatment had a differential effect on the course of disease depending on how CsA was given relative to infection. Daily administration of CsA for 7 days beginning 1 day before primary infection with E. tenella enhanced disease resistance, whereas a single dose of CsA given before primary infection enhanced disease susceptibility compared with that of untreated controls. Chickens treated with CsA during the primary infection were resistant to reinfection at 5 weeks post-primary infection. Treatment of chickens immune to E. tenella with CsA at the time of secondary infection abrogated their resistance to reinfection despite the presence of high levels of coccidia-specific secretory immunoglobulin A and serum immunoglobulin G. Splenic lymphocytes obtained after CsA treatment demonstrated a substantially depressed concanavalin A response, but not a depressed lipopolysaccharide response. Because CsA was not directly toxic to parasites in vivo when administered during the secondary infection, these results suggest that CsA interacts with the immune system to allow priming during the primary infection, while interfering with the effector function of CMI during the secondary infection. Taken together, present findings indicate that CMI plays a major role in host protective immunity to E. tenella. PMID:3496277

  13. Production and characterization of monoclonal antibodies specific to pangasius catfish, basa, and tra.

    Science.gov (United States)

    Gajewski, K G; Chen, Y-T; Hsieh, Y-H P

    2009-04-01

    Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra (Pangasius hypophthalmus) and basa (Pangasius bocourti), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.

  14. Alkylation of Histidine Residues of Bothrops jararacussu Venom Proteins and Isolated Phospholipases A2: A Biotechnological Tool to Improve the Production of Antibodies

    Directory of Open Access Journals (Sweden)

    C. L. S. Guimarães

    2014-01-01

    Full Text Available Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2 of this toxin (BthTX-I and BthTX-II were chemically modified (alkylation by p-bromophenacyl bromide (BPB in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated. BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2 diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

  15. Rapid optimization of antibotulinum toxin antibody fragment production by an integral approach utilizing RC-SELDI mass spectrometry and statistical design.

    Science.gov (United States)

    Park, Jun T; Bradbury, Lisa; Kragl, Frank J; Lukens, Dennis C; Valdes, James J

    2006-01-01

    A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

  16. A comprehensive comparison of mixing, mass transfer, Chinese hamster ovary cell growth, and antibody production using Rushton turbine and marine impellers.

    Science.gov (United States)

    Sandadi, Sandeepa; Pedersen, Henrik; Bowers, John S; Rendeiro, Dennis

    2011-09-01

    Large scale production of monoclonal antibodies has been accomplished using bioreactors with different length to diameter ratios, and diverse impeller and sparger designs. The differences in these physical attributes often result in dissimilar mass transfer, mechanical stresses due to turbulence and mixing inside the bioreactor that may lead to disparities in cell growth and antibody production. A rational analysis of impeller design parameters on cell growth, protein expression levels and subsequent antibody production is needed to understand such differences. The purpose of this study was to examine the impact of Rushton turbine and marine impeller designs on Chinese hamster ovary (CHO) cell growth and metabolism, and antibody production and quality. Experiments to evaluate mass transfer and mixing characteristics were conducted to determine if the nutrient requirements of the culture would be met. The analysis of mixing times indicated significant differences between marine and Rushton turbine impellers at the same power input per unit volume of liquid (P/V). However, no significant differences were observed between the two impellers at constant P/V with respect to oxygen and carbon dioxide mass transfer properties. Experiments were conducted with CHO cells to determine the impact of different flow patterns arising from the use of different impellers on cell growth, metabolism and antibody production. The analysis of cell culture data did not indicate any significant differences in any of the measured or calculated variables between marine and Rushton turbine impellers. More importantly, this study was able to demonstrate that the quality of the antibody was not altered with a change in the impeller geometry.

  17. Blockade of invariant TCR-CD1d interaction specifically inhibits antibody production against blood group A carbohydrates

    Science.gov (United States)

    Tazawa, Hirofumi; Irei, Toshimitsu; Tanaka, Yuka; Igarashi, Yuka; Tashiro, Hirotaka

    2013-01-01

    Previously, we detected B cells expressing receptors for blood group A carbohydrates in the CD11b+CD5+ B-1a subpopulation in mice, similar to that in blood group O or B in humans. In the present study, we demonstrate that CD1d-restricted natural killer T (NKT) cells are required to produce anti-A antibodies (Abs), probably through collaboration with B-1a cells. After immunization of wild-type (WT) mice with human blood group A red blood cells (A-RBCs), interleukin (IL)-5 exclusively and transiently increased and the anti-A Abs were elevated in sera. However, these reactions were not observed in CD1d−/− mice, which lack NKT cells. Administration of anti-mouse CD1d blocking monoclonal Abs (mAb) prior to immunization abolished IL-5 production by NKT cells and anti-A Ab production in WT mice. Administration of anti-IL-5 neutralizing mAb also diminished anti-A Ab production in WT mice, suggesting that IL-5 secreted from NKT cells critically regulates anti-A Ab production by B-1a cells. In nonobese diabetic/severe combined immunodeficient (NOD/SCID/γcnull) mice, into which peripheral blood mononuclear cells from type O human volunteers were engrafted, administration of anti-human CD1d mAb prior to A-RBC immunization completely inhibited anti-A Ab production. Thus, anti-CD1d treatment might constitute a novel approach that could help in evading Ab-mediated rejection in ABO-incompatible transplant recipients. PMID:23943651

  18. Lipoxin A4 decreases human memory B cell antibody production via an ALX/FPR2-dependent mechanism: A link between resolution signals and adaptive immunity

    Science.gov (United States)

    Ramon, Sesquile; Bancos, Simona; Serhan, Charles N.; Phipps, Richard P.

    2013-01-01

    Summary Specialized proresolving mediators (SPMs) are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. SPMs are classified into lipoxins, resolvins, protectins and maresins. Lipoxins and other SPMs have been identified in important immunological tissues including bone marrow, spleen and blood. Lipoxins regulate functions of the innate immune system including the promotion of monocyte recruitment and increase macrophage phagocytosis of apoptotic neutrophils. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we analyzed the actions of lipoxin A4 (LXA4) and its receptor ALX/FPR2 on human B cells. LXA4 decreased IgM and IgG production on activated B cells through ALX/FPR2-dependent signaling, which downregulated NF-κB p65 nuclear translocation. LXA4 also inhibited human memory B cell antibody production and proliferation, but not naïve B cell function. Lastly, LXA4 decreased antigen-specific antibody production in vivo. To our knowledge, this is the first description of the actions of lipoxins on human B cells, which shows a link between resolution signals and adaptive immunity. Regulating antibody production is crucial to prevent unwanted inflammation. Harnessing the ability of lipoxins to decrease memory B cell antibody production can be beneficial to threat inflammatory and autoimmune disorders. PMID:24166736

  19. Thermal influences on antibody production and metabolism in chicken lines divergently selected for immune responsiveness.

    NARCIS (Netherlands)

    Donker, R.

    1989-01-01

    IntroductionThe international character of the poultry industry requires that poultry bred under temperate conditions should be able to perform under tropical conditions as well. However, abrupt changes in environmental temperature or continuous high temperatures may affect production traits, im

  20. Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site.

    Science.gov (United States)

    Kawabe, Yoshinori; Inao, Takanori; Komatsu, Shodai; Huang, Guan; Ito, Akira; Omasa, Takeshi; Kamihira, Masamichi

    2017-03-01

    Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production.

  1. PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    何凤田; 聂勇战; 陈宝军; 乔太东; 韩者艺; 樊代明

    2002-01-01

    Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti -Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL)genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13KO7 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. Thetypesoftheanti-IdScFvdisplayedontheselectedphagecloneswerepreliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γtype anti-Id ScFv.Conclsion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

  2. Does the antibody production ability affect the serum anti-Helicobacter pylori Ig G titer?

    Institute of Scientific and Technical Information of China (English)

    Hyun Ah Chung; Sun-Young Lee; Hee Won Moon; Jeong Hwan Kim; In-Kyung Sung; Hyung Seok Park; Chan Sup Shim; Hye Seung Han

    2016-01-01

    AIM: To investigate the relationship between serum titers of anti-Helicobacter pylori(H.pylori) immunoglobulin G(IgG) and hepatitis B virus surface antibody(HBsA b).METHODS: Korean adults were included whose samples had positive Giemsa staining on endoscopic biopsy and were studied in the hepatitis B virus surface antigen(HBsA g)/HBsA b serologic assay,pepsinogen(PG) assay,and H.pylori serologic test on the same day.Subjects were excluded if they were positive for HBs Ag,had a recent history of medication,or had other medical condition(s).We analyzed the effects of the following factors on serum titers of HBsA b and the anti-H.pylori IgG : Age,density of H.pylori infiltration in biopsy samples,serum concentrations of PG Ⅰ and PG Ⅱ,PG Ⅰ/Ⅱ ratio,and white blood cell count.RESULTS: Of 111 included subjects,74(66.7%) exhibited a positive HBsA b finding.The serum anti-H.pylori IgG titer did not correlate with the serum HBsA b titer(P = 0.185); however,it correlated with the degree of H.pylori infiltration on gastric biopsy(P < 0.001) and serum PG Ⅱ concentration(P = 0.042).According to the density of H.pylori infiltration on gastric biopsy,subjects could be subdivided into those with a marked(median: 3.95,range 0.82-4.00)(P = 0.458),moderate(median: 3.37,range 1.86-4.00),and mild H.pylori infiltrations(median: 2.39,range 0.36-4.00)(P < 0.001).Subjects with a marked H.pylori infiltration on gastric biopsy had the highest serological titer,whereas in subjects with moderate and mild H.pylori infiltrations titers were correspondingly lower(P < 0.001).After the successful eradication,significant decreases of the degree of H.pylori infiltration(P < 0.001),serum anti-H.pylori IgG titer(P < 0.001),and serum concentrations of PG I(P = 0.028) and PG Ⅱ(P = 0.028) were observed.CONCLUSION: The anti-H.pylori IgG assay can be used to estimate the burden of bacteria in immunocompetent hosts with H.pylori infection,regardless of the HBsA b titer after HBV vaccination.

  3. 9 CFR 113.452 - Erysipelothrix Rhusiopathiae Antibody.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Erysipelothrix Rhusiopathiae Antibody... REQUIREMENTS Antibody Products § 113.452 Erysipelothrix Rhusiopathiae Antibody. Erysipelothrix Rhusiopathiae Antibody is a specific antibody product containing antibodies directed against one or more somatic...

  4. ANTIBODY POLYCLONAL PRODUCTION ON RABBIT ANTI-OVINE PREGNANCY-ASSOCIATED GLYCOPROTEIN (Rabbit anti-ovPAG

    Directory of Open Access Journals (Sweden)

    E.T. Setiatin

    2011-09-01

    Full Text Available The aim of the study was to produce polyclonal antibody (rabbit anti-ovPAG which could detect PAG in the urine of pregnant ewes. Twelve rabbits were immunized against ovPG DEAE-TrisHCl (DT, DEAE-NaCl 20mM (DN2, DEAE-NaCl 40mM (DN4, DEAE-NaCl 80mM (DN8, DEAE-NaCl 160mM (DN16, DEAE-NaCl 320mM (DN32 and DEAE-NaCl 1M (DN1 and NaCl 0.9 % as a placebo. The 0.5 ml of isolate (purified from ovine cotyledon was emulsified in equal volume with complete and incomplete Freud’s adjuvant. The mixture of each isolate and adjuvant was injected at mutiple sites along the dorsal area of rabbits by subcutaneous route. Blood were collected from marginal ear vein, starting before first injection (baseline and every 14 days. Rabbit anti-ovPAG were measured using Modified ELISA Technique. By using Western Blot Technique, DN32 showed the best immune response among others and also could differenciate ovPAG in the urine of pregnant ewes It could be concluded that ovPAG DN32 is a specific source of rabbit anti-ovPAG production. Protein of ovPAG at molecular weight 31 kDa is a pregnancy protein marker of garut sheep and could be developed as a major protein for producing antibodi.

  5. Determination for multiple mycotoxins in agricultural products using HPLC-MS/MS via a multiple antibody immunoaffinity column.

    Science.gov (United States)

    Zhang, Zhaowei; Hu, Xiaofeng; Zhang, Qi; Li, Peiwu

    2016-05-15

    Mycotoxins usually found in agricultural products such as peanut, corn, and wheat, are a serious threat to human health and their detection requires multiplexed and sensitive analysis methods. Herein, a simultaneous determination for aflatoxin B1, B2, G1, G2, ochratoxin A, zearalanone and T-2 toxin was investigated using high performance liquid chromatography coupled with tandem mass spectrometry in a single run via a home-made multiple immunoaffinity column. Four monoclonal antibodies were produced in our lab against aflatoxins, ochratoxin A, zearalanone and T-2 toxin, respectively, then combined as a pool and bound to Sepharose-4B for affinity chromatography. Seven mycotoxins were effectively extracted from the agricultural product samples by using acetonitrile/water/acetic acid (80:19:1, v/v/v) Then, the extraction was cleanup by multiple immunoaffinity column. This method demonstrated a considerable linear range of 0.30-25, 0.12-20, 0.30-20, 0.12-20, 0.60-30, 0.30-25, and 1.2-40μgkg(-1)and lower limits of detection at 0.1, 0.04, 0.1, 0.04, 0.2, 0.1 and 0.4μgkg(-1) for AFB1, AFB2, AFG1, AFG2, OTA, ZEN and T-2, respectively, in comparison with previously reported methods, as well as excellent recoveries. The mIAC capacity for AFB1, AFB2, AFG1, AFG2, OTA, ZEN, and T-2 were 187, 181, 153, 151, 105, 130, 88ng, respectively. It was found that all of the 7 mycotoxins were present in 90 agricultural product samples. The proposed method meets the requirements for rapid sample preparation and highly sensitive identification of multiple mycotoxins in agricultural product and food safety. This method provides a promising alternative with high throughput and high sensitivity for rapid analysis of seven mycotoxins in the monitoring of food safety.

  6. Antibody-dependent enhancement infection facilitates dengue virus-regulated signaling of IL-10 production in monocytes.

    Directory of Open Access Journals (Sweden)

    Tsung-Ting Tsai

    2014-11-01

    Full Text Available Interleukin (IL-10 levels are increased in dengue virus (DENV-infected patients with severe disorders. A hypothetical intrinsic pathway has been proposed for the IL-10 response during antibody-dependent enhancement (ADE of DENV infection; however, the mechanisms of IL-10 regulation remain unclear.We found that DENV infection and/or attachment was sufficient to induce increased expression of IL-10 and its downstream regulator suppressor of cytokine signaling 3 in human monocytic THP-1 cells and human peripheral blood monocytes. IL-10 production was controlled by activation of cyclic adenosine monophosphate response element-binding (CREB, primarily through protein kinase A (PKA- and phosphoinositide 3-kinase (PI3K/PKB-regulated pathways, with PKA activation acting upstream of PI3K/PKB. DENV infection also caused glycogen synthase kinase (GSK-3β inactivation in a PKA/PI3K/PKB-regulated manner, and inhibition of GSK-3β significantly increased DENV-induced IL-10 production following CREB activation. Pharmacological inhibition of spleen tyrosine kinase (Syk activity significantly decreased DENV-induced IL-10 production, whereas silencing Syk-associated C-type lectin domain family 5 member A caused a partial inhibition. ADE of DENV infection greatly increased IL-10 expression by enhancing Syk-regulated PI3K/PKB/GSK-3β/CREB signaling. We also found that viral load, but not serotype, affected the IL-10 response. Finally, modulation of IL-10 expression could affect DENV replication.These results demonstrate that, in monocytes, IL-10 production is regulated by ADE through both an extrinsic and an intrinsic pathway, all involving a Syk-regulated PI3K/PKB/GSK-3β/CREB pathway, and both of which impact viral replication.

  7. IL-4 abrogates TH17 cell-mediated inflammation by selective silencing of IL-23 in antigen-presenting cells

    Science.gov (United States)

    Guenova, Emmanuella; Skabytska, Yuliya; Hoetzenecker, Wolfram; Weindl, Günther; Sauer, Karin; Tham, Manuela; Kim, Kyu-Won; Park, Ji-Hyeon; Seo, Ji Hae; Ignatova, Desislava; Cozzio, Antonio; Levesque, Mitchell P.; Volz, Thomas; Köberle, Martin; Kaesler, Susanne; Thomas, Peter; Mailhammer, Reinhard; Ghoreschi, Kamran; Schäkel, Knut; Amarov, Boyko; Eichner, Martin; Schaller, Martin; Clark, Rachael A.; Röcken, Martin; Biedermann, Tilo

    2015-01-01

    Interleukin 4 (IL-4) can suppress delayed-type hypersensitivity reactions (DTHRs), including organ-specific autoimmune diseases in mice and humans. Despite the broadly documented antiinflammatory effect of IL-4, the underlying mode of action remains incompletely understood, as IL-4 also promotes IL-12 production by dendritic cells (DCs) and IFN-γ–producing TH1 cells in vivo. Studying the impact of IL-4 on the polarization of human and mouse DCs, we found that IL-4 exerts opposing effects on the production of either IL-12 or IL-23. While promoting IL-12–producing capacity of DCs, IL-4 completely abrogates IL-23. Bone marrow chimeras proved that IL-4–mediated suppression of DTHRs relies on the signal transducer and activator of transcription 6 (STAT6)-dependent abrogation of IL-23 in antigen-presenting cells. Moreover, IL-4 therapy attenuated DTHRs by STAT6- and activating transcription factor 3 (ATF3)-dependent suppression of the IL-23/TH17 responses despite simultaneous enhancement of IL-12/TH1 responses. As IL-4 therapy also improves psoriasis in humans and suppresses IL-23/TH17 responses without blocking IL-12/TH1, selective IL-4–mediated IL-23/TH17 silencing is promising as treatment against harmful inflammation, while sparing the IL-12–dependent TH1 responses. PMID:25646481

  8. Occurrence of antibodies against natalizumab in relapsing multiple sclerosis patients treated with natalizumab

    DEFF Research Database (Denmark)

    Sørensen, Per Soelberg; Jensen, Poul Erik Hyldgaard; Haghikia, Aiden;

    2011-01-01

    In the clinical trials about 9% of natalizumab treated multiple sclerosis (MS) patients generated anti-natalizumab antibodies, of which 6% were persistent and 3% transient. The occurrence of antibodies reduced serum levels of natalizumab, decreased bio-efficacy, and abrogated the therapeutic...

  9. The Role of TLR4 on B Cell Activation and Anti-β2GPI Antibody Production in the Antiphospholipid Syndrome

    Science.gov (United States)

    2016-01-01

    High titer of anti-β2-glycoprotein I antibodies (anti-β2GPI Ab) plays a pathogenic role in antiphospholipid syndrome (APS). Numerous studies have focused on the pathological mechanism in APS; however, little attention is paid to the immune mechanism of production of anti-β2GPI antibodies in APS. Our previous study demonstrated that Toll-like receptor 4 (TLR4) plays a vital role in the maturation of bone marrow-derived dendritic cells (BMDCs) from the mice immunized with human β2-glycoprotein I (β2GPI). TLR4 is required for the activation of B cells and the production of autoantibody in mice treated with β2GPI. However, TLR4 provides a third signal for B cell activation and then promotes B cells better receiving signals from both B cell antigen receptor (BCR) and CD40, thus promoting B cell activation, surface molecules expression, anti-β2GPI Ab production, and cytokines secretion and making B cell functioning like an antigen presenting cell (APC). At the same time, TLR4 also promotes B cells producing antibodies by upregulating the expression of B-cell activating factor (BAFF). In this paper, we aim to review the functions of TLR4 in B cell immune response and antibody production in autoimmune disease APS and try to find a new way for the prevention and treatment of APS. PMID:27868072

  10. Antimitochondrial antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  11. Produção de anticorpos policlonais anti-ricina Production of polyclonal anti-ricin antibodies

    Directory of Open Access Journals (Sweden)

    Roselayne Ferro Furtado

    2011-02-01

    rabbit, with one half in the begin of the experiment and the other half after 21 days of immunization. The ELISA method indicated that the lower doses (100 e 180 µg induced primary and secondary immunological response with production of specific antibodies, while the higher dose of ricin (400 µg showed a primary response with increase of the antibody titre, followed of immunological suppression. This profile suggests immunological tolerance. By Western blotting technique it was verified that polyclonal antibodies are too specific to ricin, however, they detected ricin in native and denaturated form and are not recommended for the monitoring of ricin in detoxificated castor bean cake by heat treatment.

  12. Upregulation of neurovascular communication through filamin abrogation promotes ectopic periventricular neurogenesis

    Science.gov (United States)

    Houlihan, Shauna L; Lanctot, Alison A; Guo, Yan; Feng, Yuanyi

    2016-01-01

    Neuronal fate-restricted intermediate progenitors (IPs) are derived from the multipotent radial glia (RGs) and serve as the direct precursors for cerebral cortical neurons, but factors that control their neurogenic plasticity remain elusive. Here we report that IPs’ neuron production is enhanced by abrogating filamin function, leading to the generation of periventricular neurons independent of normal neocortical neurogenesis and neuronal migration. Loss of Flna in neural progenitor cells (NPCs) led RGs to undergo changes resembling epithelial-mesenchymal transition (EMT) along with exuberant angiogenesis that together changed the microenvironment and increased neurogenesis of IPs. We show that by collaborating with β-arrestin, Flna maintains the homeostatic signaling between the vasculature and NPCs, and loss of this function results in escalated Vegfa and Igf2 signaling, which exacerbates both EMT and angiogenesis to further potentiate IPs’ neurogenesis. These results suggest that the neurogenic potential of IPs may be boosted in vivo by manipulating Flna-mediated neurovascular communication. DOI: http://dx.doi.org/10.7554/eLife.17823.001 PMID:27664421

  13. Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A.

    Science.gov (United States)

    de Medina, Philippe; Paillasse, Michael R; Segala, Gregory; Al Saati, Talal; Boyes, Jeannine; Delsol, Georges; Allal, Cuider; Marsili, Sabrina; Silvente-Poirot, Sandrine; Poirot, Marc

    2013-03-01

    We have recently discovered the existence of 5α-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-o-hemisuccinate with a carboxylic spacer arm attached to the 3β-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.

  14. Production of anti-fullerene C{sub 60} polyclonal antibodies and study of their interaction with a conjugated form of fullerene

    Energy Technology Data Exchange (ETDEWEB)

    Hendrickson, O. D., E-mail: odhendrick@gmail.com; Fedyunina, N. S. [Russian Academy of Sciences, Institute of Biochemistry (Russian Federation); Martianov, A. A. [Moscow State University (Russian Federation); Zherdev, A. V.; Dzantiev, B. B. [Russian Academy of Sciences, Institute of Biochemistry (Russian Federation)

    2011-09-15

    The aim of this study was to produce anti-fullerene C{sub 60} antibodies for the development of detection systems for fullerene C{sub 60} derivatives. To produce anti-fullerene C{sub 60} antibodies, conjugates of the fullerene C{sub 60} carboxylic derivative with thyroglobulin, soybean trypsin inhibitor, and bovine serum albumin were synthesized by carbodiimide activation and characterized. Immunization of rabbits by the conjugates led to the production of polyclonal anti-fullerene antibodies. The specificity of the immune response to fullerene was investigated. Indirect competitive immunoenzyme assay was developed for the determination of conjugated fullerene with detection limits of 0.04 ng/mL (calculated for coupled C{sub 60}) and 0.4 ng/mL (accordingly to total fullerene-protein concentration).

  15. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  16. An alternative chemical redox method for the production of bispecific antibodies: implication in rapid detection of food borne pathogens.

    Directory of Open Access Journals (Sweden)

    Mohammad Owais

    Full Text Available Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC and the food borne pathogen Listeria monocytogenes (L. monocytogenes were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches.

  17. Purification of Herpes Simplex Virus Type 1 for Production of High Titer Polyclonal Antibody against the Virus

    Directory of Open Access Journals (Sweden)

    Z Meshkat

    2008-12-01

    Full Text Available ABSTRACT: Introduction & Objective: Herpes simplex virus type 1 infection is one of the most prevalent viral infections worldwide. Different methods are being investigated for the virus’ detection, prevention and therapy. The aim of the present study was to purify the virus and to produce a high titer polyclonal antibody against the virus. Materials & Methods: This experimental study was done in the Virology Department of Tarbiat Modares University from 2001 to 2002. Virus purification was done using serial dilution and plaque purification protocols. A single plaque was chosen and propagated, and the virus titer was determined. In inoculated animals, the titer of produced antibody against the virus was measured by virus neutralization test. Results: Using virus neutralization test, it was found that the high level of antibody has been raised in animals against the virus. Conclusion: Considering the preparation of high titer antibody against the virus, the produced antibody can be used for the development and optimization of different diagnostic methods.

  18. Cryptorchidism is not a risk factor for antisperm antibody production in post-orchidopexy males with infertility.

    Science.gov (United States)

    Jiang, Huan; Zhu, Wei-Jie

    2013-01-01

    Infertility in adulthood is a well-recognized consequence of cryptorchidism, even after successful orchidopexy. Autoimmune reactions against spermatozoa are associated with infertility and often found in cryptorchids. The purposes of this study were to evaluate the linkage between antisperm antibody (ASA) and cryptorchidism, and furthermore, to clarify whether ASA is involved in cryptorchidism-associated infertility. We investigated a total of 48 infertile males with a history of unilateral (n = 30) or bilateral (n = 18) cryptorchidism who had undergone successful orchidopexy in prepuberty, and 20 age-matched fertile and healthy males were collected as controls. ASA in sperm samples was detected by the direct immunobead test, and semen analysis was performed concomitantly. No infertile case satisfied the diagnostic criteria of ASA-mediated infertility set forth by the World Health Organization. Decreases in both sperm concentration and motility accompanied by increases in abnormal morphology were seen in infertile cryptorchids when compared with the healthy controls. Testicular heat exposure in prepuberty is not a risk factor for ASA production. It is evident that the mechanisms that underlie cryptorchidism-associated infertility do not involve ASA. Poor sperm characteristics in cryptorchids resulting from thermal damage to the testes seem to be responsible for their infertility, even after successful orchidopexy. Copyright © 2013 S. Karger AG, Basel.

  19. ASCT2 (SLC1A5)-Deficient Mice Have Normal B-Cell Development, Proliferation, and Antibody Production

    Science.gov (United States)

    Masle-Farquhar, Etienne; Bröer, Angelika; Yabas, Mehmet; Enders, Anselm; Bröer, Stefan

    2017-01-01

    SLC1A5 (solute carrier family 1, member 5) is a small neutral amino acid exchanger that is upregulated in rapidly proliferating lymphocytes but also in many primary human cancers. Furthermore, cancer cell lines have been shown to require SLC1A5 for their survival in vitro. One of SLC1A5’s primary substrates is the immunomodulatory amino acid glutamine, which plays an important role in multiple key processes, such as energy supply, macromolecular synthesis, nucleotide biosynthesis, redox homeostasis, and resistance against oxidative stress. These processes are also essential to immune cells, including neutrophils, macrophages, B and T lymphocytes. We show here that mice with a stop codon in Slc1a5 have reduced glutamine uptake in activated lymphocytes and primary fibroblasts. B and T cell populations and maturation in resting mice were not affected by absence of SLC1A5. Antibody production in resting and immunized mice and the germinal center response to immunization were also found to be normal. SLC1A5 has been recently described as a novel target for the treatment of a variety of cancers, and our results indicate that inhibition of SLC1A5 in cancer therapy may be tolerated well by the immune system of cancer patients. PMID:28553292

  20. ASCT2 (SLC1A5-Deficient Mice Have Normal B-Cell Development, Proliferation, and Antibody Production

    Directory of Open Access Journals (Sweden)

    Etienne Masle-Farquhar

    2017-05-01

    Full Text Available SLC1A5 (solute carrier family 1, member 5 is a small neutral amino acid exchanger that is upregulated in rapidly proliferating lymphocytes but also in many primary human cancers. Furthermore, cancer cell lines have been shown to require SLC1A5 for their survival in vitro. One of SLC1A5’s primary substrates is the immunomodulatory amino acid glutamine, which plays an important role in multiple key processes, such as energy supply, macromolecular synthesis, nucleotide biosynthesis, redox homeostasis, and resistance against oxidative stress. These processes are also essential to immune cells, including neutrophils, macrophages, B and T lymphocytes. We show here that mice with a stop codon in Slc1a5 have reduced glutamine uptake in activated lymphocytes and primary fibroblasts. B and T cell populations and maturation in resting mice were not affected by absence of SLC1A5. Antibody production in resting and immunized mice and the germinal center response to immunization were also found to be normal. SLC1A5 has been recently described as a novel target for the treatment of a variety of cancers, and our results indicate that inhibition of SLC1A5 in cancer therapy may be tolerated well by the immune system of cancer patients.

  1. High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

    Science.gov (United States)

    Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

    2013-01-01

    Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

  2. Production of IgG antibodies to pneumococcal polysaccharides is associated with expansion of ICOS+ circulating memory T follicular-helper cells which is impaired by HIV infection.

    Science.gov (United States)

    Abudulai, Laila N; Fernandez, Sonia; Corscadden, Karli; Burrows, Sally A; Hunter, Michael; Tjiam, M Christian; Kirkham, Lea-Ann S; Post, Jeffrey J; French, Martyn A

    2017-01-01

    Dysfunction of T follicular-helper (TFH) cells is a possible cause of impaired germinal centre (GC) and IgG antibody responses in individuals with human immunodeficiency virus-1 (HIV-1) infection and might contribute to decreased magnitude and isotype diversification of IgG antibodies to pneumococcal polysaccharides (PcPs). We examined the production of IgG1 and IgG2 antibodies to PcPs 4, 6B, 9V and 14 by enumerating antibody secreting cells (ASCs) at day (D) 7 and determining fold-increase in serum antibody levels at D28 after vaccination with unconjugated PcPs in HIV seronegative subjects (n = 20) and in HIV patients who were receiving antiretroviral therapy (ART) (n = 28) or who were ART-naive (n = 11) and determined their association with ICOS+ and ICOS- circulating memory TFH (cmTFH) cells (CD4+CD45RA-CD27+CXCR5+PD-1+) and short lived plasmablasts (SPBs) at D7, and with PcP-specific and total IgM+ and IgG+ memory B cells at D0. In HIV seronegative subjects, production of IgG1+ and IgG2+ ASCs was consistently associated with the frequency of ICOS+ cmTFH cells but not ICOS- cmTFH cells or memory B cells. In contrast, post-vaccination ASCs in HIV patients, regardless of ART status, were lower than in HIV seronegative subjects and not associated with ICOS+ cmTFH cells, the expansion of which was absent (ART-naive patients) or much lower than in HIV seronegative subjects (ART-treated patients). Production of SPBs was also lower in ART-naive patients. Fold-increase in IgG2 antibodies at D28 also correlated with ICOS+ cmTFH cells at D7 in HIV seronegative subjects but not in HIV patients. These novel findings provide evidence that ICOS+ cmTFH cells contribute to the regulation of PcP-specific IgG antibody responses, including isotype diversification, and that TFH cell dysfunction may be a cause of impaired PcP-specific IgG antibody responses and increased susceptibility to pneumococcal disease in HIV patients.

  3. Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function.

    Science.gov (United States)

    Minkoff, Marjorie Sue

    A monoclonal antibody to an eosinophil specific determinant is very important in assessing eosinophil function during helminthic infection. Eosinophils induced by Schistosoma mansoni infection in BALB/c mice were used to induce C57B1/6 immunocytes for production of hybridomas secreting eosinophil monoclonal antibodies. These antibodies were shown to react with an eosinophil surface epitope but not with neutrophils or macrophages as determined by ELISA, immunodiffusion, immunofluorescence, and immunoblot assay. Affinity chromatography with eosinophil chemotactic factor-sepharose consistently selected out a { rm M_ R} 67,000 protein from solubilized eosinophil membrane antigens but not from neutrophil and macrophage antigens. In vitro studies showed that the eosinophil-specific monoclonal antibodies abrogated antibody-dependent eosinophil -mediated killing of S. mansoni schistosomula using mouse, rat or human eosinophils. Neutrophil and macrophage killing activities were unaffected. The monoclonal antibodies effected complement-dependent lysis of mouse and rat eosinophils but not of human eosinophils. ECF-treated eosinophils showed enhanced killing of schistosomula which was blocked by the monoclonal antibody. Murine and human eosinophils preincubated with monoclonal antibody exhibited decreased chemotaxis to ECF at optimal chemotactic concentrations. The monoclonal antibody also blocked eosinophil binding to ECF- sepharose beads. In vivo induction of peripheral blood eosinophilia by injection of S. mansoni eggs was suppressed by injections of monoclonal antibodies 2CD13 and 2QD45 in mouse and rat experimental models. Eosinophilia induced by keyhole limpet hemocyanin- cyclophosphamide treatment was also suppressed by monoclonal antibody in both murine and rat systems. Pulmonary granulomas in mice given egg injection and monoclonal antibody were smaller and contained fewer eosinophils than those granulomas from mice given eggs only. In immuno-biochemical studies, the

  4. Adjuvant effect of Asparagus racemosus Willd. derived saponins in antibody production, allergic response and pro-inflammatory cytokine modulation.

    Science.gov (United States)

    Tiwari, Nimisha; Gupta, Vivek Kumar; Pandey, Pallavi; Patel, Dinesh Kumar; Banerjee, Suchitra; Darokar, Mahendra Pandurang; Pal, Anirban

    2017-02-01

    The study manifests the immunoadjuvant potential of saponin rich fraction from Asparagus racemosus in terms of cellular and humoral immune response that can be exploited against microbial infections. Asparagus racemosus (AR) has been attributed as an adaptogen and rasayana in traditional medication systems for enhancing the host defence mechanism. Spectrophotometric and HPTLC analysis ensured the presence of saponins. The saponin rich fractions were tested for immunoadjuvant property in ovalbumin immunised mice for the humoral response, quantified in terms of prolonged antibody production upto a duration of 56days. Proinflammatory cytokines (IL-6 and TNF) were estimated for the cellular immune response in LPS stimulated primary murine macrophages. The safety evaluation in terms of cytotoxicity and allergic response has also been evaluated through in-vitro (MTT) and in-vivo (IgE) respectively. ARS significantly inhibited the pro-inflammatory cytokines, in LPS stimulated murine macrophages with no intrinsic cytotoxicity. The significant increase in IgG production infers the utility of ARS for prolonged humoral response. Further, the antigen specific response of IL-12 at early stage and IgE titres also suggests the generation of cellular immune response and low allergic reaction respectively, as compared to conventional adjuvants. IL-6 and TNF fluctuations in LPS stimulated and non-stimulated macrophages along with IgG and IL-12 also confirmed the Th1/Th2 modulating effect of ARS. The study indicates potential effect of ARS as an adjuvant for the stimulation of cellular immune response in addition to generating a sustained adaptive response without any adverse effects paving way for further validation with pathogenic organisms. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. Aging-dependent decline of IL-10 producing B cells coincides with production of antinuclear antibodies but not rheumatoid factors.

    Science.gov (United States)

    van der Geest, Kornelis S M; Lorencetti, Pedro G; Abdulahad, Wayel H; Horst, Gerda; Huitema, Minke; Roozendaal, Caroline; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M H

    2016-03-01

    Aging is associated with development of autoimmunity. Loss of B cell tolerance in the elderly is suggested by an increased prevalence of anti-nuclear antibodies (ANAs) and rheumatoid factors (RFs). Accumulating evidence indicates that B cells also impact autoimmunity via secretion of cytokines. So far, few studies have directly assessed the effect of aging on the latter B cell function. Here, we determined if and how human aging influences the production of cytokines by B cells. In a cross-sectional study, we found that absolute numbers of circulating B cells were similar in 31 young (ages 19-39) and 73 old (age ≥ 60) individuals. Numbers of transitional B cells (CD19(+)CD27(-)CD38(High)CD24(High)) were decreased in old individuals, whereas numbers of naive and memory B cell subsets were comparable in young and old individuals. Short-term in vitro stimulation of whole blood samples revealed that numbers of B cells capable of producing TNF-α were similar in young and old individuals. In contrast, B cells capable of IL-10 production were decreased in old subjects. This decline of IL-10(+) B cells was observed in old individuals that were ANA positive, and in those that were negative for both ANAs and RFs. However, IL-10(+) B cells were remarkably well retained in the circulation of old subjects that were RF positive. Thus, pro-inflammatory TNF-α(+) B cells are retained in the elderly, whereas IL-10(+) B cells generally decline. In addition, our findings indicate that IL-10(+) B cells may differentially impact the development of ANAs and RFs in the elderly.

  6. Monensin, a small molecule ionophore, can be used to increase high mannose levels on monoclonal antibodies generated by Chinese hamster ovary production cell-lines.

    Science.gov (United States)

    Pande, Sandhya; Rahardjo, Ayu; Livingston, Brittney; Mujacic, Mirna

    2015-07-01

    Asparagine-linked glycosylation of the constant region of monoclonal antibodies (mAbs) plays an important role in their stability and efficacy and is a critical product quality attribute that needs to be consistent between various process changes and production lots. Exact product quality match is also of the utmost importance for the development of biosimilar protein therapeutics. This poses a process development challenge since mAb glycosylation profiles can fluctuate easily with changes in process parameters. Therefore, there is a need to identify methods to modulate glycosylation levels on therapeutic antibodies during a production run in order to maintain consistent product quality profiles between different drug lots. Here, we demonstrate the use of a small molecule ionophore, monensin, to increase high mannose levels on multiple therapeutic human immunoglobulins (IgGs) in both plate-based small scale production models as well as in production bioreactors. This method is simple to implement and readily applicable for multiple production cell lines. Moreover, high mannose levels can be increased without significant negative impact on titer or cell culture performance. As such, monensin gives us a manipulable product quality lever. © 2015 Wiley Periodicals, Inc.

  7. Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim

    Full Text Available Burkholderia pseudomallei (BP and B. mallei (BM are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7 were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min, sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1 reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM. The cMAb BP7 2C6 (cMAb CK2 recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3 reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis. Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.

  8. Triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production.

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsushita

    Full Text Available Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs in transchromosomic (Tc cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC comprising the entire unrearranged human immunoglobulin (Ig heavy-chain (hIGH, kappa-chain (hIGK, and lambda-chain (hIGL germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO. However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK and lambda-chain (bIGL genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ. To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J and constant (C gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5 by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ.

  9. Prokaryotic expression, purification, and production of polyclonal antibody against novel human serum inhibited related protein I (SI1).

    Science.gov (United States)

    Ma, Mingxing; Ma, Jie; Shi, Yinghui; Wu, Hong; Zhao, Wenxiu; Huang, Weiwei; Jiao, Yang; Tan, Deyong

    2010-02-01

    A novel serum inhibited related gene (SI1) has been cloned in our lab by using mRNA differential display analysis of U251 cells in the presence or absence of serum, the expression of SI1 was dramatically inhibited by the addition of serum to serum starved cells. Previous reports suggested the potential significance of SI1 in regulating the cell cycle. In this study, the plasmid construction, protein expression and purification, as well as the generation of anti-SI1 polyclonal antibody are described. A full-length cDNA of Si1 was inserted in a prokaryotic expression plasmid pET28-b(+) and efficiently expressed in E. coli Rosetta (DE3) strain after induction by isopropyl-b-D: -thiogalactoside. The expressed 6His-tagged SI1 fusion protein was purified by Ni(+) affinity column and then used to immunize Balb/C mice, and the anti-SI1 polyclonal antibody was purified by protein A column. To determine the sensitivity and specificity of the antibody against SI1, a cell lysate of pEGFP-N2-SI1 plasmid transiently transfected Hela cell was identified by anti-GFP monoclonal antibody and anti-SI1 polyclonal antibody. Both the GFP-SI1 fusion protein and endogenous SI1 protein in Hela cell can be recognized by the anti-SI1 polyclonal antibody. The anti-SI1 polyclonal antibody will provide a useful tool for further characterization of SI1.

  10. Fcγ receptor-induced soluble vascular endothelial growth factor receptor-1 (VEGFR-1) production inhibits angiogenesis and enhances efficacy of anti-tumor antibodies.

    Science.gov (United States)

    Justiniano, Steven E; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P; Byrd, John C; Tridandapani, Susheela

    2013-09-13

    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

  11. Fcγ Receptor-induced Soluble Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1) Production Inhibits Angiogenesis and Enhances Efficacy of Anti-tumor Antibodies*

    Science.gov (United States)

    Justiniano, Steven E.; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M.; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D.; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P.; Byrd, John C.; Tridandapani, Susheela

    2013-01-01

    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy. PMID:23902770

  12. Expression of recombinant antibodies.

    Science.gov (United States)

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  13. Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

    Science.gov (United States)

    Abdolahi Khabisi, Samaneh; Sarkari, Bahador; Moshfe, Abdolali; Jalali, Sedigheh

    2017-02-01

    Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately

  14. Production of high titre antibody response against Russell's viper venom in mice immunized with ethanolic extract of fruits of Piper longum L. (Piperaceae) and piperine.

    Science.gov (United States)

    Shenoy, P A; Nipate, S S; Sonpetkar, J M; Salvi, N C; Waghmare, A B; Chaudhari, P D

    2014-01-15

    Piper longum L. fruits have been traditionally used against snakebites in north-eastern and southern region of India. The aim of the study was to assess the production of antibody response against Russell's viper venom in mice after prophylactic immunization with ethanolic extract of fruits of Piper longum L. and piperine. The mice sera were tested for the presence of antibodies against Russell's viper venom by in vitro lethality neutralization assay and in vivo lethality neutralization assay. Polyvalent anti-snake venom serum (antivenom) manufactured by Haffkine Bio-Pharmaceutical Corporation Ltd. was used as standard. Further confirmation of presence of antibodies against the venom in sera of mice immunized with PLE and piperine was done using indirect enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion test. Treatment with PLE-treated mice serum and piperine-treated mice serum was found to inhibit the lethal action of venom both in the in vitro lethality neutralization assay and in vivo lethality neutralization assay. ELISA testing indicated that there were significantly high (ppiperine treated mice serum and the venom antigens. In double immunodiffusion test, a white band was observed between the two wells of antigen and antibodies for both the PLE-treated and piperine-treated mice serum. Thus it can be concluded that immunization with ethanolic extract of fruits of Piper longum and piperine produced a high titre antibody response against Russell's viper venom in mice. The antibodies against PLE and piperine could be useful in antivenom therapy of Russell's viper bites. PLE and piperine may also have a potential interest in view of the development of antivenom formulations used as antidote against snake bites.

  15. Evaluation of a Fluorescent Antibody-Enrichment Serology Combination Procedure for the Detection of Salmonellae in Condiments, Food Products, Food By-Products, and Animal Feeds1

    Science.gov (United States)

    Hilker, John S.; Solberg, Myron

    1973-01-01

    The reliability of the enrichment serology (ES), fluorescent antibody (FA), and a combination of the FA and ES procedures for the detection of salmonellae were compared to the Salmonella cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). A total of 126 subsamples from 22 different products were analyzed. By utilizing the BAM procedure as the reference standard, a total of 66 samples were positive for salmonellae. Within 44 h approximately 65% of the Salmonella-negative samples could be cleared by the FA test. At the end of 50 h 97% of the Salmonella-negative samples could be cleared by the combination FA-ES test. The FA procedure detected all 66 BAM positives but exhibited a high incidence of presumptive positives which were cultural negatives. The ES procedure detected 64 of the 66 BAM positives but exhibited a low incidence of presumptive positives which were cultural negatives. Incorporating positive FA and positive ES results in a combination FA-ES technique revealed that FA-ES positives were statistically equivalent to BAM positives. PMID:4586931

  16. A high-yielding, generic fed-batch process for recombinant antibody production of GS-engineered cell lines

    DEFF Research Database (Denmark)

    Fan, Li; Zhao, Liang; Sun, Yating;

    2009-01-01

    An animal component-free and chemically defined fed-batch process for GS-engineered cell lines producing recombinant antibodies has been developed. The fed-batch process relied on supplying sufficient nutrients to match their consumption, simultaneously minimizing the accumulation of byproducts....... This generic and high-yielding fed-batch process would shorten development time, and ensure process stability, thereby facilitating the manufacture of therapeutic antibodies by GS-engineered cell lines....

  17. Bone marrow fibrosis and production of anti—rhEPO antibody induced by long—term abministration of rhEPO

    Institute of Scientific and Technical Information of China (English)

    LuQJ; WenLQ

    2002-01-01

    Recombinant human erythropoietin(rhEPO) is used extensively in anemic patients on dialysis and other patients,and is considered as very safety and effective in the correction of anemia in these patients.In a safety evaluation recently done in Beagles dogs,mild or moderate bone marrow fibrosis and neutralizing anti-rhEPO IgG production were found in the high dosage group of 1800 U·kg-1 after 13-week subcutaneous administration of rhEPO.The results suggest that long-term administration of rhEPO may result in the occurrence of bone marrow fibrosis and the development of neutralizing antibodies to rhEPO.In clinical practice,it is important to choose an appropriate dose regimen to reduce the risk of antibody production and adverse effects associated with long-term administration of high dose of rhEPO while achieving target hematocrit hevel.

  18. Production of polyclonal antibody against Tehran strain influenza virus (A/H1N1/2009 hemagglutinin conserved domain (HA2: brief report

    Directory of Open Access Journals (Sweden)

    Somayeh Zamani

    2015-10-01

    Full Text Available Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2 for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID in both forms, Single RID (SRID and Double RID (DRID. Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.

  19. The production of antibody by invading B cells is required for the clearance of rabies virus from the central nervous system.

    Directory of Open Access Journals (Sweden)

    D Craig Hooper

    2009-10-01

    Full Text Available The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell-deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus-neutralizing antibody reach infected cells in the cerebellum of B cell-deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus-specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues.

  20. Influence of Exogenous Reproductive Hormones on Specific Antibody Production in Genital Secretions after Vaginal Vaccination with Recombinant Cholera Toxin B Subunit in Humans

    OpenAIRE

    Wassen, Lotta; Jertborn, Marianne

    2006-01-01

    The objective of this study was to investigate the influence of exogenous reproductive hormones on the local and systemic production of specific immunoglobulin A (IgA) and IgG antibodies after vaginal vaccination with recombinant cholera toxin subunit B (CTB). Three groups of women using either progesterone-containing intrauterine devices (n = 9), oral contraceptives (n = 8), or no hormonal contraceptive methods (n = 9) were vaginally immunized twice, 2 weeks apart. Cervical secretions, vagin...

  1. Serotype-specific anti-Dengue virus NS1 mouse antibodies cross-react with prM and are potentially involved in virus production.

    Science.gov (United States)

    Masrinoul, Promsin; Omokoko, Magot Diata; Pambudi, Sabar; Ikuta, Kazuyoshi; Kurosu, Takeshi

    2013-08-01

    Dengue virus (DENV) infection induces a strong B-cell immune response against the viral nonstructural protein 1 (NS1). Anti-NS1 antibodies (Abs) may affect virus production because they coexist with the virus in the patients' blood. The present study examined whether ten mouse monoclonal antibodies (MAbs) raised against NS1 affected production of the DENV-2. Three MAbs, 4C2, 4G11, and 4E5, showed weak neutralizing activity in a focus reduction assay. In addition, two serotype-specific MAbs, 4C2 and 4G11, protected suckling mice from lethal infection with DENV-2. An immunoprecipitation assay with DENV-2 showed that these MAbs, which were specific for the NS1 of DENV-4 and DENV-1, cross-reacted with the DENV-2 pre-membrane (prM) protein, but not with DENV-2 NS1. Interestingly, high concentrations of MAb 4G11 showed antibody-dependent enhancement of DENV-2 infection in human monocyte THP-1 cells. Taken together, these observations suggest that serotype-specific anti-NS1 MAbs are potentially involved in virus production.

  2. Wheat germ cell-free system-based production of hemagglutinin-neuraminidase protein of human parainfluenza virus type 3: generation and characterization of monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Satoko eMatsunaga

    2014-05-01

    Full Text Available Human parainfluenza virus 3 (HPIV3 commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of monoclonal antibodies against hemagglutinin-neuraminidase (HN of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we newly identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. This study provides the availability of our newly-developed MAbs as a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

  3. Production and Application of Polyclonal Antibodies Against Recombinant Capsid Protein of Extra Small Virus of Macrobrachium rosenbergii.

    Science.gov (United States)

    Neethi, V; Sivakumar, N; Kumar, Kundan; Rajendran, K V; Makesh, M

    2012-12-01

    Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein.

  4. Determinants of mitotic catastrophe on abrogation of the G2 DNA damage checkpoint by UCN-01.

    Science.gov (United States)

    On, Kin Fan; Chen, Yue; Ma, Hoi Tang; Chow, Jeremy P H; Poon, Randy Y C

    2011-05-01

    Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G(2) DNA damage checkpoint. The CHK1 inhibitor 7-hydroxystaurosporine (UCN-01) can bypass the checkpoint and induce unscheduled mitosis in irradiated cells. Precisely, how cells behave following checkpoint abrogation remains to be defined. In this study, we tracked the fates of individual cells after checkpoint abrogation, focusing in particular on whether they undergo mitotic catastrophe. Surprisingly, while a subset of UCN-01-treated cells were immediately eliminated during the first mitosis after checkpoint abrogation, about half remained viable and progressed into G(1). Both the delay of mitotic entry and the level of mitotic catastrophe were dependent on the dose of radiation. Although the level of mitotic catastrophe was specific for different cell lines, it could be promoted by extending the mitosis. In supporting this idea, weakening of the spindle-assembly checkpoint, by either depleting MAD2 or overexpressing the MAD2-binding protein p31(comet), suppressed mitotic catastrophe. Conversely, delaying of mitotic exit by depleting either p31(comet) or CDC20 tipped the balance toward mitotic catastrophe. These results underscore the interplay between the level of DNA damage and the effectiveness of the spindle-assembly checkpoint in determining whether checkpoint-abrogated cells are eliminated during mitosis.

  5. Enhancement and abrogation : modifications of host immune status influence IL-2 and LAK cell immunotherapy

    NARCIS (Netherlands)

    E.P. Steller (Erick)

    1988-01-01

    textabstractThis thesis will discuss the role immune cells and the host immune system can play in enhancement and abrogation of this novel immunotherapy with interleukin 2 and lymphokine-activated killer cells. Chapter 3 and 4 will discuss the scoring methods in this intraperitoneal cancer and immun

  6. Drug-induced reactivation of apoptosis abrogates HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Hartmut M Hanauske-Abel

    Full Text Available HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of

  7. Two-dimensional capillary zone electrophoresis-mass spectrometry for the characterization of intact monoclonal antibody charge variants, including deamidation products.

    Science.gov (United States)

    Jooß, Kevin; Hühner, Jens; Kiessig, Steffen; Moritz, Bernd; Neusüß, Christian

    2017-08-12

    Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE-CZE-MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE-CZE-MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained.

  8. RNA sensors enable human mast cell anti-viral chemokine production and IFN-mediated protection in response to antibody-enhanced dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Michael G Brown

    Full Text Available Dengue hemorrhagic fever and/or dengue shock syndrome represent the most serious pathophysiological manifestations of human dengue virus infection. Despite intensive research, the mechanisms and important cellular players that contribute to dengue disease are unclear. Mast cells are tissue-resident innate immune cells that play a sentinel cell role in host protection against infectious agents via pathogen-recognition receptors by producing potent mediators that modulate inflammation, cell recruitment and normal vascular homeostasis. Most importantly, mast cells are susceptible to antibody-enhanced dengue virus infection and respond with selective cytokine and chemokine responses. In order to obtain a global view of dengue virus-induced gene regulation in mast cells, primary human cord blood-derived mast cells (CBMCs and the KU812 and HMC-1 mast cell lines were infected with dengue virus in the presence of dengue-immune sera and their responses were evaluated at the mRNA and protein levels. Mast cells responded to antibody-enhanced dengue virus infection or polyinosiniċpolycytidylic acid treatment with the production of type I interferons and the rapid and potent production of chemokines including CCL4, CCL5 and CXCL10. Multiple interferon-stimulated genes were also upregulated as well as mRNA and protein for the RNA sensors PKR, RIG-I and MDA5. Dengue virus-induced chemokine production by KU812 cells was significantly modulated by siRNA knockdown of RIG-I and PKR, in a negative and positive manner, respectively. Pretreatment of fresh KU812 cells with supernatants from dengue virus-infected mast cells provided protection from subsequent infection with dengue virus in a type I interferon-dependent manner. These findings support a role for tissue-resident mast cells in the early detection of antibody-enhanced dengue virus infection via RNA sensors, the protection of neighbouring cells through interferon production and the potential recruitment of

  9. Evaluation of cysticercus-specific IgG (total and subclasses) and IgE antibody responses in cerebrospinal fluid samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies.

    Science.gov (United States)

    Suzuki, Lisandra Akemi; Rossi, Cláudio Lúcio

    2013-02-01

    In the present study, an enzyme-linked immunosorbent assay (ELISA) standardized with vesicular fluid of Taenia solium cysticerci was used to screen for IgG (total and subclasses) and IgE antibodies in cerebrospinal fluid (CSF) samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies and patients with other neurological disorders. The following results were obtained: IgG-ELISA: 100% sensitivity (median of the ELISA absorbances (MEA)=1.17) and 100% specificity; IgG1-ELISA: 72.7% sensitivity (MEA=0.49) and 100% specificity; IgG2-ELISA: 81.8% sensitivity (MEA=0.46) and 100% specificity; IgG3-ELISA: 63.6% sensitivity (MEA=0.12) and 100% specificity; IgG4-ELISA: 90.9% sensitivity (MEA=0.85) and 100% specificity; IgE-ELISA 93.8% sensitivity (MEA=0.60) and 100% specificity. There were no significant differences between the sensitivities and specificities in the detection of IgG-ELISA and IgE-ELISA, although in CSF samples from patients with neurocysticercosis the MEA of the IgG-ELISA was significantly higher than that of the IgE-ELISA. The sensitivity and MEA values of the IgG4-ELISA were higher than the corresponding values for the other IgG subclasses. Future studies should address the contribution of IgG4 and IgE antibodies to the physiopathology of neurocysticercosis.

  10. Evaluation of cysticercus-specific IgG (total and subclasses and IgE antibody responses in cerebrospinal fluid samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies

    Directory of Open Access Journals (Sweden)

    Lisandra Akemi Suzuki

    Full Text Available In the present study, an enzyme-linked immunosorbent assay (ELISA standardized with vesicular fluid of Taenia solium cysticerci was used to screen for IgG (total and subclasses and IgE antibodies in cerebrospinal fluid (CSF samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies and patients with other neurological disorders. The following results were obtained: IgG-ELISA: 100% sensitivity (median of the ELISA absorbances (MEA=1.17 and 100% specificity; IgG1-ELISA: 72.7% sensitivity (MEA=0.49 and 100% specificity; IgG2-ELISA: 81.8% sensitivity (MEA=0.46 and 100% specificity; IgG3-ELISA: 63.6% sensitivity (MEA=0.12 and 100% specificity; IgG4-ELISA: 90.9% sensitivity (MEA=0.85 and 100% specificity; IgE-ELISA 93.8% sensitivity (MEA=0.60 and 100% specificity. There were no significant differences between the sensitivities and specificities in the detection of IgG-ELISA and IgE-ELISA, although in CSF samples from patients with neurocysticercosis the MEA of the IgG-ELISA was significantly higher than that of the IgE-ELISA. The sensitivity and MEA values of the IgG4-ELISA were higher than the corresponding values for the other IgG subclasses. Future studies should address the contribution of IgG4 and IgE antibodies to the physiopathology of neurocysticercosis.

  11. Kinetics of the avian influenza-specific humoral responses in lung are indicative of local antibody production

    NARCIS (Netherlands)

    Geus, de E.D.; Rebel, J.M.J.; Vervelde, L.

    2012-01-01

    The role and kinetics of respiratory immunoglobulins in AIV infection has not been investigated. In this study we determined the numbers of both total antibody secreting cells (ASC) and virus-specific ASC in lung, spleen, blood and bone marrow (BM) following low-pathogenic AIV infection. Antiviral h

  12. Production and characterization of a camelid single domain antibody-urease enzyme conjugate for the treatment of cancer.

    Science.gov (United States)

    Tian, Baomin; Wong, Wah Yau; Hegmann, Elda; Gaspar, Kim; Kumar, Praveen; Chao, Heman

    2015-06-17

    A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.

  13. Immunization with Protein D from Non-Typeable Haemophilus influenzae (NTHi) Induced Cytokine Responses and Bioactive Antibody Production

    Science.gov (United States)

    Davoudi Vijeh Motlagh, Atefeh; Siadat, Seyed Davar; Abedian Kenari, Saeid; Mahdavi, Mehdi; Behrouzi, Ava; Asgarian-Omran, Hossein

    2016-01-01

    Background Outer membrane protein D (PD) is a highly conserved and stable protein in the outer membrane of both encapsulated (typeable) and non-capsulated (non-typeable) strains of Haemophilus influenzae. As an immunogen, PD is a potential candidate vaccine against non-typeable H. influenzae (NTHi) strains. Objectives The aim of this study was to determine the cytokine pattern and the opsonic antibody response in a BALB/c mouse model versus PD from NTHi as a vaccine candidate. Methods Protein D was formulated with Freund’s and outer membrane vesicle (OMV) adjuvants and injected into experimental mice. Sera from all groups were collected. The bioactivity of the anti-PD antibody was determined by opsonophagocytic killing test. To evaluate the cytokine responses, the spleens were assembled, suspension of splenocytes was recalled with antigen, and culture supernatants were analyzed by ELISA for IL-4, IL-10, and IFN-γ cytokines. Results Anti-PD antibodies promoted phagocytosis of NTHi in both immunized mice groups (those administered PD + Freund’s and those administered PD + OMV adjuvants, 92.8% and 83.5%, respectively, compared to the control group). In addition, the concentrations of three cytokines were increased markedly in immunized mice. Conclusions We conclude that immunization with PD protects mice against NTHi. It is associated with improvements in both cellular and humoral immune responses and opsonic antibody activity. PMID:27942362

  14. Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Daohong [Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062 (China); Li Peiwu [Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062 (China); Quality Inspection and Test Center for Oilseeds and Products of Ministry of Agriculture, Wuhan 430062 (China)], E-mail: peiwuli@oilcrops.cn; Zhang Qi; Zhang Wen; Huang Yanling; Ding Xiaoxia; Jiang Jun [Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062 (China); Quality Inspection and Test Center for Oilseeds and Products of Ministry of Agriculture, Wuhan 430062 (China)

    2009-03-16

    Monoclonal antibodies (McAbs) cross-reactive with four major aflatoxins were achieved using a modified two-step screening procedure. The first step was twice modified indirect enzyme-linked immunosorbent assay (ELISA) and resulted in positive hybridomas and hapten-specific antibodies. The modified indirect competitive ELISA (ciELISA) was the second step, in which the competition incubation time was decreased to 30 min, aflatoxin B{sub 1}, B{sub 2}, G{sub 1} and G{sub 2} were all used as competitors, the concentrations of four aflatoxins were gradiently decreased in each screening. 2-3 subclonings were performed after every modified fusion and resulted in eight hybridomas that secreted antibodies with good cross-reactivity and high affinity to four aflatoxins. Five McAbs were chosen for further analysis. Of the five, two antibodies had similar reaction efficiency with aflatoxin B{sub 1}, B{sub 2} and G{sub 1} but showed a weak cross-reaction to G{sub 2}. Another two had almost identical reaction capability with four aflatoxins. One clone 1C11 exhibited the highest sensitivity for all four aflatoxins. The concentrations of aflatoxin B{sub 1}, B{sub 2}, G{sub 1} and G{sub 2} at 50% inhibition for 1C11 were 1.2, 1.3, 2.2 and 18.0 pg mL{sup -1} respectively. This is the most sensitive for all four major aflatoxins described so far. The results indicated that the modified two-step screening procedure had superiority and these antibodies could be used for simultaneous analysis of total aflatoxins.

  15. Immunization of chickens with quail and turkey perivitelline membrane proteins: production of antibodies and their effects on fertility.

    Science.gov (United States)

    Kapoor, P; Compton, M M; Howarth, B

    2000-02-01

    The cross-reactivity of antibodies developed against zona pellucida proteins and their subsequent deleterious effect on fertility in heterologous species is well documented. However, similar investigations have not been undertaken in avian species. In Experiment 1, White Leghorn hens were immunized with intact germinal discs (GD) of quail and chickens. Chicken GD proteins did not elicit an immune response in chicken hens, whereas quail GD proteins were highly immunogenic. Anti-quail GD antibodies did not bind with chicken inner perivitelline membrane (IPM) proteins as determined by immunoblot analysis. To examine the fertility of immunized hens, artificial insemination was performed at weekly intervals for 4 wk following the booster immunization. No significant differences were detected in fertility or hatchability of immunized hens when compared with unimmunized control hens. In Experiment 2, White Leghorn hens were immunized with intact turkey GD, solubilized turkey perivitelline membrane (PM) modified with dinitrophenol (DNP), and solubilized chicken IPM modified with DNP. High titers of antibodies were detected against the turkey GD and the DNP-modified turkey PM proteins. A weak immune response was observed in hens immunized with modified chicken IPM proteins. The fertility and hatchability of eggs laid by immunized hens, however, were not significantly different from those of unimmunized hens. Antibodies from immunized hens were further analyzed using an in vitro assay that assesses sperm penetration of intact IPM. Sperm penetration of intact IPM was inhibited to the same extent when IPM was preincubated with preimmune as well as anti-PM immunoglobulins. Collectively, these results suggested that the antibodies developed in these hens did not cross-react with the chicken IPM proteins involved in sperm-egg interaction and thus did not influence the fertility.

  16. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  17. L-carnitine, a diet component and organic cation transporter OCTN ligand, displays immunosuppressive properties and abrogates intestinal inflammation.

    Science.gov (United States)

    Fortin, G; Yurchenko, K; Collette, C; Rubio, M; Villani, A-C; Bitton, A; Sarfati, M; Franchimont, D

    2009-04-01

    Allele variants in the L-carnitine (LCAR) transporters OCTN1 (SLC22A4, 1672 C --> T) and OCTN2 (SLC22A5, -207 G --> C) have been implicated in susceptibility to Crohn's disease (CD). LCAR is consumed in the diet and transported actively from the intestinal lumen via the organic cation transporter OCTN2. While recognized mainly for its role in fatty acid metabolism, several lines of evidence suggest that LCAR may also display immunosuppressive properties. This study sought to investigate the immunomodulatory capacity of LCAR on antigen-presenting cell (APC) and CD4+ T cell function by examining cytokine production and the expression of activation markers in LCAR-supplemented and deficient cell culture systems. The therapeutic efficacy of its systemic administration was then evaluated during the establishment of colonic inflammation in vivo. LCAR treatment significantly inhibited both APC and CD4+ T cell function, as assessed by the expression of classical activation markers, proliferation and cytokine production. Carnitine deficiency resulted in the hyperactivation of CD4+ T cells and enhanced cytokine production. In vivo, protection from trinitrobenzene sulphonic acid colitis was observed in LCAR-treated mice and was attributed to the abrogation of both innate [interleukin (IL)-1beta and IL-6 production] and adaptive (T cell proliferation in draining lymph nodes) immune responses. LCAR therapy may therefore represent a novel alternative therapeutic strategy and highlights the role of diet in CD.

  18. From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants.

    Science.gov (United States)

    Sack, Markus; Rademacher, Thomas; Spiegel, Holger; Boes, Alexander; Hellwig, Stephan; Drossard, Juergen; Stoger, Eva; Fischer, Rainer

    2015-10-01

    The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority.

  19. Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells.

    Science.gov (United States)

    Ren, Weicheng; Grimsholm, Ola; Bernardi, Angelina I; Höök, Nina; Stern, Anna; Cavallini, Nicola; Mårtensson, Inga-Lill

    2015-04-01

    Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

  20. Effect of ambient light on monoclonal antibody product quality during small-scale mammalian cell culture process in clear glass bioreactors.

    Science.gov (United States)

    Mallaney, Mary; Wang, Szu-Han; Sreedhara, Alavattam

    2014-01-01

    During a small-scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small-scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production.

  1. Monoclonal Antibodies Production Platforms: An Opportunity Study of a Non-Protein-A Chromatographic Platform Based on Process Economics.

    Science.gov (United States)

    Grilo, António L; Mateus, Marília; Aires-Barros, Maria R; Azevedo, Ana M

    2017-09-13

    Monoclonal antibodies currently dominate the biopharmaceutical market with growing sales having reached 80 billion USD in 2016. As most top-selling mAbs are approaching the end of their patent life, biopharmaceutical companies compete fiercely in the biosimilars market. These two factors present a strong motivation for alternative process strategies and process optimization. In this work a novel purification strategy for monoclonal antibodies comprising phenylboronic acid multimodal chromatography for capture followed by polishing by ion-exchange monolithic chromatography and packed bed hydrophobic interaction chromatography is presented and compared to the traditional protein-A-based process. Although the capital investment is similar for both processes, the operation cost is 20% lower for the novel strategy. This study shows that the new process is worthwhile investing in and could present a viable alternative to the platform process used by most industrial players. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Gamma ray-induced mutants as a tool for the production and characterisation of monoclonal antibodies against HLA-alloantigens

    Energy Technology Data Exchange (ETDEWEB)

    Spring, B.; Pawelec, G.; Ziegler, A.

    1986-01-01

    To simplify the screening procedure for murine monoclonal antibodies specific for polymorphic HLA determinants, spleen cells from a mouse immunized with the human cell line BJAB-B95.8.6 were fused with NS1 mouse myeloma cells, and hybridoma supernatants were screened for their reactivity on BJAB-B95.8.6 and two gamma ray-induced HLA-loss mutants of this line. The use of these HLA-loss mutants allowed the rapid identification of two new allospecific MOABs designated TU160 and TU161. Serological as well as biochemical studies revealed TU160 to be specific for HLA=A2, and TU161 for HLA-B13 molecules, respectively. Both MOABs were determined to be antibodies of the IgG class and were able to precipitate their antigens from lysates of radioactively labeled cells.

  3. Oral Application of T4 Phage Induces Weak Antibody Production in the Gut and in the Blood

    Directory of Open Access Journals (Sweden)

    Joanna Majewska

    2015-08-01

    Full Text Available A specific humoral response to bacteriophages may follow phage application for medical purposes, and it may further determine the success or failure of the approach itself. We present a long-term study of antibody induction in mice by T4 phage applied per os: 100 days of phage treatment followed by 112 days without the phage, and subsequent second application of phage up to day 240. Serum and gut antibodies (IgM, IgG, secretory IgA were analyzed in relation to microbiological status of the animals. T4 phage applied orally induced anti-phage antibodies when the exposure was long enough (IgG day 36, IgA day 79; the effect was related to high dosage. Termination of phage treatment resulted in a decrease of IgA again to insignificant levels. Second administration of phage induces secretory IgA sooner than that induced by the first administrations. Increased IgA level antagonized gut transit of active phage. Phage resistant E. coli dominated gut flora very late, on day 92. Thus, the immunological response emerges as a major factor determining phage survival in the gut. Phage proteins Hoc and gp12 were identified as highly immunogenic. A low response to exemplary foreign antigens (from Ebola virus presented on Hoc was observed, which suggests that phage platforms can be used in oral vaccine design.

  4. Influence of single nucleotide polymorphisms of cytokine genes on anti-HBs antibody production after hepatitis B vaccination in a Japanese young adult population.

    Science.gov (United States)

    Yukimasa, Nobuyasu; Sato, Shoichi; Oboshi, Wataru; Watanabe, Toru; Uzawa, Ryuichi

    2016-01-01

    Hepatitis B (HB) vaccination is one of the most efficient tools to prevent the transmission of the virus. Considerable variability exists in HB vaccine responses, with 5-10% of healthy Japanese adults demonstrating no response following a standard vaccination. Recently, polymorphisms of immune-regulatory genes, such as cytokine genes, have been reported to influence the immune response to HB vaccine. The aim of this study was to investigate the underlying mechanisms of the genetic association between several cytokine gene polymorphisms and the immune response to HB vaccination in a Japanese population. One hundred and twenty three vaccinated young adults were classified according to the level of antibody-titer (anti-HBs). Single nucleotide polymorphism typing for IFN-γ (+874, 3'-UTR), IL-10 (-591, -819, -1082), and TNF-α (-308, -857), was accomplished using the PCR-RFLP or SSP-PCR method. The TNF-α (-857) CC type and the IL-10 (-1082) AG type were present more frequently in the low titer group than in the high titer group. The TNF-α (-857) CC type was found to be significantly associated with low response of serum anti-HBs. The anti-HBs antibody was not readily produced in the IL-10 (-1082) AG and TNF-α (-857) CC haplotype. Conversely, the antibody was readily produced in the IL-10 (-1082) AA and TNF-α (-857) CC haplotype, and the IL-10 (-1082) AA and TNF-α (-857) CT haplotype, suggesting a high likelihood of the IL-10 (-1082) AG type to be included in the low anti-HBs group, and high anti-HBs antibody production in those with the TNF-α (-857) CT type. These SNPs may produce ethnically-specific differences in the immune response to HB vaccine in the Japanese population. J. Med. Invest. 63: 256-261, August, 2016.

  5. The influence of beryllium on cell survival rates in theIn-vitro culture system, on intracellular DNA synthesis and on SRBC-IgM antibody production responses

    OpenAIRE

    Yoshida, Tsutomu; Shima , Syogo; Kurita , Hideki; Nagaoka, Kaoru; Taniwaki, Hiroshige; Asada, Yasuki; Shai , Kai-ping; Koike, Mitsumasa; Morita, Kunihiko

    1997-01-01

    Immunocytotoxicity of beryllium (Be) was evaluated by studying cell viability, intracellular DNA synthesis and SRBC-IgM response in an in-vitro culture system using non-sensitized spleen cells of a C57BL mouse. Be addition showed a suppressive effect on cell viability, an enhancing effect on DNA synthesis and on IgM antibody production. The suppressive effect on cell viability manifested itself markedly as the concentration of Be was increased or the culture time was prolonged. The DNA synthe...

  6. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis.

    Science.gov (United States)

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C

    2008-07-01

    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  7. Optimisation of production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular chaperones and functional immobilisation on a mesoporous silicate support.

    Science.gov (United States)

    Hu, Xuejun; O'Hara, Liam; White, Simon; Magner, Edmond; Kane, Marian; Wall, J Gerard

    2007-03-01

    Domoic acid is a potent neurotoxin that can lead to amnesic shellfish poisoning in humans through ingestion of contaminated shellfish. We have produced and purified an anti-domoic acid single-chain Fragment variable (scFv) antibody fragment from the Escherichia coli periplasm. Yields of functional protein were increased by up to 100-fold upon co-production of E. coli DnaKJE molecular chaperones but co-overproduction of GroESL led to a reduction in solubility of the scFv. Co-production of the peptidyl-prolyl isomerase trigger factor resulted in accumulation of unprocessed scFv in the E. coli cytoplasm. This was due to an apparent bottleneck in translocation of the cytoplasmic membrane by the recombinant polypeptide. Co-expression of the E. coli disulfide bond isomerase dsbC increased scFv yields by delaying lysis of the host bacterial cells though this effect was not synergistic with molecular chaperone co-production. Meanwhile, use of a cold-shock promoter for protein production led to accumulation of greater amounts of scFv polypeptide which was predominantly in insoluble form and could not be rescued by chaperones. Purification of the scFv was achieved using an optimised metal affinity chromatography procedure and the purified protein bound domoic acid when immobilised on a mesoporous silicate support. The work outlines the potential benefit of applying a molecular chaperone/folding catalyst screening approach to improve antibody fragment production for applications such as sensor development.

  8. Oral Vitamin A and Retinoic Acid Supplementation Stimulates Antibody Production and Splenic Stra6 Expression in Tetanus Toxoid–Immunized Mice12

    Science.gov (United States)

    Tan, Libo; Wray, Amanda E.; Ross, A. Catharine

    2012-01-01

    Coadministration of retinoic acid (RA) and polyinosinic acid:polycytidylic acid (PIC) has been shown to cooperatively enhance the anti–tetanus toxoid (anti-TT) vaccine response in adult mice. Germinal center formation in the spleen is critical for a normal antibody response. Recent studies have identified Stimulated by Retinoic Acid-6 (Stra6) as the cell membrane receptor for retinol-binding protein (RBP) in many organs, including spleen. The objectives of the present studies were to test whether orally administered vitamin A (VA) itself, either alone or combined with RA, and/or treatment with PIC regulates Stra6 gene expression in mouse spleen and, concomitantly, antibody production. Eight-week-old C57BL/6 mice were immunized with TT. In an initial kinetic study, oral VA (6 mg/kg) increased anti-TT IgM and IgG production as well as splenic Stra6 mRNA expression. In treatment studies that were analyzed 9 d postimmunization, retinoids including VA, RA, VA and RA combined, and PIC significantly increased plasma anti-TT IgM and IgG (P Treatments that included PIC elevated plasma anti-TT IgM and IgG concentrations >20-fold (P treatment. In conclusion, retinoid treatments that included VA, RA, VA and RA combined, and the combination of retinoid and PIC stimulated the expression of Stra6 in spleen, which potentially could increase the local uptake of retinol. Concomitantly, these treatments increased the systemic antigen-specific antibody response. The ability of oral retinoids to stimulate systemic immunity has implications for public health and therapeutic use of VA. PMID:22739370

  9. Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

    OpenAIRE

    Hiroaki Matsushita; Akiko Sano; Hua Wu; Jin-An Jiao; Poothappillai Kasinathan; Eddie J. Sullivan; Zhongde Wang; Yoshimi Kuroiwa

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM−/− , bIGHM...

  10. Triple Immunoglobulin Gene Knockout Transchromosomic (Tc) Cattle: Bovine Lambda Cluster Deletion and its Effect on Fully Human Polyclonal Antibody Production

    OpenAIRE

    Matsushita, H.; Sano, A.; Wu, H.; J. Jiao; Kasinathan, P.; Sullivan, E. J.; Wang, Zhongde; Kuroiwa, K

    2014-01-01

    Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML...

  11. Oral delivery of Brucella spp. recombinant protein U-Omp16 abrogates the IgE-mediated milk allergy

    Science.gov (United States)

    Smaldini, Paola Lorena; Ibañez, Andrés Esteban; Fossati, Carlos Alberto; Cassataro, Juliana; Docena, Guillermo Horacio

    2014-01-01

    Food allergies are increasingly common disorders and no therapeutic strategies are yet approved. The unlipidated Omp16 (U-Omp16) is the outer membrane protein of 16 kDa from B. abortus and possesses a mucosal adjuvant property. In this study, we aimed to examine the U-Omp16 capacity to abrogate an allergen-specific Th2 immune response when it is administered as an oral adjuvant in a mouse model of food allergy.   Balb/c mice were sensitized with cholera toxin and cow’s milk proteins (CMP) by gavage and simultaneously treated with U-Omp16 and CMP. Oral challenge with CMP was performed to evaluate the allergic status of mice. Symptoms, local (small bowel cytokine and transcription factor gene expression) and systemic (specific isotypes and spleen cell-secreted cytokines) parameters, and skin tests were done to evaluate the immune response. We found that the oral administration of U-Omp16 with CMP during sensitization dampened the allergic symptoms, with negativization of immediate skin test and increased skin DTH response. Serum specific IgE and IL-5 were inhibited and a Th1 response was promoted (specific IgG2a antibodies and CMP-induced IFN-γ secretion). We found at the mucosal site an inhibition of the gene expression corresponding to IL-13 and Gata-3, with an induction of IFN-γ and T-bet. These results indicated that the oral administration of U-Omp16 significantly controlled the allergic response in sensitized mice with a shift of the balance of Th1- and Th2-T cells toward Th1 predominance. These findings suggest that U-Omp16 may be useful as a Th1-directing adjuvant in an oral vaccine. PMID:25424811

  12. High zinc ion supplementation of more than 30 μM can increase monoclonal antibody production in recombinant Chinese hamster ovary DG44 cell culture.

    Science.gov (United States)

    Kim, Bong Gyun; Park, Hong Woo

    2016-03-01

    Effects of high ZnSO4·7H2O supplementation on cell growth and monoclonal antibody (mAb) production in chemically defined suspension cultures of recombinant Chinese hamster ovary (rCHO) DG44 cells were examined. The supplementation of ZnSO4·7H2O up to 120 μM gradually increased specific mAb production rate of rCHO DG44 cells in the early growth phase (0-4 days of culture). The ZnSO4·7H2O concentration for enhancing mAb production without any cytotoxic effects on cell growth was 30-60 μM. In addition of 60 μM ZnSO4·7H2O to in-house protein-free medium and in-house chemically defined medium, mAb production was increased 2.0-fold and 6.5-fold, respectively. Moreover, addition of ZnSO4·7H2O to three kinds of commercial chemically defined media yielded a greater than 1.2-fold enhancement of mAb production. These data indicate that simple supplementation of a relatively high zinc ion concentration to cell culture media without significant changes of rCHO DG44 cell culture process can be useful for achieving high production of mAb.

  13. Production of two hemopoietic growth factors is differentially regulated in single T lymphocytes activated with an anti-T cell receptor antibody

    DEFF Research Database (Denmark)

    Kelso, A; Owens, T

    1988-01-01

    by micromanipulation into wells coated with the monoclonal anti-T cell receptor antibody F23.1, up to 90% of cells produced CSF as detected by CSF-dependent hemopoietic cell lines. Production occurred in the absence of proliferation and did not require the addition of accessory cells or IL-2. Both the frequency of CSF......-producing cells and the average production per positive cell depended on the density of the immobilized stimulating ligand, indicating that the response of each cell is not an all-or-none phenomenon but varies with the strength of stimulation. Individual cells of the clone varied over a 100-fold range...... based since such cells could give rise to progeny that synthesized multi-CSF. These results suggest that the synthesis of these two lymphokines can be differentially regulated at the level of the single cell....

  14. Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells.

    Science.gov (United States)

    Lee, Yang-Jin; Kim, Jong-Hyun; Jeong, Seok-Ryoul; Song, Kyoung-Ju; Kim, Kyongmin; Park, Sun; Park, Moon-Sung; Shin, Ho-Joon

    2007-10-01

    Naegleria fowleri, agent of fatal primary amoebic meningoencephalitis, appears to induce cytotoxicity mechanically through its contact with the cell. The nfa1 gene cloned from a cDNA library of pathogenic N. fowleri by immunoscreening consists of 360 bp and expresses a 13.1-kDa recombinant protein (rNfa1) that demonstrated localization in the pseudopodia when examined using immunocytochemistry. To study the mechanisms involved in N. fowleri cytotoxicity, we developed a large volume of rNfa1-specific monoclonal antibody (McAb) against a 17-kDa His-tag fusion rNfa1 protein using a cell fusion technique. We established eight McAb-producing hybridoma cells. The antibodies were all immunoglobulin G2b and reacted strongly with a 17-kDa band representing the rNfa1 fusion protein in Western blotting, demonstrating immunoreactivity to the Nfa1 protein in pseudopodia (especially in the food cups) of N. fowleri trophozoites. A 51Cr-release assay indicated N. fowleri cytotoxicity by demonstrating that it eliminated 37.8, 60.6, and 98.8% of the target (microglial) cells 6, 12, and 24 h after co-incubation, respectively. When an anti-Nfa1 McAb was added to the coculture system, N. fowleri cytotoxicity decreased to 29.8, 44.1, and 66.3%, respectively.

  15. Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Caroline A Menezes

    2006-12-01

    Full Text Available Strains of enterotoxigenic Escherichia coli (ETEC are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

  16. Ibuprofen and other widely used non-steroidal anti-inflammatory drugs inhibit antibody production in human cells.

    Science.gov (United States)

    Bancos, Simona; Bernard, Matthew P; Topham, David J; Phipps, Richard P

    2009-01-01

    The widely used non-steroidal anti-inflammatory drugs (NSAIDs) function mainly through inhibition of cyclooxygenases 1 and 2 (Cox-1 and Cox-2). Unlike Cox-1, Cox-2 is considered an inducible and pro-inflammatory enzyme. We previously reported that Cox-2 is upregulated in activated human B lymphocytes and using Cox-2 selective inhibitors that Cox-2 is required for optimal antibody synthesis. It is not known whether commonly used non-prescription and non-Cox-2 selective drugs also influence antibody synthesis. Herein, we tested a variety of Cox-1/Cox-2 non-selective NSAIDs, namely ibuprofen, tylenol, aspirin and naproxen and report that they blunt IgM and IgG synthesis in stimulated human peripheral blood mononuclear cells (PBMC). Ibuprofen had its most profound effects in inhibiting human PBMCs and purified B lymphocyte IgM and IgG synthesis when administered in the first few days after activation. As shown by viability assays, ibuprofen did not kill B cells. The implications of this research are that the use of widely available NSAIDs after infection or vaccination may lower host defense. This may be especially true for the elderly who respond poorly to vaccines and heavily use NSAIDs.

  17. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  18. [Production of a recombinant CagA protein for the detection of Helicobacter pylori CagA antibodies].

    Science.gov (United States)

    Akgüç, Miray; Karatayli, Ersin; Çelik, Esra; Koyuncu, Duygu; Çelik, İnci; Karatayli, Senem Ceren; Özden, Ali; Bozdayi, A Mithat

    2014-07-01

    At present, Helicobacter pylori infections affect approximately 50% of the world population. It is known that H.pylori is related with several gastric diseases including chronic atrophic gastritis, peptic and gastric ulcers as well as gastric carcinomas. CagA (Cytotoxin-associated gene A) protein which is one of the most important virulence factors of H.pylori, is thought to be responsible for the development of gastric cancer. CagA is a 128 kDa hydrophilic protein which binds to the epitelial stomach cells and is known to be phosphorylated on its EPIYA regions. The EPIYA regions are highly variable and carry a higher risk of developing gastric cancer than CagA negative strains. The aim of this study was to construct a prokaryotic expression system expressing a recombinant CagA protein, which can be used for the detection of anti-CagA antibodies. For the isolation of H.pylori genomic DNA, a total of 112 gastric biopsy samples obtained from patients who were previously found positive for rapid urease (CLO) test, were used. H.pylori DNAs were amplified from 57 of those samples by polymerase chain reaction (PCR) and of them 35 were found positive in terms of cagA gene. Different EPIYA motifs were detected in 25 out of 35 cagA positive samples, and one of those samples that contained the highest number of EPIYA motif, was chosen for the cloning procedure. Molecular cloning and expression of the recombinant fragment were performed with Champion Pet151/D expression vector (Invitrogen, USA), the expression of which was induced by the addition of IPTG (Isopropyl-beta-D-thiogalactopyranoside) into the E.coli culture medium. Expression was observed with anti-histidin HRP (Horse Radish Peroxidase) antibodies by SDS-PAGE and Western Blot (WB) analysis. In our study, two clones possessing different fragments from the same H.pylori strain with three different EPIYA motifs were succesfully expressed. Since CagA antigen plays a signicant role in the pathogenesis of H

  19. Use of a Plackett-Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells.

    Science.gov (United States)

    González-Leal, I J; Carrillo-Cocom, L M; Ramírez-Medrano, A; López-Pacheco, F; Bulnes-Abundis, D; Webb-Vargas, Y; Alvarez, M M

    2011-01-01

    Culture media design is central to the optimization of monoclonal antibody (mAb) production. Although general strategies do not currently exist for optimization of culture media, the combined use of statistical design and analysis of experiments and strategies based on simple material balances can facilitate culture media design. In this study, we evaluate the effect of selected amino acids on the growth rate and monoclonal antibody production of a Chinese hamster ovary DG-44 (CHO-DG44) cell line. These amino acids were selected based on their relative mass fraction in the specific mAb produced in this study, their consumption rate during bioreactor experiments, and also through a literature review. A Plackett-Burman statistical design was conducted to minimize the number of experiments needed to obtain statistically relevant information. The effect of this set of amino acids was evaluated during exponential cell culture (considering viable cell concentration and the specific growth rate as main output variables) and during the high cell-density stage (considering mAb final concentration and specific productivity as relevant output variables). For this particular cell line, leucine (Leu) and arginine (Arg) had the highest negative and positive effects on cell viability, respectively; Leu and threonine (Thr) had the highest negative effect on growth rate, and valine (Val) and Arg demonstrated the highest positive impact on mAb final concentration. Results suggest the pertinence of a two-stage strategy for amino acid supplementation, with a mixture optimized for cell growth and a different amino acid mixture for mAb production at high density.

  20. Frequencies of the expression of main protein antigens from Helicobacter pylori isolates and production of specific serum antibodies in infected patients

    Science.gov (United States)

    Yan, Jie; Mao, Ya-Fei; Shao, Zhe-Xin

    2005-01-01

    AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori (H pylori) isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori. METHODS: H pylori strains in biopsy specimens from 157 patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected. The target recombinant proteins rUreB, rVacA, rCagA1, rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography. Rabbit antisera against rUreB, rVacA, rCagA1, rHpaA, rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody, expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed. RESULTS: In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2% respectively. In the 125 serum samples from the H pylori-infected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FlaA and

  1. Conjugation of R-Phycoerythrin to a Polyclonal Antibody and F (ab')2 Fragment of a Polyclonal Antibody by Two Different Methods.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Zarnani, Amir Hassan; Goli, Leila Balaei; Babaei, Mahdokht; Ghods, Roya

    2010-04-01

    R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab')2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity.

  2. A monoclonal antibody against leptin.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.

  3. Identification of IgE-binding proteins from Lepidoglyphus destructor and production of monoclonal antibodies to a major allergen.

    Science.gov (United States)

    Ventas, P; Carreira, J; Polo, F

    1991-08-01

    The allergen composition of one of the most important storage mites, Lepidoglyphus destructor, has been studied by immunodetection after SDS-PAGE with individual patient sera. An allergenic polypeptide of 14 kDa was identified with 95% of the sera. This major allergen was isolated in the supernatant of 60% ammonium sulfate salt precipitation of the whole extract, which was subsequently used to immunize BALB/c mice so as to produce monoclonal antibodies. Four mAbs recognizing molecules with IgE-binding ability were obtained. The specificity of the mAbs was assayed against different allergenic extracts, and the molecules recognized by them were characterized by immunoblotting. Two mAbs (Le5B5 and Le9E4) were directed to the 14-kDa allergen; the other two to several proteins of lesser allergenic significance.

  4. Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection

    Science.gov (United States)

    Dowall, Stuart David; Callan, Jo; Zeltina, Antra; Al-Abdulla, Ibrahim; Strecker, Thomas; Fehling, Sarah K.; Krähling, Verena; Bosworth, Andrew; Rayner, Emma; Taylor, Irene; Charlton, Sue; Landon, John; Cameron, Ian; Hewson, Roger; Nasidi, Abdulsalami; Bowden, Thomas A.; Carroll, Miles W.

    2016-01-01

    The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease. PMID:26715676

  5. Efficient production of a bioactive Bevacizumab monoclonal antibody using the 2A self-cleavage peptide in transgenic rice callus

    Directory of Open Access Journals (Sweden)

    Lei Chen

    2016-08-01

    Full Text Available Bevacizumab, a humanized monoclonal antibody (mAb targeting to the vascular endothelial growth factor (VEGF, has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC and heavy chain (BHC genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus (FMDV, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme linked immunosorbent assay (ELISA analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7-242.8 mg kg-1FW in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target hVEGF antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment.

  6. Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus

    Science.gov (United States)

    Chen, Lei; Yang, Xiaoyu; Luo, Da; Yu, Weichang

    2016-01-01

    Bevacizumab, a humanized monoclonal antibody (mAb) targeting to the vascular endothelial growth factor (VEGF), has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC) and Bevacizumab heavy chain (BHC) genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme-linked immunosorbent assay (ELISA) analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7–242.8 mg/Kg) in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target human VEGF (hVEGF) antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin) were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment. PMID:27555853

  7. A role for uric acid and the Nalp3 inflammasome in antiphospholipid antibody-induced IL-1β production by human first trimester trophoblast.

    Directory of Open Access Journals (Sweden)

    Melissa J Mulla

    Full Text Available Women with antiphospholipid syndrome (APS are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR. Antiphospholipid antibodies (aPL directly target the placenta by binding beta2-glycoprotein I (β2GPI expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β2GPI antibodies (Abs induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β2GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β2GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.

  8. Inhibition of IL-10 production by maternal antibodies against Group B Streptococcus GAPDH confers immunity to offspring by favoring neutrophil recruitment.

    Directory of Open Access Journals (Sweden)

    Pedro Madureira

    2011-11-01

    Full Text Available Group B Streptococcus (GBS is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10 by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab'(2 fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/- mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a

  9. A natural bacterial-derived product, the metalloprotease arazyme, inhibits metastatic murine melanoma by inducing MMP-8 cross-reactive antibodies.

    Directory of Open Access Journals (Sweden)

    Felipe V Pereira

    Full Text Available The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

  10. Effect of sow and piglet porcine circovirus type 2 (PCV2) vaccination on piglet mortality, viraemia, antibody titre and production parameters.

    Science.gov (United States)

    Fraile, Lorenzo; Sibila, Marina; Nofrarías, Miquel; López-Jimenez, Rosa; Huerta, Eva; Llorens, Anna; López-Soria, Sergio; Pérez, Diego; Segalés, Joaquim

    2012-12-28

    The present study describes the effects of sow and/or piglet porcine circovirus type 2 (PCV2) vaccination on viraemia, antibody response and production parameters (average daily weight gain [ADWG] and mortality) of piglets from a PCV2 subclinically infected farm. Four hundred seventy-six piglets born from vaccinated (V) or non-vaccinated (NV) sows were further subdivided in a total of four groups: NV sows-NV pigs (NV-NV, n=134), NV sows-V pigs (NV-V, n=135);V sows-NV pigs (V-NV, n=104) and V sows-V pigs (V-V, n=103). A single vaccination of sows before mating was able to confer significantly higher antibody titres to their piglets at 4 weeks of age and a different PCV2 dynamics infection compared to piglets coming from NV sows. Piglet vaccination (independently of sow treatment) caused an earlier seroconversion and lower percentages of PCV2 infected pigs compared to the NV ones throughout their life. The double PCV2 vaccination strategy was able to reduce PCV2 infection but apparently caused some interference in piglet humoral response development. PCV2 vaccination was able to overcome this interference since the ADWG was improved in both groups of vaccinated piglets, independently of the sow treatment, being the highest ones obtained in the double vaccination group.

  11. An empirical approach towards the efficient and optimal production of influenza-neutralizing ovine polyclonal antibodies demonstrates that the novel adjuvant CoVaccine HT™ is functionally superior to Freund's adjuvant.

    Science.gov (United States)

    Stevens, Natalie E; Fraser, Cara K; Alsharifi, Mohammed; Brown, Michael P; Diener, Kerrilyn R; Hayball, John D

    2013-01-01

    Passive immunotherapies utilising polyclonal antibodies could have a valuable role in preventing and treating infectious diseases such as influenza, particularly in pandemic situations but also in immunocompromised populations such as the elderly, the chronically immunosuppressed, pregnant women, infants and those with chronic diseases. The aim of this study was to optimise current methods used to generate ovine polyclonal antibodies. Polyclonal antibodies to baculovirus-expressed recombinant influenza haemagglutinin from A/Puerto Rico/8/1934 H1N1 (PR8) were elicited in sheep using various immunisation regimens designed to investigate the priming immunisation route, adjuvant formulation, sheep age, and antigen dose, and to empirically ascertain which combination maximised antibody output. The novel adjuvant CoVaccine HT™ was compared to Freund's adjuvant which is currently the adjuvant of choice for commercial production of ovine polyclonal Fab therapies. CoVaccine HT™ induced significantly higher titres of functional ovine anti-haemagglutinin IgG than Freund's adjuvant but with fewer side effects, including reduced site reactions. Polyclonal hyperimmune sheep sera effectively neutralised influenza virus in vitro and, when given before or after influenza virus challenge, prevented the death of infected mice. Neither the age of the sheep nor the route of antigen administration appeared to influence antibody titre. Moreover, reducing the administrated dose of haemagglutinin antigen minimally affected antibody titre. Together, these results suggest a cost effective way of producing high and sustained yields of functional ovine polyclonal antibodies specifically for the prevention and treatment of globally significant diseases.

  12. Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Choudhary, Nandlal; Roy, Avijit; Govindarajulu, A; Nakhla, M K; Levy, L; Brlansky, R H

    2014-09-01

    Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing. Published by Elsevier B.V.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylationrelated product quality. In this work, different fed-batch processes with two chemically defined proprietary media...... and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality....... energy and recombinant product, respectively. Accumulation of by-products such as NH4+ and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled...

  14. Educational paper: Primary antibody deficiencies

    NARCIS (Netherlands)

    G.J.A. Driessen (Gertjan); M. van der Burg (Mirjam)

    2011-01-01

    textabstractPrimary antibody deficiencies (PADs) are the most common primary immunodeficiencies and are characterized by a defect in the production of normal amounts of antigen-specific antibodies. PADs represent a heterogeneous spectrum of conditions, ranging from often asymptomatic selective IgA a

  15. Application of a quality by design approach to the cell culture process of monoclonal antibody production, resulting in the establishment of a design space.

    Science.gov (United States)

    Nagashima, Hiroaki; Watari, Akiko; Shinoda, Yasuharu; Okamoto, Hiroshi; Takuma, Shinya

    2013-12-01

    This case study describes the application of Quality by Design elements to the process of culturing Chinese hamster ovary cells in the production of a monoclonal antibody. All steps in the cell culture process and all process parameters in each step were identified by using a cause-and-effect diagram. Prospective risk assessment using failure mode and effects analysis identified the following four potential critical process parameters in the production culture step: initial viable cell density, culture duration, pH, and temperature. These parameters and lot-to-lot variability in raw material were then evaluated by process characterization utilizing a design of experiments approach consisting of a face-centered central composite design integrated with a full factorial design. Process characterization was conducted using a scaled down model that had been qualified by comparison with large-scale production data. Multivariate regression analysis was used to establish statistical prediction models for performance indicators and quality attributes; with these, we constructed contour plots and conducted Monte Carlo simulation to clarify the design space. The statistical analyses, especially for raw materials, identified set point values, which were most robust with respect to the lot-to-lot variability of raw materials while keeping the product quality within the acceptance criteria.

  16. Product and process understanding to relate the effect of freezing method on glycation and aggregation of lyophilized monoclonal antibody formulations.

    Science.gov (United States)

    Awotwe-Otoo, David; Agarabi, Cyrus; Read, Erik K; Lute, Scott; Brorson, Kurt A; Khan, Mansoor A

    2015-07-25

    The objective of the study was to analyze the effect of controlled and uncontrolled freezing step of a lyophilization process on the extent of non-enzymatic glycation and aggregation of an IgG1 formulation at two concentrations (1mg/ml and 20mg/ml). The degree of glycation (%) was determined through boronate affinity chromatography and its effect on the formation of soluble aggregates and higher molecular weight species was studied using dynamic light scattering (DLS) and size exclusion chromatography with multi-angle light scattering (SEC-MALS). The effect of non-enzymatic glycation on the secondary structure of the formulations was also studied using circular dichroism (CD) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. Results indicated that controlled nucleation yielded higher residual moisture contents and significantly lower specific surface areas for the two monoclonal antibody concentrations compared with uncontrolled nucleation cycle (p<0.05). For the two concentrations, uncontrolled nucleation resulted in significantly higher levels of glycation compared with controlled nucleation samples (p<0.05). Further, it was observed that higher storage temperatures (25°C/60% RH) versus 5°C resulted in higher glycation. Even though SEC-MALS analyses of the low concentrated formulations did not reveal the formation of higher molecular weight species, DLS analyses at two storage conditions revealed increases in the hydrodynamic radii and polydispersity index of the reconstituted formulations, suggesting the onset of formation of smaller species in the formulations. CD spectroscopy did not reveal any differences in the secondary structure of the mAb for the two concentrations after lyophilization. In conclusion, the freezing step method impacted the extent of glycation in lyophilized samples and the hydrolyzed component of sucrose was critical for increasing glycation. Even though some level of glycation was observed in lyophilized samples, the

  17. Liposome-based polymer complex as a novel adjuvant: enhancement of specific antibody production and isotype switch

    Directory of Open Access Journals (Sweden)

    Chen CH

    2012-02-01

    Full Text Available Chia-Hung Chen1,*, Yu-Ling Lin1,*, Yen-Ku Liu1, Pei-Juin He2, Ching-Min Lin1, Yi-Han Chiu2, Chang-Jer Wu3, Tian-Lu Cheng4, Shih-Jen Liu5,6,**, Kuang-Wen Liao1,2,**1Institute of Molecular Medicine and Bioengineering, 2Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, 3Department of Food Science, National Taiwan Ocean University, Keelung, 4Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, 5National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, 6Graduate Institute of Immunology, China Medical University, Taichung, Taiwan, , *Chia-Hung Chen and Yu-Ling Lin contributed equally to this work**Kuang-Wen Liao and Shih-Jen Liu contributed equally to this workAbstract: The aim of vaccination is to induce appropriate immunity against pathogens. Antibody-mediated immunity is critical for protection against many virus diseases, although it is becoming more evident that coordinated, multifunctional immune responses lead to the most effective defense. Specific antibody (Ab isotypes are more efficient at protecting against pathogen invasion in different locations in the body. For example, compared to other Ab isotypes, immunoglobulin (Ig A provides more protection at mucosal areas. In this study, we developed a cationic lipopolymer (liposome-polyethylene glycol-polyethyleneimine complex [LPPC] adjuvant that strongly adsorbs antigens or immunomodulators onto its surface to enhance or switch immune responses. The results demonstrate that LPPC enhances uptake ability, surface marker expression, proinflammatory cytokine release, and antigen presentation in mouse phagocytes. In contrast to Freund's adjuvant, LPPC preferentially activates Th1-immunity against antigens in vivo. With lipopolysaccharides or CpG oligodeoxynucleotides, LPPC dramatically enhances the IgA or IgG2A proportion of total Ig, even in hosts that have developed

  18. Human anti-mouse antibodies.

    Science.gov (United States)

    Klee, G G

    2000-06-01

    Human anti-mouse antibodies (HAMA) are human immunoglobulins with specificity for mouse immunoglobulins. This topic currently is of interest because of the increased use of monoclonal mouse antibodies as diagnostic reagents both for in vitro laboratory measurements and for in vivo imaging studies. Monoclonal mouse antibodies also are being used therapeutically. This short article reviews the production of HAMA in patients receiving monoclonal antibodies and illustrates the potential ways that HAMA can interfere with immunoassay measurements. Methods for measuring and neutralizing HAMA also are discussed.

  19. Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies.

    Science.gov (United States)

    Chan, Kah Fai; Shahreel, Wahyu; Wan, Corrine; Teo, Gavin; Hayati, Noor; Tay, Shi Jie; Tong, Wen Han; Yang, Yuansheng; Rudd, Pauline M; Zhang, Peiqing; Song, Zhiwei

    2016-03-01

    Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor FcγRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity activity. While previous works have shown that inactivation of fucosyltransferase 8 results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in Chinese hamster ovary (CHO) cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies.

  20. Antibody Engineering for Pursuing a Healthier Future

    Science.gov (United States)

    Saeed, Abdullah F. U. H.; Wang, Rongzhi; Ling, Sumei; Wang, Shihua

    2017-01-01

    Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.

  1. Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

    Energy Technology Data Exchange (ETDEWEB)

    Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.; Reynard, Olivier; Volchkova, Valentina A.; Reid, St. Patrick; Ramanan, Parameshwaran; Cárdenas, Washington B.; Amarasinghe, Gaya K.; Volchkov, Viktor E.; Basler, Christopher F. (CNRS-INSERM); (Mount Sinai Hospital); (LB-Ecuador); (Iowa State)

    2010-10-11

    Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.

  2. Up-regulation of T lymphocyte and antibody production by inflammatory cytokines released by macrophage exposure to multi-walled carbon nanotubes.

    Science.gov (United States)

    Grecco, Ana Carolina P; Paula, Rosemeire F O; Mizutani, Erica; Sartorelli, Juliana C; Milani, Ana M; Longhini, Ana Leda F; Oliveira, Elaine C; Pradella, Fernando; Silva, Vania D R; Moraes, Adriel S; Peterlevitz, Alfredo C; Farias, Alessandro S; Ceragioli, Helder J; Santos, Leonilda M B; Baranauskas, Vitor

    2011-07-01

    Our data demonstrate that multi-walled carbon nanotubes (MWCNTs) are internalized by macrophages, subsequently activating them to produce interleukin (IL)-12 (IL-12). This cytokine induced the proliferative response of T lymphocytes to a nonspecific mitogen and to ovalbumin (OVA). This increase in the proliferative response was accompanied by an increase in the expression of pro-inflammatory cytokines, such as interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα) and IL-6, in mice inoculated with MWCNTs, whether or not they had been immunized with OVA. A decrease in the expression of transforming growth factor-beta (TGFβ) was observed in the mice treated with MWCNTs, whereas the suppression of the expression of both TGFβ and IL-10 was observed in mice that had been both treated and immunized. The activation of the T lymphocyte response by the pro-inflammatory cytokines leads to an increase in antibody production to OVA, suggesting the important immunostimulatory effect of carbon nanotubes.

  3. Production of anti-horse antibodies induced by IgG, F(ab')2 and Fab applied repeatedly to rabbits. Effect on antivenom pharmacokinetics.

    Science.gov (United States)

    Vázquez, Hilda; Olvera, Felipe; Alagón, Alejandro; Sevcik, Carlos

    2013-12-15

    We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.

  4. Up-regulation of T lymphocyte and antibody production by inflammatory cytokines released by macrophage exposure to multi-walled carbon nanotubes

    Science.gov (United States)

    Grecco, Ana Carolina P.; Paula, Rosemeire F. O.; Mizutani, Erica; Sartorelli, Juliana C.; Milani, Ana M.; Longhini, Ana Leda F.; Oliveira, Elaine C.; Pradella, Fernando; Silva, Vania D. R.; Moraes, Adriel S.; Peterlevitz, Alfredo C.; Farias, Alessandro S.; Ceragioli, Helder J.; Santos, Leonilda M. B.; Baranauskas, Vitor

    2011-07-01

    Our data demonstrate that multi-walled carbon nanotubes (MWCNTs) are internalized by macrophages, subsequently activating them to produce interleukin (IL)-12 (IL-12). This cytokine induced the proliferative response of T lymphocytes to a nonspecific mitogen and to ovalbumin (OVA). This increase in the proliferative response was accompanied by an increase in the expression of pro-inflammatory cytokines, such as interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα) and IL-6, in mice inoculated with MWCNTs, whether or not they had been immunized with OVA. A decrease in the expression of transforming growth factor-beta (TGFβ) was observed in the mice treated with MWCNTs, whereas the suppression of the expression of both TGFβ and IL-10 was observed in mice that had been both treated and immunized. The activation of the T lymphocyte response by the pro-inflammatory cytokines leads to an increase in antibody production to OVA, suggesting the important immunostimulatory effect of carbon nanotubes.

  5. The role of Campylobacter jejuni cytolethal distending toxin in gastroenteritis: toxin detection, antibody production, and clinical outcome

    Energy Technology Data Exchange (ETDEWEB)

    Boisen, Nadia [University of Virginia School of Medicine

    2011-01-01

    The role of Campylobacter jejuni cytolethal distending toxin (CDT) on clinical outcome after gastroenteritis was investigated. Clinical data, blood serum samples, and Campylobacter spp. isolated, from each of 30 patients were collected over a period of 6 months. The CDT encoding genes, cdtABC, characterized by PCR, revealed that all but one of the C. jejuni strains had the wild-type sequence. Sequencing of cdtABC from this strain showed two major deletions. From all of the strains, CDT titers were determined, and toxin neutralizing antibodies were documented using an in vitro assay. Three of the thirty clinical isolates, including the one with the mutant cdtABC coding genes, did not have a detectable CDT activity. Analyzing the relationship between CDT titer, serum neutralization of CDT, and the clinical outcome showed that campylobacteriosis caused by CDT-negative strains was clinically indistinguishable from that of patients infected with an isolate that produced high levels of CDT. These results suggest that CDT does not solely determine severity of infection and clinical outcome.

  6. Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope.

    Science.gov (United States)

    Zhang, Limeng; Zhang, Hua; Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Zhu, Zhanbo; Cui, Yudong

    2015-02-01

    Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine.

  7. Intranasal administration of HIV-DNA vaccine formulated with a polymer, carboxymethylcellulose, augments mucosal antibody production and cell-mediated immune response.

    Science.gov (United States)

    Hamajima, K; Sasaki, S; Fukushima, J; Kaneko, T; Xin, K Q; Kudoh, I; Okuda, K

    1998-08-01

    We previously reported that intramuscular (i.m.) immunization of DNA vaccine encoding human immunodeficiency virus type 1 (HIV-1)IIIB env and rev genes alone or in combination with appropriate adjuvant induces substantial and enhanced immune response against HIV-1. In the present study, we examined whether a polymer, low-viscosity carboxymethylcellulose sodium salt (CMCS-L), has an adjuvant effect on immune response induced by DNA vaccination. BALB/c mice were immunized with HIV-DNA vaccine formulated with CMCS-L via the intranasal (i.n.) and i.m. routes. The combination with the polymer elicited higher levels of antigen-specific serum IgG and fecal IgA antibodies than DNA vaccine alone. For cell-mediated immunity, HIV-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were measured by the footpad-swelling test and the 51Cr-release assay, respectively. Both were enhanced by the combination with CMCS-L via i.n. and i.m. inoculation. Cytokine analysis in culture media of bulk splenocytes harvested from immunized animals showed higher levels of IL-4 production in i.n. -immunized mice compared with i.m.-immunized mice. Nevertheless, the increased IFN-gamma production resulting from the combination with CMCS-L was observed only in i.n.-immunized mice. These data indicate that i.n. immunization of HIV-DNA vaccine formulated with CMCS-L enhances HIV-specific mucosal antibody (Ab) and systemic Ab and cell-mediated immune response.

  8. Tyrosine phosphorylation of Munc18c on residue 521 abrogates binding to Syntaxin 4

    Directory of Open Access Journals (Sweden)

    Bryant Nia J

    2011-05-01

    Full Text Available Abstract Background Insulin stimulates exocytosis of GLUT4 from an intracellular store to the cell surface of fat and muscle cells. Fusion of GLUT4-containing vesicles with the plasma membrane requires the SNARE proteins Syntaxin 4, VAMP2 and the regulatory Sec1/Munc18 protein, Munc18c. Syntaxin 4 and Munc18c form a complex that is disrupted upon insulin treatment of adipocytes. Munc18c is tyrosine phosphorylated in response to insulin in these cells. Here, we directly test the hypothesis that tyrosine phosphorylation of Munc18c is responsible for the observed insulin-dependent abrogation of binding between Munc18c and Syntaxin 4. Results We show that Munc18c is directly phosphorylated by recombinant insulin receptor tyrosine kinase in vitro. Using pull-down assays, we show that phosphorylation abrogates binding of Munc18c to both Syntaxin 4 and the v-SNARE VAMP2, as does the introduction of a phosphomimetic mutation into Munc18c (Y521E. Conclusion Our data indicate that insulin-stimulated tyrosine phosphorylation of Munc18c impairs the ability of Munc18c to bind its cognate SNARE proteins, and may therefore represent a regulatory step in GLUT4 traffic.

  9. The culture of referendum in Albania: Technical and theoritecal reflections on the abrogative referendum

    Directory of Open Access Journals (Sweden)

    Valbona Pajo Bala

    2014-01-01

    Full Text Available The aim of this paper is to analyse the Albanian constitutional and legal framework on referenda, in general, focusing special attention to the abrogative referenda of a law or part thereof. Given the absence of any concrete case of an abrogative referenda held in Albania, which does not creates very much room for discussion in that regard, the paper, through a comparative approach on the referenda culture in other european states, aims at offering to the reader a more complete view on the mechanisms and guarantees enjoyed by voters and the effective way of their use, in order to give life to the direct democracy, but without replacing the representative one. In addition, part of the analyses will be the powers of the Constitutional Court for the ex ante constitutional review of the issue subject to a referendum, the review of constitutionality of the referndum and of its results. In this context, the paper will focus on the constitutional case-law as a tool for increasing the referenda culture and shaping the constitional order, as well as a source of standards and values. Another objective of the paper is to open a discussion on the need for the reception of referenda-related standards elaborated in those European countries, where the culture of helding a referenda and the case-law on the regard is enriched and may serve as a qualitative basis for further reference.

  10. Plasma membrane Toll-like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon-β.

    Science.gov (United States)

    Boye, Louise; Welsby, Iain; Lund, Lisbeth Drozd; Goriely, Stanislas; Frøkiaer, Hanne

    2016-11-01

    Lactobacillus acidophilus induces a potent interferon-β (IFN-β) response in dendritic cells (DCs) by a Toll-like receptor 2 (TLR2) -dependent mechanism, in turn leading to strong interleukin-12 (IL-12) production. In the present study, we investigated the involvement of different types of endocytosis in the L. acidophilus-induced IFN-β and IL-12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN-β and IL-12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place in the immature cells as well as by spleen tyrosine kinase (Syk) -dependent phagocytosis but without involvement of plasma membrane TLR2. Stimulation with TLR2 or TLR4 ligands increased macropinocytosis in a Syk-independent manner. As a consequence, incubation of DCs with TLR ligands before incubation with L. acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN-β and IL-12 was strongly inhibited. As L. acidophilus-induced IFN-β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading to increased macropinocytosis abrogates IFN-β induction we conclude that plasma membrane TLR stimulation leading to increased macropinocytosis decreases endosomal induction of IFN-β and speculate that this is due to competition between compartments for molecules involved in the signal pathways. In summary, endosomal signalling by L. acidophilus that leads to IFN-β and IL-12 production is inhibited by TLR stimulation from the plasma membrane.

  11. Sclerostin antibody inhibits skeletal deterioration due to reduced mechanical loading.

    Science.gov (United States)

    Spatz, Jordan M; Ellman, Rachel; Cloutier, Alison M; Louis, Leeann; van Vliet, Miranda; Suva, Larry J; Dwyer, Denise; Stolina, Marina; Ke, Hua Zhu; Bouxsein, Mary L

    2013-04-01

    Sclerostin, a product of the SOST gene produced mainly by osteocytes, is a potent negative regulator of bone formation that appears to be responsive to mechanical loading, with SOST expression increasing following mechanical unloading. We tested the ability of a murine sclerostin antibody (SclAbII) to prevent bone loss in adult mice subjected to hindlimb unloading (HLU) via tail suspension for 21 days. Mice (n = 11-17/group) were assigned to control (CON, normal weight bearing) or HLU and injected with either SclAbII (subcutaneously, 25 mg/kg) or vehicle (VEH) twice weekly. SclAbII completely inhibited the bone deterioration due to disuse, and induced bone formation such that bone properties in HLU-SclAbII were at or above values of CON-VEH mice. For example, hindlimb bone mineral density (BMD) decreased -9.2% ± 1.0% in HLU-VEH, whereas it increased 4.2% ± 0.7%, 13.1% ± 1.0%, and 30.6% ± 3.0% in CON-VEH, HLU-SclAbII, and CON-SclAbII, respectively (p bone volume, assessed by micro-computed tomography (µCT) imaging of the distal femur, was lower in HLU-VEH versus CON-VEH (p bone outcomes appeared to be enhanced by normal mechanical loading. Altogether, these results confirm the ability of SclAbII to abrogate disuse-induced bone loss and demonstrate that sclerostin antibody treatment increases bone mass by increasing bone formation in both normally loaded and underloaded environments.

  12. Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture.

    Science.gov (United States)

    Yang, Jeng-Dar; Lu, Canghai; Stasny, Brad; Henley, Joseph; Guinto, Woodrow; Gonzalez, Carlos; Gleason, Joseph; Fung, Monica; Collopy, Brett; Benjamino, Michael; Gangi, Jennifer; Hanson, Melissa; Ille, Elisabeth

    2007-09-01

    This case study focuses on the scale-up of a Sp2/0 mouse myeloma cell line based fed-batch bioreactor process, from the initial 3-L bench scale to the 2,500-L scale. A stepwise scale-up strategy that involved several intermediate steps in increasing the bioreactor volume was adopted to minimize the risks associated with scale-up processes. Careful selection of several available mixing models from literature, and appropriately applying the calculated results to our settings, resulted in successful scale-up of agitation speed for the large bioreactors. Consideration was also given to scale-up of the nutrient feeding, inoculation, and the set-points of operational parameters such as temperature, pH, dissolved oxygen, dissolved carbon dioxide, and aeration in an integrated manner. It has been demonstrated through the qualitative and the quantitative side-by-side comparison of bioreactor performance as well as through a panel of biochemical characterization tests that the comparability of the process and the product was well controlled and maintained during the process scale-up. The 2,500-L process is currently in use for the routine clinical production of Epratuzumab in support of two global Phase III clinical trials in patients with lupus. Today, the 2,500 L, fed-batch production process for Epratuzumab has met all scheduled batch releases, and the quality of the antibody is consistent and reproducible, meeting all specifications, thus confirming the robustness of the process.

  13. Impact of thiamine deficiency on T-cell dependent and T-cell independent antibody production in lake trout.

    Science.gov (United States)

    Ottinger, Christopher A; Honeyfield, Dale C; Densmore, Christine L; Iwanowicz, Luke R

    2012-12-01

    Lake trout Salvelinus namaycush on thiamine-replete and thiamine-depleted diets were evaluated for the effects of thiamine status on in vivo responses to the T-dependent antigen trinitophenol (TNP)-keyhole limpet hemocyanin (TNP-KLH), the T-independent antigen trinitrophenol-lipolysaccaharide (TNP-LPS), or Dulbecco's phosphate-buffered saline (DPBS; negative control fish). Plasma antibody concentrations were evaluated for possible differences in total anti-TNP activity as well as differences in response kinetics. Associations between anti-TNP activity and muscle and liver thiamine concentrations as well as ratios of muscle-to-liver thiamine to anti-TNP activity were also examined. Thiamine-depleted lake trout that were injected with TNP-LPS exhibited significantly more anti-TNP activity than thiamine-replete fish. The depleted fish injected with TNP-LPS also exhibited significantly different response kinetics relative to thiamine-replete lake trout. No differences in activity or kinetics were observed between the thiamine-replete and -depleted fish injected with TNP-KLH or in the DPBS negative controls. Anti-TNP activity in thiamine-depleted lake trout injected with TNP-KLH was positively associated with muscle thiamine pyrophosphate (thiamine diphosphate; TPP) concentration. A negative association was observed between the ratio of muscle-to-liver TPP and T-independent responses. No significant associations between anti-TNP activity and tissue thiamine concentration were observed in the thiamine-replete fish. We demonstrated that thiamine deficiency leads to alterations in both T-dependent and T-independent immune responses in lake trout.

  14. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    Science.gov (United States)

    He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

    2016-06-15

    Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection.

  15. Interference of daratumumab in monitoring multiple myeloma patients using serum immunofixation electrophoresis can be abrogated using the daratumumab IFE reflex assay (DIRA).

    Science.gov (United States)

    van de Donk, Niels W C J; Otten, Henny G; El Haddad, Omar; Axel, Amy; Sasser, A Kate; Croockewit, Sandra; Jacobs, Joannes F M

    2016-06-01

    Daratumumab is a fully human anti-CD38 IgG1-κ monoclonal antibody (mAb) currently being evaluated in several Phase 2 and 3 clinical studies for the treatment of multiple myeloma (MM). In this clinical case study we demonstrate that daratumumab can be detected as an individual monoclonal band in serum immunofixation electrophoresis (IFE). M-protein follow-up by IFE is part of the International Myeloma Working Group (IMWG) criteria to assess treatment response. Therefore, it is crucial that the daratumumab band is not confused with the endogenous M-protein of the patient during IFE interpretation. Moreover, a significant number of IgG-κ M-proteins co-migrate with daratumumab. Co-migration introduces a bias in the M-protein quantification since pharmacokinetic studies show that daratumumab peak plasma concentrations reach up to 1 g/L. More importantly, co-migration can mask clearance of the M-protein by IFE which is necessary for classification of complete response by IMWG criteria (negative serum IFE). For optimal M-protein monitoring the laboratory specialist needs to be informed when patients receive daratumumab, and it is essential that the laboratory specialist is aware that a slow migrating band in the γ-region in those patients may be derived from the daratumumab. A daratumumab specific IFE reflex assay (DIRA) has been developed and can be utilized to abrogate interference. The here described mAb interference is not limited to daratumumab, and as therapeutic antibodies gain approval and enter into common clinical practice, laboratory specialists will need additional processes to characterize IFE interference and distinguish endogenous M-protein from therapeutic antibodies.

  16. Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

    DEFF Research Database (Denmark)

    Kim, Che Lin; Kwang Ha, Tae; Min Lee, Gyun

    2016-01-01

    Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (qp)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), ...

  17. Faecal Salmonella shedding in fattening pigs in relation to the presence of Salmonella antibodies in three pig production systems

    DEFF Research Database (Denmark)

    Bonde, Marianne Kjær; Sørensen, Jan Tind

    2012-01-01

    Human salmonellosis originating from pork is an important zoonotic disease, and the production of outdoor pigs may increase the risk of contaminating the food chain with Salmonella from environmental sources. The prevalence of faecal Salmonella shedding has therefore been compared in organic...

  18. Nanoparticles for the delivery of therapeutic antibodies

    DEFF Research Database (Denmark)

    Sousa, Flávia; Castro, Pedro; Fonte, Pedro;

    2016-01-01

    INTRODUCTION: Over the past two decades, therapeutic antibodies have demonstrated promising results in the treatment of a wide array of diseases. However, the application of antibody-based therapy implies multiple administrations and a high cost of antibody production, resulting in costly therapy...

  19. Effects of T mitogens on in vivo antibody production to a T-dependent antigen in lines of mice genetically selected for high or low in vitro responsiveness to PHA.

    Science.gov (United States)

    Stiffel, C; Decreusefond, C; Liacopoulos-Briot, M

    1984-01-01

    The influence of genes which regulate the in vitro T-cell proliferative response to T mitogen upon in vivo antibody production to a T-dependent antigen was studied in two lines of mice genetically selected for a high or a low in vitro lymphocyte response to phytohaemagglutinin (PHA). Kinetics of agglutinin production to increasing doses of sheep erythrocytes was similar in the two lines, except for the titres of mercaptoethanol-resistant antibodies, which were slightly lower in the low-responder line. Treatment with mitogen prior to immunization modified the antibody response in the two lines differently. This finding would indicate that the genes which regulate in vitro stimulation of T cells by PHA also control in vivo activation of T-cell subsets involved in immunoresponsiveness.

  20. Abrogation of E-cadherin-mediated cellular aggregation allows proliferation of pluripotent mouse embryonic stem cells in shake flask bioreactors.

    Directory of Open Access Journals (Sweden)

    Lisa Mohamet

    Full Text Available BACKGROUND: A fundamental requirement for the exploitation of embryonic stem (ES cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/- or the neutralising antibody DECMA-1 (EcadAb, allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are

  1. Prenatal exposure to non-ionizing radiation: effects of WiFi signals on pregnancy outcome, peripheral B-cell compartment and antibody production.

    Science.gov (United States)

    Sambucci, Manolo; Laudisi, Federica; Nasta, Francesca; Pinto, Rosanna; Lodato, Rossella; Altavista, Pierluigi; Lovisolo, Giorgio Alfonso; Marino, Carmela; Pioli, Claudio

    2010-12-01

    During embryogenesis, the development of tissues, organs and systems, including the immune system, is particularly susceptible to the effects of noxious agents. We examined the effects of prenatal (in utero) exposure to WiFi signals on pregnancy outcome and the immune B-cell compartment, including antibody production. Sixteen mated (plug-positive) female mice were assigned to each of the following groups: cage control, sham-exposed and microwave-exposed (WiFi signals at 2.45 GHz, whole body, SAR 4 W/kg, 2 h/day, 14 consecutive days starting 5 days after mating). No effects due to exposure to WiFi signals during pregnancy on mating success, number of newborns/mother and body weight at birth were found. Newborn mice were left to grow until 5 or 26 weeks of age, when immunological analyses were performed. No differences due to exposure were found in spleen cell number, B-cell frequency or antibody serum levels. When challenged in vitro with LPS, B cells from all groups produced comparable amounts of IgM and IgG, and proliferated at a similar level. All these findings were consistently observed in the female and male offspring at both juvenile (5 weeks) and adult (26 weeks) ages. Stress-associated effects as well as age- and/or sex-related differences were observed for several parameters. In conclusion, our results do not show any effect on pregnancy outcome or any early or late effects on B-cell differentiation and function due to prenatal exposure to WiFi signals.

  2. Immunosuppression abrogates resistance of young rabbits to Rabbit Haemorrhagic Disease (RHD).

    Science.gov (United States)

    Marques, Raquel M; Teixeira, Luzia; Aguas, Artur P; Ribeiro, Joana C; Costa-e-Silva, António; Ferreira, Paula G

    2014-02-04

    Rabbit Haemorrhagic Disease (RHD) is caused by a calicivirus (RHDV) that kills 90% of infected adult European rabbits within 3 days. Remarkably, young rabbits are resistant to RHD. We induced immunosuppression in young rabbits by treatment with methylprednisolone acetate (MPA) and challenged the animals with RHDV by intramuscular injection. All of these young rabbits died within 3 days of infection due to fulminant hepatitis, presenting a large number of RHDV-positive dead or apoptotic hepatocytes, and a significant seric increase in cytokines, features that are similar to those of naïve adult rabbits infected by RHDV. We conclude that MPA-induced immunosuppression abrogates the resistance of young rabbits to RHD, indicating that there are differences in the innate immune system between young and adult rabbits that contribute to their distinct resistance/susceptibility to RHDV infection.

  3. HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding

    Institute of Scientific and Technical Information of China (English)

    Guo-Zhong Gong; Yong-Fang Jiang; Yan He; Li-Ying Lai; Ying-Hua Zhu; Xian-Shi Su

    2004-01-01

    AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function.METHODS: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter.Western blot experiment was used for detection of HCV NS5A and p53 proteins expression.RESULTS: Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV NS5A expression vector, there was no difference in the p53 protein expression.CONCLUSION: HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.

  4. Frequencies of the expression of main protein antigens from Helicobacter pylori isolates and production of specific serum antibodies in infected patients

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Ya-Fei Mao; Zhe-Xin Shao

    2005-01-01

    AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori(H pylori)isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori.METHODS: H pylori strains in biopsy specimens from 157patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected.The target recombinant proteins rUreB, rVacA, rCagA1,rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography.Rabbit antisera against rUreB, rVacA, rCagA1, rHpaA,rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody,expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed.RESULTS: In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagA1, HpaA, NapA, FlaA and Flab were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2%respectively. In the 125 serum samples from the H pyloriinfected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FlaA and Flab were

  5. Production and characterization of monoclonal antibodies specific for canine CD138 (syndecan-1) for nuclear medicine preclinical trials on spontaneous tumours

    OpenAIRE

    Diab, Maya; Nguyen, Frédérique; Berthaud, Maxime; Maurel, Catherine; Gaschet, J.; Verger, Elise; Ibisch, Catherine; Chérel, Michel; Abadie, Jérôme; Davodeau, François; Rousseau, Caroline

    2016-01-01

    International audience; We isolated 11 antibodies specific for canine CD138 (cCD138) to validate the interest of CD138 antigen targeting in dogs with spontaneous mammary carcinoma. The affinity of the monoclonal antibodies in the nanomolar range is suitable for immunohistochemistry and nuclear medicine applications. Four distinct epitopes were recognized on cCD138 by this panel of antibodies. CD138 expression in canine healthy tissues is comparable to that reported in humans. CD138 is frequen...

  6. Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette

    2004-01-01

    This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

  7. Single-chain antibody-based gene therapy: Inhibition of tumor growth by in situ production of phage-derived antibodies blocking functionally active sites of cell-associated matrices

    DEFF Research Database (Denmark)

    Sanz, Laura; Kristensen, Peter; Blanco, Belén

    2002-01-01

    . This scFv inhibits angiogenesis in vivo in the chick embryo chorioallantoic membrane assay and prevents the establishment and growth of subcutaneous tumors in mice, either when administered as bolus protein therapy or when produced locally by gene-modified tumor cells. Our work represents the first...... demonstration of a direct in vivo therapeutic effect of a single-chain antibody secreted by gene-modified mammalian cells. These results open the way for a new antibody-based gene therapy strategy of cancer....

  8. Antiparietal cell antibody test

    Science.gov (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

  9. Lack of gender-specific antibody recognition of products from domains of a var gene implicated in pregnancy-associated Plasmodium falciparum malaria

    DEFF Research Database (Denmark)

    Jensen, Anja T R; Zornig, Hanne D; Buhmann, Caecilie

    2003-01-01

    Gender-specific and parity-dependent acquired antibody recognition is characteristic of variant surface antigens (VSA) expressed by chondroitin sulfate A (CSA)-adherent Plasmodium falciparum involved in pregnancy-associated malaria (PAM). However, antibody recognition of recombinant products...... of a specific VSA gene (2O2var1) implicated in PAM and transcribed by a CSA-adhering parasite line did not have these characteristics. Furthermore, we could not demonstrate preferential transcription of 2O2var1 in the CSA-adhering line versus the unselected, parental isolate. Our data call for circumspection...... regarding the molecular identity of the parasite ligand mediating adhesion to CSA in PAM....

  10. Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Alexandra M Fajardo

    Full Text Available Tocopherylquinone (TQ, the oxidation product of alpha-tocopherol (AT, is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells, whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.

  11. Antibody directed enzyme prodrug therapy: Discovery of novel genes, isolation of novel gene variants and production of long acting drugs for efficient cancer treatment

    NARCIS (Netherlands)

    Goda, S.K.; AlQahtani, A.; Rashidi, F.A.; Dömling, A.

    2015-01-01

    Background: Cancer accounts for 13% of the mortality rate worldwide. Antibody-Directed Enzyme Prodrug Therapy (ADEPT) is a novel strategy to improve the selectivity of cancer treatment. The ADEPT uses the bacterial enzyme, glucarpidase to produce the antibody-enzyme complex. Also the glucarpidase is

  12. Improved specificity of in vitro anti-HIV antibody production: implications for diagnosis and timing of transmission in infants born to HIV-seropositive mothers.

    Science.gov (United States)

    Wang, X P; Paul, M; Tetali, S; Abrams, E; Bamji, M; Gulick, L; Chirmule, N; Oyaizu, N; Bakshi, S; Pahwa, S

    1994-06-01

    In vitro anti-HIV antibody production (IVAP), initially introduced as a method for diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in infants, has been limited in its application because of poor specificity and sensitivity early in life. The aims of this study were to improve the specificity of the IVAP assay and to evaluate its sensitivity in conjunction with assays of HIV culture, polymerase chain reaction (PCR), and p24 antigen. To prevent false-positive reactions resulting from maternal serum-derived cytophilic anti-HIV IgG, additional preculture and washing steps for peripheral blood mononuclear cells (PBMCs) were introduced that resulted in dramatic improvement in specificity of IVAP. The sensitivity of the revised IVAP at age < 3 months in 20 infected infants was, however, only 25%; of 15 infected infants initially negative in IVAP, 13 became positive at a mean estimated age of 4.4 +/- 1.8 months. When correlated with virological assays, a failure to respond in IVAP at age < 1 month was often associated with negative virological identification, whereas a positive IVAP response at age < 3 months was always associated with positive results in all virological assays. Moreover, conversion from negative IVAP to positive responses occurred subsequent to, and not concurrently with, a positive virological identification of infected infants. The revised IVAP methodology renders this assay potentially useful as an additional tool not only for the diagnosis of HIV infection, but for estimating timing of maternal-infant HIV transmission as well.

  13. Up-regulation of T lymphocyte and antibody production by inflammatory cytokines released by macrophage exposure to multi-walled carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Grecco, Ana Carolina P; Mizutani, Erica; Peterlevitz, Alfredo C; Ceragioli, Helder J; Baranauskas, Vitor [Faculdade de Engenharia Eletrica e Computacao, Universidade de Campinas, Campinas, SP (Brazil); Paula, Rosemeire F O; Sartorelli, Juliana C; Milani, Ana M; Longhini, Ana Leda F; Oliveira, Elaine C; Pradella, Fernando; Silva, Vania D R; Moraes, Adriel S; Farias, Alessandro S; Santos, Leonilda M B, E-mail: leonilda@unicamp.br [Laboratorio de Neuroimunologia, Departamento Genetica, Evolucao e Bioagentes, Instituto de Biologia, Universidade de Campinas, Campinas, SP (Brazil)

    2011-07-01

    Our data demonstrate that multi-walled carbon nanotubes (MWCNTs) are internalized by macrophages, subsequently activating them to produce interleukin (IL)-12 (IL-12). This cytokine induced the proliferative response of T lymphocytes to a nonspecific mitogen and to ovalbumin (OVA). This increase in the proliferative response was accompanied by an increase in the expression of pro-inflammatory cytokines, such as interferon-gamma (IFN{gamma}), tumor necrosis factor-alpha (TNF{alpha}) and IL-6, in mice inoculated with MWCNTs, whether or not they had been immunized with OVA. A decrease in the expression of transforming growth factor-beta (TGF{beta}) was observed in the mice treated with MWCNTs, whereas the suppression of the expression of both TGF{beta} and IL-10 was observed in mice that had been both treated and immunized. The activation of the T lymphocyte response by the pro-inflammatory cytokines leads to an increase in antibody production to OVA, suggesting the important immunostimulatory effect of carbon nanotubes.

  14. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  15. Production of a monoclonal antibody by in vitro immunization that recognizes a native chondroitin sulfate epitope in the embryonic chick limb and heart.

    Science.gov (United States)

    Capehart, A A; Wienecke, M M; Kitten, G T; Solursh, M; Krug, E L

    1997-11-01

    We report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22-23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart.

  16. The bioactivity of teleost IL-6: IL-6 protein in orange-spotted grouper (Epinephelus coioides) induces Th2 cell differentiation pathway and antibody production.

    Science.gov (United States)

    Chen, Hsin-Hung; Lin, Han-Tso; Foung, Yi-Fan; Han-You Lin, John

    2012-10-01

    Interleukin 6 (IL-6) is a protein secreted by T cells and macrophages and plays an important role in immune response. IL-6 regulates the proliferation and differentiation of T cells, and elicits immunoglobulin production in B cells. In this study, the cDNA il-6 (gil-6) sequence of the orange spotted grouper (Epinephelus coioides) was obtained. The deduced IL-6 (gIL-6) protein comprised 223 amino acids, the sequence shared approximately 30% similarity with mammalian IL-6, and between 47% and 69% similarity with other available teleost IL-6. The protein comprises the signal peptide, the IL-6 family signature, and conserved amino acid residues found in IL-6 sequences of other teleost. In order to understand the bioactivity and influence of gIL-6 on humoral immune response, recombinant gIL-6 (rgIL-6) synthesized by prokaryotes was injected into orange spotted groupers, and the immune-related gene expression at various times in various organs was observed. Our results revealed that the Th1 specific transcription factor t-bet was down-regulated and Th2 specific transcription factors gata3, and c-maf were up-regulated in immune organs, following IL-6 stimulation. Additionally, higher levels of igm mRNA and translated protein were detected in rgIL-6 stimulated fish. These results indicate that IL-6 in groupers regulates the differentiation of naїve T helper cells into Th2 cells and elicits the production of antibodies.

  17. MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica.

    Science.gov (United States)

    Valero, M Adela; Ubeira, Florencio M; Khoubbane, Messaoud; Artigas, Patricio; Muiño, Laura; Mezo, Mercedes; Pérez-Crespo, Ignacio; Periago, M Victoria; Mas-Coma, Santiago

    2009-01-22

    During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4-7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3-6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33-66 adults), while it was for F. gigantica infection (burden of 17-69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.

  18. Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    David Bradley

    2011-03-31

    During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

  19. Herpesvirus telomerase RNA (vTR) with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis.

    Science.gov (United States)

    Kaufer, Benedikt B; Arndt, Sina; Trapp, Sascha; Osterrieder, Nikolaus; Jarosinski, Keith W

    2011-10-01

    Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus

  20. Tetrandrine: A Potent Abrogator of G2 Checkpoint Function in Tumor Cells and Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To assess the ability of tetrandrine (Tet) to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice. Methods MCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry, Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo. Results Clonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio (SER) of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells,whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis

  1. Diabetes abrogates sex differences and aggravates cardiometabolic risk in postmenopausal women

    Science.gov (United States)

    2013-01-01

    Background The aim of this study is to evaluate the effect of gender and menopause in cardiometabolic risk in a type 2 diabetes mellitus (T2DM) population, based on classical and non-traditional markers. Methods Seventy four volunteers and 110 T2DM patients were enrolled in the study. Anthropometric data, blood pressure, body mass index (BMI), waist circumference (WC) and the following serum markers were analyzed: glucose, Total-c, TGs, LDL-c, Oxidized-LDL, total HDL-c and large and small HDL-c subpopulations, paraoxonase 1 activity, hsCRP, uric acid, TNF-α, adiponectin and VEGF. Results Non-diabetic women, compared to men, presented lower glycemia, WC, small HDL-c, uric acid, TNF-α and increased large HDL-c. Diabetes abrogates the protective effect of female gender, since diabetic women showed increased BMI, WC, small HDL-c, VEGF, uric acid, TNF-α and hsCRP, as well as reduced adiponectin, when compared with non-diabetic. In diabetic females, but not in males, WC is directly and significantly associated with TNF-α, VEGF, hsCRP and uric acid; TNF-α is directly associated with VEGF and hsCRP, and inversely with adiponectin. Postmenopausal females presented a worsen cardiometabolic profile, viewed by the increased WC, small HDL-c, VEGF, uric acid, TNF-α and hsCRP. In this population, WC is directly and significantly associated with TNF-α, VEGF, hsCRP; TNF-α is directly associated with VEGF; and uric acid is inversely associated with large HDL-c and hsCRP with adiponectin, also inversely. Conclusions Diabetes abrogates the protective effect of gender on non-diabetic women, and postmenopausal diabetic females presented worsen cardiometabolic risk, including a more atherogenic lipid sketch and a pro-inflammatory and pro-angiogenic profile. The classical cardiovascular risk factors (CVRFs) fail to completely explain these differences, which are better clarified using “non-traditional” factors, such as HDL-c subpopulations, rather than total HDL-c content, and

  2. Herpesvirus telomerase RNA (vTR with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis.

    Directory of Open Access Journals (Sweden)

    Benedikt B Kaufer

    2011-10-01

    Full Text Available Telomerase reverse transcriptase (TERT and telomerase RNA (TR represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5 by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1 that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2 that this strategy could be used to generate novel vaccine candidates

  3. Intravenous Bone Marrow Stem Cell Grafts Preferentially Migrate to Spleen and Abrogate Chronic Inflammation in Stroke.

    Science.gov (United States)

    Acosta, Sandra A; Tajiri, Naoki; Hoover, Jaclyn; Kaneko, Yuji; Borlongan, Cesar V

    2015-09-01

    Adult stem cell therapy is an experimental stroke treatment. Here, we assessed homing and anti-inflammatory effects of bone marrow stromal cells (hBMSCs) in chronic stroke. At 60 days post stroke, adult Sprague-Dawley rats received intravenous hBMSCs (4×10(6) labeled or nonlabeled cells) or vehicle (saline). A sham surgery group served as additional control. In vivo imaging was conducted between 1 hour and 11 days post transplantation, followed by histological examination. Labeled hBMSCs migrated to spleen which emitted significantly higher fluorescent signal across all time points, especially during the first hour, and were modestly detected in the head region at the 12 hours and 11 days, compared with nonlabeled hBMSCs and vehicle-infused stroke animals, or sham (Pcells in gray and white matter, and a 43% diminution in tumor necrosis factor-α cell density in the spleen of transplanted stroke animals compared with vehicle-infused stroke animals (Pstroke possibly by abrogating the inflammation-plagued secondary cell death. © 2015 The Authors.

  4. Neuroblastoma-targeted nanocarriers improve drug delivery and penetration, delay tumor growth and abrogate metastatic diffusion.

    Science.gov (United States)

    Cossu, Irene; Bottoni, Gianluca; Loi, Monica; Emionite, Laura; Bartolini, Alice; Di Paolo, Daniela; Brignole, Chiara; Piaggio, Francesca; Perri, Patrizia; Sacchi, Angelina; Curnis, Flavio; Gagliani, Maria Cristina; Bruno, Silvia; Marini, Cecilia; Gori, Alessandro; Longhi, Renato; Murgia, Daniele; Sementa, Angela Rita; Cilli, Michele; Tacchetti, Carlo; Corti, Angelo; Sambuceti, Gianmario; Marchiò, Serena; Ponzoni, Mirco; Pastorino, Fabio

    2015-11-01

    Selective tumor targeting is expected to enhance drug delivery and to decrease toxicity, resulting in an improved therapeutic index. We have recently identified the HSYWLRS peptide sequence as a specific ligand for aggressive neuroblastoma, a childhood tumor mostly refractory to current therapies. Here we validated the specific binding of HSYWLRS to neuroblastoma cell suspensions obtained either from cell lines, animal models, or Schwannian-stroma poor, stage IV neuroblastoma patients. Binding of the biotinylated peptide and of HSYWLRS-functionalized fluorescent quantum dots or liposomal nanoparticles was dose-dependent and inhibited by an excess of free peptide. In animal models obtained by the orthotopic implant of either MYCN-amplified or MYCN single copy human neuroblastoma cell lines, treatment with HSYWLRS-targeted, doxorubicin-loaded Stealth Liposomes increased tumor vascular permeability and perfusion, enhancing tumor penetration of the drug. This formulation proved to exert a potent antitumor efficacy, as evaluated by bioluminescence imaging and micro-PET, leading to (i) delay of tumor growth paralleled by decreased tumor glucose consumption, and (ii) abrogation of metastatic spreading, accompanied by absence of systemic toxicity and significant increase in the animal life span. Our findings are functional to the design of targeted nanocarriers with potentiated therapeutic efficacy towards the clinical translation.

  5. Niacinamide abrogates the organ dysfunction and acute lung injury caused by endotoxin.

    Science.gov (United States)

    Kao, Shang-Jyh; Liu, Demeral David; Su, Chain-Fa; Chen, Hsing I

    2007-09-01

    Poly (ADP-ribose) synthabse (PARS) or polymerase (PARP) is a cytotoxic enzyme causing cellular damage. Niacinamide inhibits PARS or PARP. The present experiment tests the effects of niacinamide (NCA) on organ dysfunction and acute lung injury (ALI) following lipopolysaccharide (LPS). LPS was administered to anesthetized rats and to isolated rat lungs. In anesthetized rats, LPS caused systemic hypotension and increased biochemical factors, nitrate/nitrite (NOx), methyl guanidine (MG), tumor necrosis factoralpha (TNFalpha), and interleukin-1beta (IL-1beta). In isolated lungs, LPS increased lung weight (LW) to body weight ratio, LW gain, protein and dye tracer leakage, and capillary permeability. The insult also increased NOx, MG, TNFalpha, and IL-1beta in lung perfusate, while decreased adenosine triphosphate (ATP) content with an increase in PARP activity in lung tissue. Pathological examination revealed pulmonary edema with inflammatory cell infiltration. These changes were abrogated by posttreatment (30 min after LPS) with NCA. Following LPS, the inducible NO synthase (iNOS) mRNA expression was increased. NCA reduced the iNOS expression. Niacinamide exerts protective effects on the organ dysfunction and ALI caused by endotoxin. The mechanisms may be mediated through the inhibition on the PARP activity, iNOS expression and the subsequent suppression of NO, free radicals, and proinflammatory cytokines with restoration of ATP.

  6. Coenzyme q10 abrogated the 28 days aluminium chloride induced oxidative changes in rat cerebral cortex.

    Science.gov (United States)

    Majumdar, Anuradha S; Nirwane, Abhijit; Kamble, Rahul

    2014-05-01

    The present study was designed to elucidate the impact of oral administration of aluminium chloride for 28 days with respect to oxidative stress in the cerebral cortex of female rats. Further, to investigate the potentials of Coenzyme (Co) Q10 (4, 8, and 12 mg/kg, i.p.) in mitigating the detrimental changes. Biochemical estimations of cerebral lipid peroxidation (LPO), reduced glutathione (GSH), vitamin E and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were carried out after 28 days of aluminium chloride (AlCl3) and Co Q10 exposures along with histopathological examination of cerebral cortex of the rats. Subacute exposure to AlCl3(5 mg/kg) led to significant decrease in levels of GSH, vitamin E and activities of SOD, CAT, GPx, and an increase in LPO of cerebral cortex. These aberrations were restored by Co Q10 (12 mg/kg, i.p.). This protection offered was comparable to that of L-deprenyl (1 mg/kg, i.p.) which served as a reference standard. Histopathological evaluations confirmed that the normal cerebral morphology was maintained by Co Q10. Thus, AlCl3 exposure hampers the activities of various antioxidant enzymes and induces oxidative stress in cerebral cortex of female Wistar rats. Supplementation with intraperitoneal Co Q10 abrogated these deleterious effects of AlCl3.

  7. Telomerase expression abrogates rapamycin-induced irreversible growth arrest of uterine fibroid smooth muscle cells.

    Science.gov (United States)

    Suo, Guangli; Sadarangani, Anil; Tang, Wingchung; Cowan, Bryan D; Wang, Jean Y J

    2014-09-01

    Uterine fibroids are the most common solid tumors found in women of reproductive age. It has been reported that deregulation of the mammalian target of rapamycin (mTOR) pathway plays an important role in the etiology of leiomyoma. Here, we investigated the effect of rapamycin, an inhibitor of mTORC1, on the growth of primary fibroid smooth muscle cells (fSMCs) and human telomerase reverse transcriptase (hTERT)-transduced and immortalized fSMCs. With the primary fSMCs, a 24-hour treatment with rapamycin was sufficient to trigger a growth arrest that was not reversible upon drug removal. By contrast, the growth inhibitory effect of rapamycin on the hTERT-transduced fSMCs was readily reversible, as these cells resumed proliferation upon the withdrawal of the drug. These results suggest that rapamycin-induced irreversible growth arrest of fSMCs is dependent on the senescence barrier that is abrogated by the ectopic expression of telomerase.

  8. Galangin Abrogates Ovalbumin-Induced Airway Inflammation via Negative Regulation of NF-κB

    Directory of Open Access Journals (Sweden)

    Wang-Jian Zha

    2013-01-01

    Full Text Available Persistent activation of nuclear factor κB (NF-κB has been associated with the development of asthma. Galangin, the active pharmacological ingredient from Alpinia galanga, is reported to have a variety of anti-inflammatory properties in vitro via negative regulation of NF-κB. This study aimed to investigate whether galangin can abrogate ovalbumin- (OVA- induced airway inflammation by negative regulation of NF-κB. BALB/c mice sensitized and challenged with OVA developed airway hyperresponsiveness (AHR and inflammation. Galangin dose dependently inhibited OVA-induced increases in total cell counts, eosinophil counts, and interleukin-(IL- 4, IL-5, and IL-13 levels in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. Galangin also attenuated AHR, reduced eosinophil infiltration and goblet cell hyperplasia, and reduced expression of inducible nitric oxide synthase and vascular cell adhesion protein-1 (VCAM-1 levels in lung tissue. Additionally, galangin blocked inhibitor of κB degradation, phosphorylation of the p65 subunit of NF-κB, and p65 nuclear translocation from lung tissues of OVA-sensitized mice. Similarly, in normal human airway smooth muscle cells, galangin blocked tumor necrosis factor-α induced p65 nuclear translocation and expression of monocyte chemoattractant protein-1, eotaxin, CXCL10, and VCAM-1. These results suggest that galangin can attenuate ovalbumin-induced airway inflammation by inhibiting the NF-κB pathway.

  9. Abrogation of CC chemokine receptor 9 ameliorates ventricular remodeling in mice after myocardial infarction.

    Science.gov (United States)

    Huang, Yan; Wang, Dandan; Wang, Xin; Zhang, Yijie; Liu, Tao; Chen, Yuting; Tang, Yanhong; Wang, Teng; Hu, Dan; Huang, Congxin

    2016-01-01

    CC chemokine receptor 9 (CCR9), which is a unique receptor for CC chemokine ligand (CCL25), is mainly expressed on lymphocytes, dendritic cells (DCs) and monocytes/macrophages. CCR9 mediates the chemotaxis of inflammatory cells and participates in the pathological progression of inflammatory diseases. However, the role of CCR9 in the pathological process of myocardial infarction (MI) remains unexplored; inflammation plays a key role in this process. Here, we used CCR9 knockout mice to determine the functional significance of CCR9 in regulating post-MI cardiac remodeling and its underlying mechanism. MI was induced by surgical ligation of the left anterior descending coronary artery in CCR9 knockout mice and their CCR9+/+ littermates. Our results showed that the CCR9 expression levels were up-regulated in the hearts of the MI mice. Abrogation of CCR9 improved the post-MI survival rate and left ventricular (LV) dysfunction and decreased the infarct size. In addition, the CCR9 knockout mice exhibited attenuated inflammation, apoptosis, structural and electrical remodeling compared with the CCR9+/+ MI mice. Mechanistically, CCR9 mainly regulated the pathological response by interfering with the NF-κB and MAPK signaling pathways. In conclusion, the data reveal that CCR9 serves as a novel modulator of pathological progression following MI through NF-κB and MAPK signaling.

  10. Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.

    Science.gov (United States)

    Wang, Weimin; Kryczek, Ilona; Dostál, Lubomír; Lin, Heng; Tan, Lijun; Zhao, Lili; Lu, Fujia; Wei, Shuang; Maj, Tomasz; Peng, Dongjun; He, Gong; Vatan, Linda; Szeliga, Wojciech; Kuick, Rork; Kotarski, Jan; Tarkowski, Rafał; Dou, Yali; Rattan, Ramandeep; Munkarah, Adnan; Liu, J Rebecca; Zou, Weiping

    2016-05-19

    Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.

  11. Lipoteichoic acid synthesis inhibition in combination with antibiotics abrogates growth of multidrug-resistant Enterococcus faecium.

    Science.gov (United States)

    Paganelli, Fernanda L; van de Kamer, Tim; Brouwer, Ellen C; Leavis, Helen L; Woodford, Neil; Bonten, Marc J M; Willems, Rob J L; Hendrickx, Antoni P A

    2017-03-01

    Enterococcus faecium is a multidrug-resistant (MDR) nosocomial pathogen causing significant morbidity in debilitated patients. New antimicrobials are needed to treat antibiotic-resistant E. faecium infections in hospitalised patients. E. faecium incorporates lipoteichoic acid (LTA) (1,3-polyglycerol-phosphate linked to glycolipid) in its cell wall. The small-molecule inhibitor 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] specifically blocks the activity of Staphylococcus aureus LtaS synthase, which polymerises 1,3-glycerolphosphate into LTA polymers. Here we characterised the effects of the small-molecule inhibitor 1771 on the growth of E. faecium isolates, alone (28 strains) or in combination with the antibiotics vancomycin, daptomycin, ampicillin, gentamicin or linezolid (15 strains), and on biofilm formation (16 strains). Inhibition of LTA synthesis at the surface of the cell by compound 1771 in combination with current antibiotic therapy abrogates enterococcal growth in vitro but does not affect mature E. faecium biofilms. Targeting LTA synthesis may provide new possibilities to treat MDR E. faecium infections.

  12. Secoisolariciresinol diglucoside abrogates oxidative stress-induced damage in cardiac iron overload condition.

    Directory of Open Access Journals (Sweden)

    Stephanie Puukila

    Full Text Available Cardiac iron overload is directly associated with cardiac dysfunction and can ultimately lead to heart failure. This study examined the effect of secoisolariciresinol diglucoside (SDG, a component of flaxseed, on iron overload induced cardiac damage by evaluating oxidative stress, inflammation and apoptosis in H9c2 cardiomyocytes. Cells were incubated with 50 μ5M iron for 24 hours and/or a 24 hour pre-treatment of 500 μ M SDG. Cardiac iron overload resulted in increased oxidative stress and gene expression of the inflammatory mediators tumor necrosis factor-α, interleukin-10 and interferon γ, as well as matrix metalloproteinases-2 and -9. Increased apoptosis was evident by increased active caspase 3/7 activity and increased protein expression of Forkhead box O3a, caspase 3 and Bax. Cardiac iron overload also resulted in increased protein expression of p70S6 Kinase 1 and decreased expression of AMP-activated protein kinase. Pre-treatment with SDG abrogated the iron-induced increases in oxidative stress, inflammation and apoptosis, as well as the increased p70S6 Kinase 1 and decreased AMP-activated protein kinase expression. The decrease in superoxide dismutase activity by iron treatment was prevented by pre-treatment with SDG in the presence of iron. Based on these findings we conclude that SDG was cytoprotective in an in vitro model of iron overload induced redox-inflammatory damage, suggesting a novel potential role for SDG in cardiac iron overload.

  13. Abrogation of plasminogen activator inhibitor-1-vitronectin interaction ameliorates acute kidney injury in murine endotoxemia.

    Directory of Open Access Journals (Sweden)

    Kamlesh K Gupta

    Full Text Available Sepsis-induced acute kidney injury (AKI contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1 and vitronectin (Vn are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/- or expressing a PAI-1-mutant (PAI-1R101A/Q123K in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

  14. Production of a monoclonal antibody against aflatoxin M1 and its application for detection of aflatoxin M1 in fortified milk

    Directory of Open Access Journals (Sweden)

    Umarphorn Chadseesuwan

    2016-10-01

    Full Text Available Aflatoxin M1 (AFM1 is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17 were obtained that showed cross-reactivity with aflatoxin B1 (AFB1 and aflatoxin G1 (AFG1 but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC50 and the limit of detection (LOD values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV and % recovery of the detection was investigated for both intra- (n = 6 and inter- (n = 12 variation assays. The % CV was found in the range of 3.50–15.8% and 1.32–7.98%, respectively, while the % recovery was in the range of 92–104% and 100–103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1–33.0% and 91–109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R2 of 0.992 for the concentration of 0.2–5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples.

  15. Production and characterization of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine.

    Science.gov (United States)

    Nakajima, Tamiko; Yasuda, Toshihiro; Takeshita, Haruo; Mori, Shinjiro; Mogi, Kouichi; Kaneko, Yasushi; Nakazato, Emiko; Kishi, Koichiro

    2002-04-15

    Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

  16. Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

    Science.gov (United States)

    Cha, Sang-Ho; Kim, Sung-Hee; Bischoff, Karyn; Kim, Hyun-Jeong; Son, Seong-Wan

    2012-01-01

    A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were from 25 to 750 ppb and from 12.5 to 100 ppb, respectively. The detection limit of both methods as measured with standard solutions was 10 ppb. The intra-plate and inter-well variation of both ELISAs were less than 10%. The IC50 values for α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol compared to ZEN were 108.1, 119.3, 114.1, and 130.3% for the ZEN-coated ELISA. These values were 100.7, 120.7, 121.6, and 151.6% for the anti-ZEN antibody-coated ELISA. According to the anti-ZEN antibody-coated ELISA, the average recovery rates of ZEN from spiked animal feed containing 150 to 600 ng/mL of ZEN ranged from 106.07 to 123.00% with 0.93 to 2.28% coefficients of variation. Our results demonstrate that the mAb developed in this study could be used to simultaneously screen for ZEN and its metabolites in feed. PMID:22705733

  17. Development and characterization of a monoclonal antibody specific for human basophils and the identification of a unique secretory product of basophil activation.

    Science.gov (United States)

    McEuen, A R; Buckley, M G; Compton, S J; Walls, A F

    1999-01-01

    Despite increasing evidence that basophils can infiltrate into inflamed tissues during allergic reactions, determination of the extent of infiltration and elucidation of their role in allergic disease has been frustrated by the lack of reliable means for detecting this cell type in tissues. In the present study, we report on a new monoclonal antibody specific for basophils and on the initial characterization of the antigen it recognizes. Basophils were isolated from peripheral blood by Percoll density gradient centrifugation and a positive-selection immunomagnetic procedure and injected into mice to produce monoclonal antibodies. A hybridoma clone, designated BB1, secreted antibody of the IgG2a isotype; this antibody bound selectively to basophils on immunocytochemistry but did not react with any other cell type or tissue structure, although it did stain a proportion of cells from the basophilic cell line KU812F. In sections of mixed populations of peripheral blood cells, similar numbers of cells stained with Alcian blue dye and BB1 over a wide range of basophil purity. BB1 antibody was effective in identifying basophils in sections of mixed cells or in tissues after fixation with ethanol, Carnoy's solution, or formalin. Staining of basophils with BB1 gave a granular appearance, although flow cytometry indicated that some antigen was also present on the surface of the cell. Activation of these cells with anti-IgE antibody or with the calcium ionophore A23187 provoked release of the antigen in parallel with that of histamine. BB1 antibody did not, by itself, stimulate histamine release. The molecular mass of the antigen was determined on Hedrick-Smith gels to be 124+/-11 kd. This new monoclonal antibody will be a valuable experimental tool in future studies, allowing the reliable detection of basophils in tissues of patients with allergic and chronic inflammatory disease; in addition, the antigen it identifies has potential as a unique marker of basophil activation.

  18. Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana: influence on expression yield and antibody degradation.

    Science.gov (United States)

    Lombardi, Raffaele; Donini, Marcello; Villani, Maria Elena; Brunetti, Patrizia; Fujiyama, Kazuhito; Kajiura, Hiroyuki; Paul, Matthew; Ma, Julian K-C; Benvenuto, Eugenio

    2012-10-01

    We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which β1,2-xylosyltransferase and α1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10(XylT/FucT)) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10(XylT/FucT) demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of β1,2-xylose and α1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible 'fragile' sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.

  19. Production of Polyclonal Antibody of Morphine and Determination of Morphine in Urine by Capillary Electrophoresis Immunoassay with Laser-induced Fluorescence Detection

    Institute of Scientific and Technical Information of China (English)

    Jian Qiu MI; Xiao Hua QI; Xin Xiang ZHANG; Wen Bao CHANG

    2004-01-01

    N-Conjugated antigen was synthesized and polyclonal antibody with high specificity was obtained from immunizing animals.With this polyclonal antibody, a rapid and efficient CEIA-LIF method was developed to determine the free morphine in urine of abusers.The detection limit was calculated to be 40 ng/mL.Simulated urine samples were analyzed with good recoveries, which showed the feasibility of its application in specific morphine determination in urine of morphine abusers.

  20. Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

    Science.gov (United States)

    Tsuruta, Lilian Rumi; Lopes Dos Santos, Mariana; Yeda, Fernanda Perez; Okamoto, Oswaldo Keith; Moro, Ana Maria

    2016-12-01

    Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

  1. Therapeutic effects of antigen affinity-purified polyclonal anti-receptor of advanced glycation end-product (RAGE) antibodies on cholestasis-induced liver injury in rats.

    Science.gov (United States)

    Xia, Peng; Deng, Qing; Gao, Jin; Yu, Xiaolan; Zhang, Yang; Li, Jingjing; Guan, Wen; Hu, Jianjun; Tan, Quanhui; Zhou, Liang; Han, Wei; Yuan, Yunsheng; Yu, Yan

    2016-05-15

    Cholestasis leads to acute hepatic injury, fibrosis/cirrhosis, inflammation, and duct proliferation. We investigated whether blocking receptor of advanced glycation end-products (RAGE) with polyclonal anti-RAGE antibodies (anti-RAGE) could regulate acute liver injury and fibrosis in a rat bile duct ligation (BDL) model. Male Wister rats received 0.5mg/kg rabbit anti-RAGE or an equal amount of rabbit IgG by subcutaneous injection twice a week after BDL. Samples of liver tissue and peripheral blood were collected at 14 days after BDL. Serum biochemistry and histology were used to analyze the degree of liver injury. Quantitative real-time PCR (qPCR) and immunohistochemical staining were used to further analyze liver injury. Anti-RAGE improved the gross appearance of the liver and the rat survival rate. Liver tissue histology and relevant serum biochemistry indicated that anti-RAGE attenuated liver necrosis, inflammation, liver fibrosis, and duct proliferation in the BDL model. qPCR and western blotting showed significant reductions in interleukin-1β expression levels in the liver by treatment with anti-RAGE. Anti-RAGE also significantly reduced the mRNA levels of α1(1) collagen (Col1α1) and cholesterol 7α-hydroxylase, and the ratio of tissue inhibitor of matrix metalloproteinase-1 to matrix metalloproteinases (MMPs) in the liver. In addition, anti-RAGE regulated the transcriptional level of Col1α1 and MMP-9 in transforming growth factor-β-induced activated LX-2 cells in vitro. Anti-RAGE was found to inhibit hepatic stellate cell proliferation in vivo and in vitro. Therefore, anti-RAGE can protect the liver from injury induced by BDL in rats. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies.

    Directory of Open Access Journals (Sweden)

    Dennis Wolf

    Full Text Available BACKGROUND: Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L--an established marker and mediator of cardiovascular disease--induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: WT or CD40L(-/- mice consumed a high fat diet (HFD for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/- mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/- mice. However, CD40L(-/- mice consuming HFD were not protected from the onset of diet-induced obesity (DIO, insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/- mice consuming a low fat diet (LFD showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels. CONCLUSION: We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.

  3. Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77.

    Science.gov (United States)

    Leblanc, Pierre; Moon, Minho; Kim, Woori; Jeong, Inhye; Kim, Chun-Hyung; Kim, Kwang-Soo

    2015-08-17

    The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson's disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.

  4. Immunization of nonautoimmune mice with DNA binding domains of the largest subunit of RNA polymerase I results in production of anti-dsDNA and anti-Sm/RNP antibodies.

    Science.gov (United States)

    Sciascia, Sandra A; Robson, Kristina; Zhu, Liangjin; Garland, Michael; Grabosch, Shannon; Kelamis, Joseph; Messamore, Will; Bradley, Todd; Sourk, Allison; Westberg, Lindsey; Goodnight, Waco; Tongson, Krystina; Holloway, Naomi; Wardak, Zabi; Sudyka, Michelle; Masrani, Shriti; Chintalapati, Sanket; Cagaanan, Manuel; Brown, John C; Stetler, Dean A

    2007-02-01

    Antibodies against the N-terminal (NT) but not the basic domain (BD), DNA binding regions of the largest subunit (S1) of RNA polymerase I (RNAPI) were detected in the sera of MRL-lpr/lpr lupus mice. Antibodies against both RNAPI(S1)-NT and -BD, as well as other systemic lupus erythematosus (SLE) autoantigens (La, ribosomal P proteins and Sm/RNP) were produced by rabbits immunized with anti-DNA antibodies that had been affinity purified from SLE patients. Immunization of nonautoimmune mice (Balb/c) with RNAPI(S1)-NT, RNAPI(S1)-BD, or La in the form of GST fusion proteins, induced production of anti-double-stranded (ds) DNA and anti-Sm/RNP. GST-P1 did not induce an anti-dsDNA response in these mice. These results demonstrate that RNAPI(S1)-NT, RNAPI(S1)-BD and La can participate in an anti-autoantigen/anti-DNA antibody loop during an SLE-like autoimmune response.

  5. Stem cell therapy for abrogating stroke-induced neuroinflammation and relevant secondary cell death mechanisms.

    Science.gov (United States)

    Stonesifer, Connor; Corey, Sydney; Ghanekar, Shaila; Diamandis, Zachary; Acosta, Sandra A; Borlongan, Cesar V

    2017-07-23

    Ischemic stroke is a leading cause of death worldwide. A key secondary cell death mechanism mediating neurological damage following the initial episode of ischemic stroke is the upregulation of endogenous neuroinflammatory processes to levels that destroy hypoxic tissue local to the area of insult, induce apoptosis, and initiate a feedback loop of inflammatory cascades that can expand the region of damage. Stem cell therapy has emerged as an experimental treatment for stroke, and accumulating evidence supports the therapeutic efficacy of stem cells to abrogate stroke-induced inflammation. In this review, we investigate clinically relevant stem cell types, such as hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), very small embryonic-like stem cells (VSELs), neural stem cells (NSCs), extraembryonic stem cells, adipose tissue-derived stem cells, breast milk-derived stem cells, menstrual blood-derived stem cells, dental tissue-derived stem cells, induced pluripotent stem cells (iPSCs), teratocarcinoma-derived Ntera2/D1 neuron-like cells (NT2N), c-mycER(TAM) modified NSCs (CTX0E03), and notch-transfected mesenchymal stromal cells (SB623), comparing their potential efficacy to sequester stroke-induced neuroinflammation and their feasibility as translational clinical cell sources. To this end, we highlight that MSCs, with a proven track record of safety and efficacy as a transplantable cell for hematologic diseases, stand as an attractive cell type that confers superior anti-inflammatory effects in stroke both in vitro and in vivo. That stem cells can mount a robust anti-inflammatory action against stroke complements the regenerative processes of cell replacement and neurotrophic factor secretion conventionally ascribed to cell-based therapy in neurological disorders. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Targeting ILK and {beta}4 integrin abrogates the invasive potential of ovarian cancer

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Pyo; Kim, Baek Gil [BK21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of); Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Gao, Ming-Qing; Kang, Suki [Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cho, Nam Hoon, E-mail: cho1988@yuhs.ac [BK21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of); Department of Pathology, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer The potential of targeting ILK and integrins for highly aggressive ovarian cancer. Black-Right-Pointing-Pointer Unanticipated synergistic effect for the combination of ILK/{beta}4 integrin. Black-Right-Pointing-Pointer Combination of ILK/{beta}4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. Black-Right-Pointing-Pointer Targeting of {beta}4 integrin/ILK had potent inhibitory effects in ovarian cancer. -- Abstract: Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of {beta}1 and {beta}4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of {beta}1 and {beta}4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of {beta}4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of {beta}4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting {beta}4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

  7. Mammalian target of rapamycin inhibitor abrogates abnormal osteoclastogenesis in neurofibromatosis type 1

    Institute of Scientific and Technical Information of China (English)

    LIU Ning; XU Ning; WEI Li-hui; CHAI Guo-lin

    2013-01-01

    Background Neurofibromatosis type 1 (NF1) is the most common genetic syndrome predisposing patients to various tumors due to dysregulation of the Ras signaling pathway.Recent research has shown NF1 patients also suffer a spectrum of bone pathologies.The pathogenesis of NF1 bone diseases is largely unknown.There is no current treatment.By Nf1 heterozygote (Nf1+/-) mice and Nf1 conditional knockout mice,we and other groups demonstrated abnormal osteoblast and osteoclast function due to dysregulation of Ras signaling.However,the specific downstream effector pathways linked to NF1 abnormal osteoblastogenesis and osteoclastogenesis have not been defined.In this study,we investigated the Ras downstream effector related with NF1 bone disease.Methods We used Nf1+/+ and Nf1+/-mice as normal and NF1 models.Bone stromal cells extracted from Nf1+/+ and Nf1+/-mice were induced osteoclasts.The osteoclast cell was stained by tartrate resistant acid phosphatase staining.The osteoclast cell number was counted and the surface area of osteoclast cells was calculated under the microscope.The mRNA of mammalian target of rapamycin (mTOR) was determined by quantitative reverse-transcription-polymerase chain reaction.The presence of ribosomal protein S6 kinase was determined by Western blotting.Results Compared with Nf1+/+ mice,Nf1+/-mice had about 20% more of osteoclast cells.These osteoclast cells werelarger in size with more nuclei.Hyperactive mTOR was detected in Nf1+/-osteoclast cells.Inhibition of mTOR signalingby rapamycin in Nf1+/-osteoclasts abrogated abnormalities in cellular size and number.Conclusion mTOR pathway inhibition may represent a viable therapy for NF1 bone diseases.

  8. Chrysin, an anti-inflammatory molecule, abrogates renal dysfunction in type 2 diabetic rats

    Energy Technology Data Exchange (ETDEWEB)

    Ahad, Amjid [Lipid Metabolism Laboratory, Department of Biochemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062 (India); Ganai, Ajaz Ahmad [Department of Biotechnology, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062 (India); Mujeeb, Mohd [Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062 (India); Siddiqui, Waseem Ahmad, E-mail: was.sid121@gmail.com [Lipid Metabolism Laboratory, Department of Biochemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062 (India)

    2014-08-15

    Diabetic nepropathy (DN) is considered as the leading cause of end-stage renal disease (ESRD) worldwide, but the current available treatments are limited. Recent experimental evidences support the role of chronic microinflammation in the development of DN. Therefore, the tumor necrosis factor-alpha (TNF-α) pathway has emerged as a new therapeutic target for the treatment of DN. We investigated the nephroprotective effects of chrysin (5, 7-dihydroxyflavone) in a high fat diet/streptozotocin (HFD/STZ)-induced type 2 diabetic Wistar albino rat model. Chrysin is a potent anti-inflammatory compound that is abundantly found in plant extracts, honey and bee propolis. The treatment with chrysin for 16 weeks post induction of diabetes significantly abrogated renal dysfunction and oxidative stress. Chrysin treatment considerably reduced renal TNF-α expression and inhibited the nuclear transcription factor-kappa B (NF-kB) activation. Furthermore, chrysin treatment improved renal pathology and suppressed transforming growth factor-beta (TGF-β), fibronectin and collagen-IV protein expressions in renal tissues. Chrysin also significantly reduced the serum levels of pro-inflammatory cytokines, interleukin-1beta (IL-1β) and IL-6. Moreover, there were no appreciable differences in fasting blood glucose and serum insulin levels between the chrysin treated groups compared to the HFD/STZ-treated group. Hence, our results suggest that chrysin prevents the development of DN in HFD/STZ-induced type 2 diabetic rats through anti-inflammatory effects in the kidney by specifically targeting the TNF-α pathway. - Highlights: • Chrysin reduced renal oxidative stress and inflammation in diabetic rats. • Chrysin reduced serum levels of pro-inflammatory in diabetic rats. • Chrysin exhibited renal protective effect by suppressing the TNF-α pathway.

  9. Antibody Request - Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    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.

  10. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  11. Cold-treated centrosome: isolation of centrosomes from mitotic sea urchin eggs, production of an anticentrosomal antibody, and novel ultrastructural imaging.

    Science.gov (United States)

    Thompson-Coffe, C; Coffe, G; Schatten, H; Mazia, D; Schatten, G

    1996-01-01

    A novel isolation of centrosomes is described and it was used to both generate a centrosome-specific monoclonal antibody and to image with high-resolution low-voltage scanning electron microscopy the surface details of the isolated centrosome. At first mitotic prometaphase, sea urchin zygotes are chilled on ice overnight. While most of the microtubules disassemble, the mitotic centrosomes collapse into aggregated masses. These centrosomes have been isolated, and used to generate a monoclonal antibody, designated 4D2, which is reactive with interphase and mitotic centrosomes. 4D2 staining of centrosomes is similar, but not identical, to that of other centrosomal antibodies like Ah6 and 5051. Centrosomal material is detected as a compact sphere after cold treatment; upon recovery the sphere expands and undergoes the shape changes previously described [Mazia et al., 1987: J. Cell Biol. 105:206a] to eventually reorganize a normal mitotic apparatus.

  12. Production and radioimmunoimaging of novel fully human phage display recombinant antibodies and growth inhibition of lung adenocarcinoma cell line overexpressing Prx I.

    Science.gov (United States)

    Luo, Yi; Pang, Hua; Li, Shujie; Cao, Hui; Peng, Zhiping; Fan, Chunbo; Li, Shaolin

    2009-07-01

    The Peroxiredoxin I (Prx I) is a member of the Peroxiredoxin family, which is overexpressed in many diverse tumor types and is an anti-apoptosis protein for tumor cell proliferation and survival. Therapeutic strategies targeting the Prx I may therefore be effective broad-spectrum anticancer agents. We constructed a phage display single-chain variable fragment (scFv) antibody library and sieve out the fully human, lung adenocarcinoma-sepcific monoclonal antibodies. The selection on Prx I was performed using above-mentioned lung adenocarcinoma-sepcific monoclonal antibodies with high affinity to Prx I overexpressing lung adenocarcinoma cells. The candidate scFv sequences, based on enzyme-linked immunosorbent assay (ELISA) screening data, were chosen for soluble expression, and a 30 kDa band was observed on polyacrylamide gel electrophoresis as predicted. The purified antibodies were characterized by immunoblotting and showed high specificity to Prx I-overexpressing lung adenocarcinoma cells A549. Radioimmunoimaging was taken to evaluate specificity and distribution of antibodies in vivo. The radiolocalization index (RI) of tumor/serum and tumor/muscle gradually increased, reaching its peak (4.06 +/- 0.13 and 5.17 +/- 0.97, respectively) at 48 h postadministration. Single photon emission computed tomography (SPECT) imaging showed the radioactivity was aggregated in tumor locations and tumor imaging was clearly observed. The internalized scFv resulted in antibody-mediated cell apoptosis and downregulation of Prx I expression. These results demonstrate that the scFv possesses strong antitumor activity on lung adenocarcinoma and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on Prx I for growth and survival.

  13. Interleukin-17 expression positively correlates with disease severity of lupus nephritis by increasing anti-double-stranded DNA antibody production in a lupus model induced by activated lymphocyte derived DNA.

    Directory of Open Access Journals (Sweden)

    Zhenke Wen

    Full Text Available Lupus nephritis is one of the most serious manifestations and one of the strongest predictors of a poor outcome in systemic lupus erythematosus (SLE. Recent evidence implicated a potential role of interlukin-17 (IL-17 in the pathogenesis of lupus nephritis. However, the correlation between IL-17 expression level and the severity of lupus nephritis still remains incompletely understood. In this study, we found that serum IL-17 expression level was associated with the severity of lupus nephritis, which was evaluated by histopathology of kidney sections and urine protein. Of note, we showed that enforced expression of IL-17 using adenovirus construct that expresses IL-17 could enhance the severity of lupus nephritis, while blockade of IL-17 using neutralizing antibody resulted in decreased severity of lupus nephritis. Consistently, we observed an impaired induction of lupus nephritis in IL-17-deficient mice. Further, we revealed that IL-17 expression level was associated with immune complex deposition and complement activation in kidney. Of interest, we found that IL-17 was crucial for increasing anti-double-stranded DNA (dsDNA antibody production in SLE. Our results suggested that IL-17 expression level positively correlated with the severity of lupus nephritis, at least in part, because of its contribution to anti-dsDNA antibody production. These findings provided a novel mechanism for how IL-17 expression level correlated with disease pathogenesis and suggested that management of IL-17 expression level was a potential and promising approach for treatment of lupus nephritis.

  14. Production and Characterization of a Monoclonal Antibody Raised Against Surface Antigens from Mycelium of Gaeumannomyces graminis var. tritici: Evidence for an Extracellular Polyphenol Oxidase.

    Science.gov (United States)

    Thornton, C R; Dewey, F M; Gilligan, C A

    1997-01-01

    ABSTRACT A murine monoclonal antibody (MAb) of immunoglobulin class M (IgM) was raised against surface antigens from Gaeumannomyces graminis var. tritici and, by enzyme-linked immunosorbent assay, recognized isolates of G. graminis var. tritici, G. graminis var. avenae and G. graminis var. graminis. Characterization of the antigen by heat and protease treatments showed that the epitope recognized by the MAb was a protein. Antigen production was detected only in live mycelia. Immunofluorescence studies showed that the antigen was associated with both the broad melanized macrohyphae and hyaline mycelia of G. graminis var. tritici. Secretion of antigen into an aqueous minimal medium was promoted only by exposure of live mycelia to certain phenolic substrates, including monophenols ortho-, para-, and meta-cresol; 3,4,5-trihydroxybenzoic acid (gallic acid); and phenolic amino acid L-3-(3,4-dihydroxyphenyl) alanine (L-DOPA). Antigen secretion was not promoted by 3-(4-hydroxyphenyl) alanine (L-tyrosine). The MAb reacted strongly with purified enzyme laccase (polyphenol oxidase, EC 1.10.3.2) but did not recognize purified tyrosinase (monophenol oxidase, EC 1.14.18.1). Moreover, chemicals that bind to copper and inhibit copper-containing enzymes such as laccase completely inhibited antigen secretion in response to L-DOPA. The MAb was tested for specificity against a wide range of fungi, common yeast species, and gram positive and negative bacteria. It did not recognize antigens from a broad range of unrelated fungi, including Gliocladium roseum, Fusarium sp., Phoma exigua, Phialophora fastigiata, Penicillium crustosum, Pythium ultimum, Rhizopus stolonifer, Rhizoctonia carotae, R. oryzae, R. tuliparum, and Trichoderma viride, nor did it recognize surface antigens from yeasts or bacteria. The MAb cross-reacted with antigens from Botrytis spp., Chaetomium globosum, R. cerealis, and R. solani. However, secretion of antigen by R. solani and R. cerealis was not promoted by L

  15. Anti-miroestrol polyclonal antibodies: a comparison of immunogen preparations used to obtain desired antibody properties.

    Science.gov (United States)

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Yusakul, Gorawit; Tanaka, Hiroyuki; Putalun, Waraporn

    2016-04-01

    Immunogen quality is one important factor that contributes to desirable antibody characteristics. Highly specific antibodies against miroestrol can be used to develop a quality control immunoassay for Pueraria candollei products. In this study, we investigated how various immunogen preparations affect antibody properties. The results show that immunogen prepared using the Mannich reaction provides antibodies with higher specificity and sensitivity against miroestrol than immunogen prepared with the periodate reaction. The results suggest the Mannich reaction maintains the original structure of miroestrol and generates useful antibodies for developing immunoassays.

  16. Selection of antibodies from synthetic antibody libraries.

    Science.gov (United States)

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  17. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  18. [Advances in the study of natural small molecular antibody].

    Science.gov (United States)

    Zhu, Lei; Zhang, Da-peng

    2012-10-01

    Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called Vnar fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC.

  19. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, van den N.; Goosen, T.; Verrips, C.T.; Hondel, van den C.A.M.J.J.; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V-HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which

  20. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which

  1. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pro

  2. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, van den N.; Goosen, T.; Verrips, C.T.; Hondel, van den C.A.M.J.J.; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V-HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pr

  3. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (V-HH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, van den N.; Goosen, T.; Verrips, C.T.; Hondel, van den C.A.M.J.J.; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5'- or 3'-terminal ends of the gene encoding llama variable heavy chain antibody fragment V-HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pr

  4. Production of bifunctional proteins by Aspergillus awamori: Llama variable heavy chain antibody fragment (VHH) R9 coupled to Arthromyces ramosus peroxidase (ARP)

    NARCIS (Netherlands)

    Joosten, V.; Roelofs, M.S.; Dries, N. van den; Goosen, T.; Verrips, C.T.; Hondel, C.A.M.J.J. van den; Lokman, B.C.

    2005-01-01

    The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment VHH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which pro

  5. Production of a monoclonal antibody specific for high molecular weight glutenin subunits (HMW-GS) in wheat and its antigenic determinant

    Institute of Scientific and Technical Information of China (English)

    WANG Hanqian; ZHANG Xueyong; WANG Hongmei; PANG Binshuang

    2005-01-01

    Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to IgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T.aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye),Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.

  6. Synthesis of an oxytetracyline-tolidin-BSA immunogen and antibodies production of anti-oxytetracyline developed for oxytetracyline residue detection with enzyme-linked immunosorbent assays technique

    Directory of Open Access Journals (Sweden)

    Widiastuti R

    2013-06-01

    Full Text Available An oxytetracycline-tolidin-bovine serum albumin (OTC-tolidin-BSA-conjugate was synthezed as immunogen for producing specific antibodies in immunized rabbits that would be used as reagent for development of OTC residue detection with enzym-linked immunoassays technique. The immunogen was prepared through diazotization tolidin and subsequently reacted with OTC. The red purple immunogen of OTC-tolidin-BSA absorbed at wave lengths of 277 nm and 488 nm under UV screening absorbances and confirmation with the high performance liquid chromatography (HPLC showed the absence of peak at retention time of 3.46 minutes. Characaterized result with SDS-PAGE showed the molecular weight of the OTC-tolidin-BSA at 69.79 kDA. Subsequently, the immunogen was immunized into New Zealand rabbits in order to produce the polyclonal antibodies. The antibodies were purified using a protein A sepharose column. The OD optimum responses of 0.92 to 1.20 were obtained from the second fractionation at dilution of 1/1000 by titrating the antibodies and OTC-tolidin-BSA coating antigen at concentration of 10 µg/mL on several bleeding times.

  7. Glycosylation profiles of therapeutic antibody pharmaceuticals.

    Science.gov (United States)

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

    2011-11-01

    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth.

  8. Production of Monoclonal Antibodies Against the Challenge Strain of Infectious Laryngotracheitis Virus of Chickens and Their Use in an Indirect Immunofluorescent Diagnostic Test

    Directory of Open Access Journals (Sweden)

    Ferhat Abbas*, James Andreasen1, Rockey Becker1, Masroor Ahmed, M Arif Awan, Abdul Wadood and Anita Sonn1

    2010-07-01

    Full Text Available The objective of the present research was to produce monoclonal antibodies (MCAs against the USDA challenge strain of infectious laryngotracheitis virus and to perform an initial investigation of their use in an indirect immunofluorescence diagnostic test. Fourteen-day old chicken embryo liver cells were grown in tissue culture plates. Confluent monolayers were obtained after 48 hours. Monolayers were infected with the USDA challenge strain of infectious laryngotracheitis virus (ILTV. Cytopethic effect of the virus in the form of syncytial formation and clumping of cells was observed after 24 hours. The virus from the tissue culture flasks was collected and purified using discontinuous sucrose gradient. A clear band of the virus from sucrose gradient was obtained. The refractory index and the density measured were 1.410 and 1.20 g/cm3, respectively. Spectrophotometry of the purified virus showed 68.117 ug/ml of protein and 9.8948 ug/ml of nucleic acid concentration. Spleen cells from immunized mice with pure virus were fused with myeloma cells and hybridomas were obtained after 10 days. Screening was performed using indirect immunofluorescence antibody test (IFAT using rabbit anti-mouse immunoglobulins as secondary antibodies. Three hybridomas, 2D1D8, 2E11G2 and 2C6C7 were found producing antibodies against ILTV. All monoclonal antibodies were of isotype IgM and reacted with different strains of ILTV (ILTV USDA, S 88 00224, 86-1169 in IFAT. None of the monoclonals reacted with Parrot herpesvirus and avian adenovirus 301 in IFAT.

  9. Role of CD4(+) T cells in the modulation of neurotrophin production in mice exposed to low-level toluene.

    Science.gov (United States)

    Fujimaki, Hidekazu; Win-Shwe, Tin-Tin; Yamamoto, Shoji; Nakajima, Daisuke; Goto, Sumio

    2009-01-01

    To investigate the role of CD4(+) T cells in neurotrophin production following toluene exposure, male C3H mice were exposed to filtered air (control) or 9 ppm of toluene in a nose-only exposure chamber for 30 min on 3 consecutive days followed by weekly sessions for 4 weeks. All the mice were immunized with ovalbumin and some groups of mice were treated with anti-CD4 antibody. BDNF content in BAL fluid and NGF content in plasma were significantly increased in toluene-exposed mice. However, treatment with anti-CD4 mAb completely abrogated these effects. These findings suggest that the CD4(+) T cells may be involved in the toluene-induced modulation of neurotrophin production.

  10. Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

    Science.gov (United States)

    Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

    2015-12-01

    Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization.

  11. Enhanced antibody production in mice to the malaria antigen AMA1 by CPG 7909 requires physical association of CpG and antigen.

    Science.gov (United States)

    Mullen, Gregory E D; Aebig, Joan A; Dobrescu, Gelu; Rausch, Kelly; Lambert, Lynn; Long, Carole A; Miles, Aaron P; Saul, Allan

    2007-07-20

    CpG oligodeoxynucleotides are potent immunostimulants. In this study, CPG 7909 was formulated with the recombinant Plasmodium falciparum protein AMA1-C1 adsorbed to Alhydrogel (aluminum hydroxide) and used to immunize mice. Mice receiving free CPG 7909 in a separate same site injection to the AMA1-C1/Alhydrogel had the same antibody responses as mice receiving AMA1-C1/Alhydrogel alone. For mice immunized with CPG 7909 bound to the AMA1-C1/Alhydrogel formulation, there was a bell shaped CPG 7909 dose-response curve with the highest antibody response co-incident with the concentration of CPG 7909 that saturated binding to the Alhydrogel. At a higher CPG 7909 dose where 74% was unbound, there was no enhancement of response over AMA1-C1/Alhydrogel alone. Our results suggest that the adjuvant effects of CpGs are optimal when adsorbed to Alhydrogel and highlight the need for careful characterization of the vaccine formulation.

  12. Bovine milk antibodies for health.

    Science.gov (United States)

    Korhonen, H; Marnila, P; Gill, H S

    2000-11-01

    The immunoglobulins of bovine colostrum provide the major antimicrobial protection against microbial infections and confer a passive immunity to the newborn calf until its own immune system matures. The concentration in colostrum of specific antibodies against pathogens can be raised by immunising cows with these pathogens or their antigens. Immune milk products are preparations made of such hyperimmune colostrum or antibodies enriched from it. These preparations can be used to give effective specific protection against different enteric diseases in calves and suckling pigs. Colostral immunoglobulin supplements designed for farm animals are commercially available in many countries. Also, some immune milk products containing specific antibodies against certain pathogens have been launched on the market. A number of clinical studies are currently in progress to evaluate the efficacy of immune milks in the prevention and treatment of various human infections, including those caused by antibiotic resistant bacteria. Bovine colostrum-based immune milk products have proven effective in prophylaxis against various infectious diseases in humans. Good results have been obtained with products targeted against rotavirus, Shigella flexneri, Escherichia coli, Clostridium difficile, Streptococcus mutans, Cryptosporidium parvum and Helicobacter pylori. Some successful attempts have been made to use immune milk in balancing gastrointestinal microbial flora. Immune milk products are promising examples of health-promoting functional foods, or nutraceuticals. This review summarises the recent progress in the development of these products and evaluates their potential as dietary supplements and in clinical nutrition.

  13. The effect of prophylaxis with chloroquine and proguanil on delayed-type hypersensitivity and antibody production following vaccination with diphtheria, tetanus, polio, and pneumococcal vaccines

    DEFF Research Database (Denmark)

    Gyhrs, A; Pedersen, B K; Bygbjerg, I;

    1991-01-01

    (1,000 mg/week), or 4) proguanil hydrochloride (200 mg/day) for six weeks. Skin testing was performed on days 0 and 28. Vaccinations with diphtheria, tetanus, polio, and pneumococcal polysaccharide antigen vaccines were performed on day 28, and the presence of specific antibodies was determined...... dosages, does not induce any detectable suppression of delayed-type hypersensitivity or vaccination responses to diphtheria, tetanus, polio, or pneumococcal polysaccharide antigens....

  14. {sup 90}Nb: potential radionuclide for application in immuno-PET. Development of appropriate production strategy and first in vivo evaluation of {sup 90}Nb-labeled monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Radchenko, Valery

    2013-07-01

    Nuclear medicine is a modern and highly effective tool for the detection and treatment of oncological disease. Molecular imaging based on radiotracers includes single photon emission tomography (SPECT) and positron emission tomography (PET), which provide non-invasive tumor visualization on nano- and picomolar level, respectively. Currently, many novel tracers for more precise discovery of small tumors and metastases have been introduced and are under investigation. Many of them are protein-based biomolecules which nature herself produces as antigens for the eradication of tumor cells. Antibodies and antibody fragments play an important role in tumor diagnostics and treatment. PET imaging with antibodies and antibody fragments is called immuno-PET. The main issue that needs to be addressed is that appropriate radiotracers with half-lives related to the half-lives of biomolecules are needed. The development of novel radiotracers is a multistep, complicated task. This task includes the evaluation of production, separation and labeling strategy for chosen radionuclide. Finally, the biomolecule-radionuclide complex should be stable in time. An equally important factor is the economic suitability of the production strategy, which will lead to a key decision for future application of the developed radionuclide. In recent work, {sup 90}Nb has been proposed as a potential candidate for application in immuno-PET. Its half-life of 14.6 hours is suitable for application with antibody fragments and some intact antibodies. {sup 90}Nb has a relatively high positron branching of 53% and an optimal energy of β{sup +} emission of 0.35 MeV that can provide high quality of imaging with low dose of used radionuclide. First proof-of-principle studies have shown that {sup 90}Nb: (i) can be produced in sufficient amount and purity by proton bombardment of natural zirconium target (ii) can be isolated from target material with appropriate radiochemical purity (iii) may be used for

  15. Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

    Science.gov (United States)

    Liu, Biing-Hui; Hung, Chun-Tse; Lu, Chuan-Chen; Chou, Hong-Non; Yu, Feng-Yih

    2014-02-12

    Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and ca