Sample records for anti-a murine monoclonal

  1. Immunohistochemical diagnosis of systemic bovine zygomycosis by murine monoclonal antibodies

    DEFF Research Database (Denmark)

    Jensen, H.E.; Aalbaek, B.; Lind, Peter


    Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with h...... for the in situ diagnosis of systemic bovine zygomycosis....

  2. Purification of Murine Monoclonal IgM Antibody

    Institute of Scientific and Technical Information of China (English)


    This paper presents the purification of a monoclonal IgM antibody against human tumor associated antigen Lewis-Y by ion exchange chromatography and gel filtration.Enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (PAGE) were used to identify purified IgM antibody.In flow cytometry analysis, the purified IgM antibody recognizes human breast tumor cell line MCF-7 which expresses Lewis-Y antigen.This work presents a new way for the purification of murine monoclonal IgM antibody.

  3. Immunotherapy of hepatoma with a monoclonal antibody against murine endoglin

    Institute of Scientific and Technical Information of China (English)

    Guang-Hong Tan; Feng-Ying Huang; Hua Wang; Yong-Hao Huang; Ying-Ying Lin; Yue-Nan Li


    AIM: To explore the capability of a monoclonal antibody(mAb) against murine endoglin to inhibit tumor angiogenesis and suppression of hepatoma growth in murine models.METHODS: A monoclonal antibody against murine endoglin was purified by affinity chromatography and passively transfused through tail veins in two murine hepatoma models. Tumor volume and survival time were observed at three-day intervals for 48 d. Microvessels in tumor tissues were detected by immunohistochemistry against CD31, and angiogenesis in vivo was determined by alginate encapsulated assay. In addition, tumor cell apoptosis was detected by TUNEL assay.RESULTS: Passive immunotherapy with anti-endoglin mAb could effectively suppress tumor growth, and prolonged the survival time of hepatoma-bearing mice.Angiogenesis was apparently inhibited within the tumor tissues, and the vascularization of alginate beads was also reduced in the mice passively transfused with antiendoglin mAb. In addition, increased apoptotic cells were observed within the tumor tissues from the mice passively transfused with anti-endoglin mAb.CONCLUSION: Passive immunotherapy with antiendoglin mAb effectively inhibits tumor growth via inhibiting tumor angiogenesis and increasing tumor cell apoptosis, which may be highly correlated with the blockage of endoglin-related signal pathway induced by anti-endoglin mAb.

  4. Porcine humoral immune responses to multiple injections of murine monoclonal antibodies

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Kamstrup, Søren;


    In humans and cattle, multiple injections of murine monoclonal antibodies (m-mAbs) induce anti-mouse antibody responses. The objectives of the present. study were to investigate whether a similar response could be seen when pigs were subjected to m-mAb therapy, and to study the kinetics of such a...

  5. Development of murine monoclonal antibodies for the immunohistochemical diagnosis of systemic bovine aspergillosis

    DEFF Research Database (Denmark)

    Jensen, H.E.; Aalbaek, B.; Lind, Peter


    Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were in.......e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle....

  6. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando


    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  7. Pharmacokinetics of indium-111-labeled antimyosin monoclonal antibody in murine experimental viral myocarditis

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, T.; Matsumori, A.; Watanabe, Y.; Tamaki, N.; Yonekura, Y.; Endo, K.; Konishi, J.; Kawai, C. (Kyoto Univ. (Japan))


    The pharmacokinetics of indium-111-labeled antimyosin monoclonal antibody Fab were investigated with use of murine experimental viral myocarditis as a model. The biodistribution of indium-111-labeled antimyosin antibody Fab on days 3, 5, 7, 14, 21 and 28 after encephalomyocarditis virus inoculation demonstrated that myocardial uptake increased significantly on days 5, 7 and 14 (maximum on day 7) in infected versus uninfected mice (p less than 0.001). In vivo kinetics in infected mice on day 7 demonstrated that the heart to blood ratio reached a maximum 48 h after the intravenous administration of indium-111-labeled antimyosin Fab, which was considered to be the optimal time for scintigraphy. The scintigraphic images obtained with indium-111-labeled antimyosin Fab demonstrated positive uptake in the cardiac lesion in infected mice. The pathologic study demonstrated that myocardial uptake correlated well with pathologic grades of myocardial necrosis. High performance liquid chromatography revealed the presence of an antigen-antibody complex in the circulation of infected mice after the injection of indium-111-labeled antimyosin Fab. This antigen bound to indium-111-labeled antimyosin Fab in the circulation might be whole myosin and this complex may decrease myocardial uptake and increase liver uptake. It is concluded that indium-111-labeled antimyosin monoclonal antibody Fab accumulates selectively in damaged heart tissue in mice with acute myocarditis and that indium-111-labeled antimyosin Fab scintigraphy may be a useful method for the visualization of acute myocarditis.

  8. Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity.

    Directory of Open Access Journals (Sweden)

    Dorota Smolarek

    Full Text Available Duffy Antigen Receptor for Chemokines (DARC plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1, but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.

  9. Mapping of Lol p I allergenic epitopes by using murine monoclonal antibodies. (United States)

    Mourad, W; Bernier, D; Jobin, M; Hébert, J


    Murine monoclonal antibodies (MAbs) against three non-overlapping epitopes of Lol p I allergen were previously produced and subsequently used for purification of the allergen. In the present study, these MAbs were further characterized, and the biological activity of the purified allergen assessed. The three MAbs were of the IgG isotype and carried a kappa light chain. Their affinity constants were in the range of 7.4-15.1 x 10(-9) mol/l. Purified Lol p I kept its biological activity, as shown by its ability to induce histamine release by basophils of Lol p I-sensitive patients. The profiles of histamine release induced by either Lol p I or crude Lolium perenne extracts were comparable. This observation suggests that human IgE bound to basophils are polyspecific which has been confirmed by immunoblot and inhibition assay. Our data indicated also that Lol p I possesses a major allergenic epitope recognized by all human serum IgE tested. This epitope seems to be partially shared by those recognized by the three MAbs. Finally, preincubation of Lol p I with either one of the Mabs did not affect significantly the basophil-histamine release induced by the purified allergen. This suggests that Lol p I possesses allergenic sites other than the one shared by MAbs and IgE Abs.

  10. Cytotoxic murine monoclonal antibody LAM8 with specificity for human small cell carcinoma of the lung. (United States)

    Stahel, R A; O'Hara, C J; Mabry, M; Waibel, R; Sabbath, K; Speak, J A; Bernal, S D


    The reactivity of the murine immunoglobulin monoclonal antibody LAM8 directed against a membrane antigen of human small cell carcinoma (SCC) of the lung was investigated on human cell lines and tissues. Indirect immunofluorescence staining, radioimmunoassays, and cytotoxicity assays showed LAM8 antibody to selectively react with SCC but not with non-SCC lung cancer cell lines and extrapulmonary tumor cell lines. Unlike other SCC antibodies, including those we have previously described, highly preferential reactivity with SCC tissues was also demonstrated by immunoperoxidase staining of deparaffinized formalin-fixed tissue sections. Membrane and cytoplasmic staining was seen in of 9 of 12 SCC tissues. No significant staining was seen in non-SCC lung cancer and a wide range of other tumors, including mesothelioma and bronchial carcinoids. Significant LAM8 reactivity was also absent in normal tissues of all major organs. Few tumors and epithelial tissues, including bronchial epithelium had rare LAM8 positive cells which were always less than 2% of the entire cell population. In vitro treatment with antibody and human complement was highly cytotoxic to SCC cells, but had not effect on bone marrow progenitor cells. Immunoblotting of membrane extracts separated on sodium dodecyl sulfate-polyacrylamide gels showed the LAM8 antigen to have a band of an approximate molecular weight of 135,000 and a cluster of bands with approximate molecular weights of 90,000. This reactivity was lost after incubation of the extracts with periodate. LAM8 antibody shows a highly preferential reactivity with SCC cell lines and formalin-fixed paraffin-embedded SCC tissues and is selectively cytotoxic to cells expressing LAM8 antigen.

  11. Detection of Neospora caninum in tissue sections using a murine monoclonal antibody. (United States)

    Cole, R A; Lindsay, D S; Dubey, J P; Blagburn, B L


    A murine monoclonal antibody (MAb 6G7), isotype IgG2a, produced against tachyzoites of Neospora caninum (isolate NC-1) reacted specifically with tachyzoites of N. caninum in an indirect immunofluorescent antibody test. MAb 6G7 did not react with tachyzoites of Toxoplasma gondii, sporozoites of Isospora suis, Eimeria bovis, or E. tenella, or merozoites of E. bovis in the indirect immunofluorescent antibody test. MAb 6G7 reacted positively with both tachyzoites and bradyzoites of N. caninum in an avidin-biotin peroxidase complex immunohistochemical test on formalin-fixed paraffin-embedded tissues. No reaction was observed with the following: tachyzoites and bradyzoites of T. gondii, T. gondii-like parasites, or Hammondia hammondi; bradyzoites of Frenkelia microti; schizonts and merozoites of Sarcocystis-like organisms; schizonts, sarcocysts, and oocysts/sporocysts of S. cruzi; schizonts and merozoites of S. canis; schizonts of S. hirsuta, S. tenella, and S. capracanis; merozoites of S. neurona and S. neurona-like organisms, E. bovis, or Haemoproteus sp.; bradyzoites and merozoites of S. montanaensis; bradyzoites of S. odocoileocanis, S. cruzi, and S. tenella; meronts, sexual stages, and caryocysts of Caryospora sp. and C. bigenetica; micromerozoites, macromerozoites, and schizonts of Hepatozoon canis; sporozoites, sexual stages, and oocysts of Cryptosporidium parvum and C. baileyi; trophozoites of Monocystis lumbrici, Tritrichomonas foetus, and Balantidium coli; tissue cysts and bradyzoites of Besnoitia sp. and B. jellisoni; amastigotes of Leishmania sp.; and trophic theronts of Ichthyopthirius multifilis. MAb 6G7 reacted with tachyzoites and bradyzoites of N. caninum in natural and experimental infections in dogs, cattle, mice, rats, sheep, and goats, indicating that host origin of the tissues did not affect the performance of the test.

  12. Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G. (United States)

    Schenk, Jörg A; Fettke, Joerg; Lenz, Christine; Albers, Katharina; Mallwitz, Frank; Gajovic-Eichelmann, Nenad; Ehrentreich-Förster, Eva; Kusch, Emely; Sellrie, Frank


    The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.

  13. High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler. (United States)

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


    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.

  14. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

    DEFF Research Database (Denmark)

    Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom


    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovar...... be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics....

  15. Generation of a murine monoclonal antibody to capsular polysaccharide Vi from Salmonella Typhi

    Directory of Open Access Journals (Sweden)

    Fátima Reyes-López


    Full Text Available The conventional hybridoma technology has enabled the development of monoclonal antibodies (Mabs against many antigens. Mabs have several applications in the field of basic research, diagnosis, immunotherapy and vaccine manufacturing processes. Mabs-producing hybridomas against the capsular polysaccharide from Salmonella Typhi were obtained, after intraperitoneal immunization of BALB/c mice with 10 µg of capsular polysaccharide Vi conjugated to diphtheria toxoid, and subsequent fusion of lymphocytes isolated of the spleen and myeloma cells SP2/O. A Mab was selected, partially characterized, and named as 4G3E11. The isotype of this Mab was IgG1. It was proved by means of a sandwich ELISA that the 4G3E11 Mab reacts with different concentrations of polysaccharide in samples of the vax-TyVi® vaccine. The Mab obtained in this research could be useful as reagent for the detection and quantitation of polysaccharide Vi in typhoid vaccines.

  16. Anti-IL-20 Monoclonal Antibody Suppresses Prostate Cancer Growth and Bone Osteolysis in Murine Models.

    Directory of Open Access Journals (Sweden)

    Yu-Hsiang Hsu

    Full Text Available Interleukin (IL-20 is a proinflammatory cytokine in the IL-10 family. IL-20 is associated with tumor promotion in the pathogenesis of oral, bladder, and breast cancer. However, little is known about the role of IL-20 in prostate cancer. We hypothesize that IL-20 promotes the growth of prostate cancer cells. Immunohistochemical staining showed that IL-20 and its receptors were expressed in human PC-3 and LNCaP prostate cancer cell lines and in prostate tumor tissue from 40 patients. In vitro, IL-20 upregulated N-cadherin, STAT3, vimentin, fibronectin, RANKL, cathepsin G, and cathepsin K, and increased the migration and colony formation of prostate cancer cells via activated p38, ERK1/2, AKT, and NF-κB signals in PC-3 cells. We investigated the effects of anti-IL-20 monoclonal antibody 7E on prostate tumor growth in vivo using SCID mouse subcutaneous and intratibial xenograft tumor models. In vivo, 7E reduced tumor growth, suppressed tumor-mediated osteolysis, and protected bone mineral density after intratibial injection of prostate cancer cells. We conclude that IL-20 is involved in the cell migration, colony formation, and tumor-induced osteolysis of prostate cancer. Therefore, IL-20 might be a novel target for treating prostate cancer.

  17. Localization of radiolabeled anti-DNA monoclonal antibodies in murine systemic lupus erythematosus (SLE)

    Energy Technology Data Exchange (ETDEWEB)

    Wahl, R.; Hahn, B.; Ebling, F.


    The diagnosis of SLE can be extremely difficult. This multi-system disease is characterized by the deposition of DNA-anti-DNA antibody (Ab) complexes in many tissues, producing glomerulonephritis and systemic vasculitis. This study evaluates an IGG monoclonal (Mo) Ab directe3d against DNA (MrSSl) for potential radioimmunodiagnosis of SLE. Six 15 wk. old F-1 female hybrids of NZB+NZW mice (an animal SLE model that develops vasculitis and nephritis) were injected with 50 of I-131 MrSSl and 15 of I-125 isotype-matched control mouse myeloma (LPC-1) (non-reactive with DNA). Imaging and tissue distribution were studied. Two animals were also imaged using I-131 LPC Ab. Images at 2 and 9 days showed no clear differences in scan patterns using MrSSl or LPC-1 Ab. Tissue distribution studies at six days, however, showed a significantly higher accumulation of MrSSl in the kidneys vs. control Ab (2.7% vs. 1.8% of injected dose) (p < .04). Similarly, higher levels of MrSS were also seen in the spleen, liver and lungs (p < .03). Blood levels tended to be higher with the specific antibody as well. These differences were not apparent at 3 days post injection. The increased concentration of MrSSl present at 9 days in several organs may be secondary to MrSSl binding to DNA containing immune complexes present in diseased tissues. Blocked clearance by immune complexes or DNA, or differences in electrical charges of the antibodies could be contributing to the higher MrSSl levels seen. Images did not suggest deiodination as responsible. Further studies are necessary to determine if the amount of MrSSl retained by diseased animals is indicative of SLE disease activity.

  18. Role of neutralizing anti-murine interleukin-17A monoclonal antibody on chronic ozone-induced airway inflammation in mice. (United States)

    Zhang, Min; Fei, Xia; Zhang, Guo-Qing; Zhang, Peng-Yu; Li, Feng; Bao, Wu-Ping; Zhang, Ying-Ying; Zhou, Xin


    Exposure to ozone has led to airway inflammation and airway hyperresponsiveness, which potential mechanisms relate to ozone-induced oxidative stress. IL-17 is a growing target for autoimmune and inflammatory diseases. The aim of the study was to examine the inhibitory effects of anti-murine interleukin-17A monoclonal antibody (IL-17mAb) on adverse effects of ozone which are noted above. After C57/BL6 mice were exposed to ozone (2.5ppm; 3h) for 12 times over 6 weeks, IL-17mAb, PBS was intraperitoneally injected into mice 1h after ozone or air exposure for 6 weeks and mice were studied 24h after final exposure, monitoring bronchial responsiveness, airway inflammatory cells, lung histology, levels of neutrophil-related chemokine and proinflammatory cytokines in bronchoalveolar lavage (BAL) fluid and serum, the expression of IL-17A mRNA and protein, glucocorticoid receptors (GR), and the phosphorylation of p38MAPK in lung tissues. The administration of IL-17mAb reduced the ozone-induced increases in total cells, especially neutrophils; decreased levels of cytokines, including IL-8 in BAL fluid, IL-8 and IL-17A in serum; mitigated the severity of airway hyperresponsiveness; attenuated lung inflammation scores and histologic analysis confirmed the suppression of lung inflammation, compared with the administration of a control PBS. Exposure to ozone results in increases in IL-17A production rate, mRNA and protein levels of IL-17A and the protein level of GR. These effects were halted and reversed by IL-17mAb treatment. Furthermore, IL-17mAb also reduced the phosphorylation of p38MAPK. Therefore, we conclude that IL-17mAb may be a useful therapy in ozone-related diseases, including COPD.

  19. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    Energy Technology Data Exchange (ETDEWEB)

    Bujak, Emil [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland); Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah [Philochem AG, Libernstrasse 3, CH-8112 Otelfingen (Switzerland); Neri, Dario, E-mail: [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland)


    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  20. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b and Its Humanized Version Rebmab200.

    Directory of Open Access Journals (Sweden)

    Sture Lindegren

    Full Text Available The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.


    Institute of Scientific and Technical Information of China (English)

    邹颖; 卞美璐; 杨子义; 连利娟; 刘文淑; 许秀英


    In the present study,four different proteases (pepsin,papain,bromelain and ficin) were screened with a murine monoclonal antibody OC859,in order to verify whether different digestion procedures could improve yield and stability of the F(ab')2 or Fab fragments.The yields of F(ab')2 or Fab fragments from digestion with pepsin,papain,bromelain and ficin were respectively 20.3+/-2.0%,50.5%+/-5.0%,74.4+/-2.7% and 82.8+/-10.2% of the theoretical maximum.Immunoreactivity in a noncompetitive solid-phase radioimmunoassay (SPRIA) of the fragments generated by the four proteases were respectively 10+/-5%,36+/-5%,60+/-6% and 75+/-6% of the intact OC859 IgG.These results suggested that the fragmentation of OC859 with ficin gave a higher yield of superior immunoreactive fragments.

  2. 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. (United States)

    Nakajima, Tamiko; Yasuda, Toshihiro; Takeshita, Haruo; Mori, Shinjiro; Mogi, Kouichi; Kaneko, Yasushi; Nakazato, Emiko; Kishi, Koichiro


    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.

  3. ING-1(heMAb, a Monoclonal Antibody to Epithelial Cell Adhesion Molecule, Inhibits Tumor Metastases in a Murine Cancer Model

    Directory of Open Access Journals (Sweden)

    Harry H. Ruan


    Full Text Available ING-1(heMAb, a human-engineered monoclonal antibody (MAb that specifically targets the epithelial cell adhesion molecule (Ep-CAM, kills adenocarcinoma cells in vitro and inhibits tumor growth in vivo. In the current study, we evaluated the efficacy of ING-1(heMAb in a murine model of cancer metastases. Mice received intravenous dosing of 1 mg/kg ING-1(heMAb, twice a week, starting on day 2 or day 5. A negative control group received 1 mg/kg human immunoglobulin G with the same dose frequency starting on day 2. A positive control group received weekly 100 mg/kg 54lurouracil/leucovorin starting on day 2. ING-1(heMAb/day 2 treatment significantly reduced both the number of visible tumor nodules in body cavities (P < .01 and the number of metastases on lung surfaces (P < .005. The treatment also resulted in a 91% reduction of micrometastases in lung tissues (P <.0001. Delaying ING-1(heMAb treatment until day 5 caused 54% reduction in micrometastases (P <.005. Our results indicate that a number of parameters, including treatment starting day, dose level, and dose frequency, are critical in achieving the optimal efficacy of ING-1(heMAb. We conclude that ING-1(heMAb effectively reduced tumor metastases in a murine cancer model. Immunotherapy with ING-1(heMAb may be beneficial in treating human metastatic diseases.

  4. Development and characterization of murine monoclonal antibody specific for the P1.4 PorA proteins from strain B:4:P1.(7b.4. of Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    María Elena Pérez


    Full Text Available Neisseria meningitidis isolates are conventionally classified by serosubtyping that characterizes the reactivities of the PorA outer membrane protein variable-region epitopes with monoclonal antibodies. Porins are outer membrane proteins (OMPs of N. meningitidis serogroup B and have attracted study principally for two reasons: their use in the classification of meningococcal isolates into serotype and subtype and as potential components of vaccines against this important pathogen. New murine hybridomas, secreting specific monoclonal antibodies against PorA serotype P1.4 of N. meningitidis serogroup B, were generated using conventional hybridoma procedures. The monoclonal antibodies obtained were characterized by Western blot and whole cell ELISA, using reference strains from different N. meningitidis serotypes and subtypes. All monoclonal antibodies belong to isotype IgG1. Others hybridomas producing MAbs against PorB and FrpB were also obtained.

  5. A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4. (United States)

    Sangawa, Takeshi; Nogi, Terukazu; Takagi, Junichi


    Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.

  6. Characterization of periplasmic protein BP26 epitopes of Brucella melitensis reacting with murine monoclonal and sheep antibodies. (United States)

    Qiu, Jinlang; Wang, Wenjing; Wu, Jingbo; Zhang, Hui; Wang, Yuanzhi; Qiao, Jun; Chen, Chuangfu; Gao, Goege F; Allain, Jean-Pierre; Li, Chengyao


    More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues ⁹³DRDLQTGGI¹⁰¹ (position 93 to 101) or residues ¹⁰⁴QPIYVYPD¹¹¹, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65-70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.

  7. Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia. (United States)

    Asada, Reiko; Wanaka, Keiko; Walenga, Jeanine; Prechel, Margaret; Miyashita, Kumiko; Escalante, Vicki; Kaneko, Chieko; Hoshino, Nobuhiro; Oosawa, Mitsuru; Matsuo, Miyako


    Quality control of the platelet activation assays to diagnose heparin-induced thrombocytopenia (HIT), (14)C-serotonin release assay (SRA) and platelet aggregation test (PAT) has yet to be established due to lack of reference standards and the difficulty of obtaining significant amounts of HIT antibodies from patients with HIT. We prepared a murine monoclonal antibody to human platelet factor 4 (hPF4)/heparin complexes (HIT-MoAb) and investigated the platelet activating action of HIT-MoAb by using SRA and PAT. The HIT-MoAb activated human platelets at low heparin concentration and the platelet activations were inhibited at high heparin concentration in both SRA and PAT. The HIT-MoAb produced a concentration-dependent effect. Moreover, the platelet activation at low heparin concentration was inhibited by anti-FcγRIIa antibody. These results indicated that HIT-MoAb has characteristics similar to human HIT antibodies regarding heparin-dependent platelet activation. Therefore, it is suggested that HIT-MoAb has the potential to be a positive control or reference standard in platelet activation assays.

  8. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay. (United States)

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K


    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

  9. Analysis of two monoclonal antibodies reactive with envelope proteins of murine retroviruses: one pan specific antibody and one specific for Moloney leukemia virus. (United States)

    Evans, Leonard H; Boi, Stefano; Malik, Frank; Wehrly, Kathy; Peterson, Karin E; Chesebro, Bruce


    Many monoclonal antibodies (MAbs) reactive with various proteins of murine leukemia viruses (MuLVs) have been developed. In this report two additional MAbs with differing and unusual specificities are described. MAb 573 is reactive with the envelope protein of all MuLVs tested including viruses in the ecotropic, xenotropic, polytropic and amphotropic classes. Notably, MAb 573 is one of only two reported MAbs that react with the envelope protein of amphotropic MuLVs. This MAb appears to recognize a conformational epitope within the envelope protein, as it reacts strongly with live virus and live infected cells, but does not react with formalin-fixed or alcohol-fixed infected cells or denatured viral envelope protein in immunoblots. In contrast, Mab 538 reacts only with an epitope unique to the envelope protein of the Moloney (Mo-) strain of MuLV, a prototypic ecotropic MuLV that is the basis for many retroviral tools used in molecular biology. MAb 538 can react with live cells and viruses, or detergent denatured or fixed envelope protein. The derivation of these antibodies as well as their characterization with regard to their isotype, range of reactivity with different MuLVs and utility in different immunological procedures are described in this study.

  10. The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells. (United States)

    Ullal, Anirudh J; Marion, Tony N; Pisetsky, David S


    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.

  11. Pretargeted Radioimmunotherapy Using Anti-CD45 Monoclonal Antibodies to Deliver Radiation to Murine Hematolymphoid Tissues and Human Myeloid Leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Pagel, John M.; Matthews, Dana C.; Kenoyer, Aimee L.; Hamlin, Donald K.; Wilbur, D. Scott; Fisher, Darrell R.; Gopal, Ajay K.; Lin, Yukang; Saganic, Laura; Appelbaum, Frederick R.; Press, Oliver W.


    The efficacy of radioimmunotherapy (RIT) for treatment of patients with hematological malignancies frequently fails because of disease recurrence. We therefore conducted pretargeted RIT studies to augment the efficacy in mice of therapy using a pretargeted anti-human (h)CD45 antibody (Ab)-streptavidin (SA) conjugate followed by delivery of a biotinylated clearing agent and radiolabeled-DOTA-biotin. Tumor-to-blood ratios at 24 hours were 20:1 using pretargeted anti-hCD45 RIT and <1:1 with conventional RIT. In vivo imaging studies confirmed that the pretargeted RIT approach provided high-contrast tumor images with minimal blood-pool activity, whereas directly-labeled anti-hCD45 Ab produced distinct tumor images but the blood pool retained a large amount of labeled antibody for a prolonged time. Therapy experiments demonstrated that 90Y-DOTA-biotin significantly prolonged survival of mice treated pretargeted with anti-hCD45 Ab-SA compared to mice treated with conventional RIT using 90Y-labeled anti-hCD45 Ab at the maximally tolerated dose (400 µCi). Since human CD45 antigens are confined to xenograft tumor cells in this model, and all murine tissues are devoid of hCD45 and will not bind anti-hCD45 Ab, we also compared one-step and pretargeted RIT using an anti-murine (m)CD45 Ab (A20 ) in a model where the target antigen is present on normal hematopoietic tissues. After 24 hours, 27.3 ± 2.8% of the injected dose of radionuclide was delivered per gram (% ID/g) of lymph node using 131I-A20-Ab compared with 40.0 ± 5.4% ID/g for pretargeted 111In-DOTA-biotin (p value). These data suggest that multi-step pretargeted methods for delivering RIT are superior to conventional RIT when targeting CD45 for the treatment of leukemia and may allow for the intensification of therapy, while minimizing toxicities.

  12. Evaluation of anti-IL-6 monoclonal antibody therapy using murine type II collagen-induced arthritis

    Directory of Open Access Journals (Sweden)

    Shealy David


    Full Text Available Abstract Interleukin-6 is a multifunctional cytokine that is critical for T/B-cell differentiation and maturation, immunoglobulin secretion, acute-phase protein production, and macrophage/monocyte functions. Extensive research into the biology of IL-6 has implicated IL-6 in the pathophysiology and pathogenesis of RA. An anti-murine IL-6 mAb that neutralizes mouse IL-6 activities was tested in animal model of collagen-induced arthritis. Prophylactic treatment with anti-IL-6 mAb significantly reduced the incidence and severity of arthritis compared to control mAb treated mice. The mitogenic response of B and T cells isolated from the lymph nodes of anti-IL-6 treated mice was significantly reduced compared to cells isolated from control mAb treated mice. The overall histopathology score for paws from the anti-IL-6 treated mice was significantly reduced when compared to paws from mice treated with control mAb, including both inflammatory (synovitis and pannus and erosive (erosions and architecture parameters. Reduced loss of cartilage matrix components was also observed in the anti-IL-6 treated mice. Collectively, these data suggest that IL-6 plays a major role in the pathophysiology of rheumatoid arthritis, and thus support the potential benefit of anti-IL-6 mAb treatment in rheumatoid arthritis patients.

  13. The effect of unlabelled monoclonal antibody (mAb) on the biodistribution of [sup 131]I-anti-idiotype mAb in murine B cell lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Schiele, J. (Stanford University (United States). Division of Laboratory Animal Medicine); Knox, S.J. (Stanford University Medical Center (United States). Department of Radiation Oncology); Ruhl, W. (Stanford University (United States). Division of Laboratory Animal Medicine Stanford Univ., CA (United States). Dept. of Radiology); Goris, M.L. (Stanford University Medical Center (United States). Department of Diagnostic Radiology and Nuclear Medicine)


    The 38C13 murine B cell lymphoma model was used to study the effect of the pre-injection of unlabelled anti-idiotype monoclonal antibody (mAb) on the subsequent biodistribution of [sup 131]I-anti-idiotype mAb. Mice with established tumors received 0-500 [mu]g of unlabelled anti-idiotype mAb 24 h prior to the administration of [sup 131]I-anti-idiotype (specific), or both [sup 125]I-anti-idiotype and [sup 131]I-isotype-matched irrelevant control (non-specific) mAb. Mice were counted daily in a gamma counter and sacrificed at 2-144 h following injection. Mice were dissected and the weight and activity of the animals and organs were measured. Mice were bled periodically and circulating idiotype levels were measured using an ELISA assay. 500 [mu]g of unlabelled anti-idiotype mAb increased the retention time of the specific but not the nonspecific mAb in all organs and tumor. Following pretreatment with unlabelled mAb, the cumulative tumor/whole body and tumor/normal organ ratios became similar to those of the nonspecific mAb, with concentration ratios (specific/nonspecific mAb) of approximately 1, which persisted until 96 h post injection when circulating idiotype reappears in antigen excess. In the absence of unlabelled mAb there was less retention in tumor and normal tissue. This is presumed to be due in part to decreased levels of circulating [sup 131]I-mAb secondary to rapid plasma clearance of antigen-body complexes and tumor cell mediated dehalogenation, which results when the specific mAb specifically binds the target antigen. Thus, the addition of unlabelled mAb increased the retention by decreasing the specific behavior of the anti-idiotype antibody. (author). 12 refs.; 1 fig.; 3 tabs.

  14. Characterization of the tumor marker muc16 (ca125 expressed by murine ovarian tumor cell lines and identification of a panel of cross-reactive monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Goodell Cara AR


    Full Text Available Abstract Objectives The ovarian tumor marker CA125 is expressed on human MUC16, a cell surface bound mucin that is also shed by proteolytic cleavage. Human MUC16 is overexpressed by ovarian cancer cells. MUC16 facilitates the binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity. Additionally, MUC16 also is a potent inhibitor of natural killer cell mediated anti-tumor cytotoxic responses. Extensive studies using human as well as murine ovarian tumor cell models are required to clearly define the function of MUC16 in the progression of ovarian tumors. The major objective of this study was to determine if the murine ovarian tumor cells, MOVCAR, express Muc16 and to characterize antibodies that recognize this mucin. Methods RT-PCR analysis was used for detecting the Muc16 message and size exclusion column chromatography for isolating Muc16 produced by MOVCAR cells. Soluble and cell-associated murine Muc16 were analyzed, respectively, by Western blotting and flow cytometry assays using a new panel of antibodies. The presence of N-linked oligosaccharides on murine Muc16 was determined by ConA chromatography. Results We demonstrate that murine Muc16 is expressed by mouse ovarian cancer cells as an ~250 kDa glycoprotein that carries both O-linked and N-linked oligosaccharides. In contrast to human MUC16, the murine ortholog is primarily released from the cells and cannot be detected on the cell surface. Since the released murine Muc16 is not detected by conventional anti-CA125 assays, we have for the first time identified a panel of anti-human MUC16 antibodies that also recognizes the murine counterpart. Conclusion The antibodies identified in this study can be used in future purification of murine Muc16 and exhaustive study of its properties. Furthermore, the initial identification and characterization of murine Muc16 is a vital preliminary step in the development of effective murine models of human ovarian cancer. These

  15. A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV. (United States)

    Zhou, E M; Dzuba-Fischer, J M; Rector, E S; Sehon, A H; Kisil, F T


    A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

  16. Selective cytotoxicity of murine monoclonal antibody LAM2 against human small-cell carcinoma in the presence of human complement: possible use for in vitro elimination of tumor cells from bone marrow. (United States)

    Stahel, R A; Mabry, M; Sabbath, K; Speak, J A; Bernal, S D


    LAM2 is a murine IgM monoclonal antibody (MAb) which binds strongly to the cell membrane of human lung small-cell carcinoma (SCC) and squamous-cell carcinoma but not to normal bone-marrow cells. The cytotoxicity of this antibody in the presence of human complement was investigated in vitro by chromium release and clonogenic assays. The optimal treatment conditions included incubation with antibody for 30 min at 37 degrees C followed by 3 additions of human complement 30 min apart. Cell lysis ranged from 94 to 98% in 4 SCC cell lines at antibody dilutions of 1:100: a lower level of lysis (60%) occurred in a lung squamous-cell carcinoma cell line. The cytotoxic effect was strictly complement-dependent. No cytotoxic effect was seen with other human cell lines including lung adenocarcinoma, lung large-cell carcinoma, myeloid leukemia, and lymphoblastic leukemia. No lysis was seen with nucleated marrow cells from healthy volunteers. Normal marrow cells in excess did not inhibit SCC cell lysis. Incubation with antibody and complement resulted in a 100-fold reduction of colony formation of SCC cells, but did not reduce the number of colonies of marrow-cell precursors, including CFU-GEMM, BFU-E, and CFU-C. The selective cytotoxicity of LAM2 antibody to SCC, but not to normal bone-marrow cells, suggests that this antibody may be useful for the in vitro elimination of SCC cells from the bone marrow.

  17. Heterogeneity of abnormal prion protein (PrP(Sc)) in murine scrapie prions determined by PrP(Sc)-specific monoclonal antibodies. (United States)

    Ushiki-Kaku, Yuko; Shimizu, Yoshihisa; Tabeta, Naoko; Iwamaru, Yoshifumi; Ogawa-Goto, Kiyoko; Hattori, Shunji; Yokoyama, Takashi


    In prion diseases, abnormal prion protein (PrP(Sc)) is considered as the main component of the infectious agent. Delineation of PrP(Sc) conformation is expected to be a critical factor in understanding properties of prions. However, practical methods to differentiate between conformers of PrP(Sc) are inadequate. Here, we used two PrP(Sc)-specific monoclonal antibodies (mAbs), 3B7 and 3H6, and found that mAb 3H6 detected a limited portion of PrP(Sc) in five mice-adapted prion strains. The quantity of mAb 3H6-precipitated PrP(Sc) was significantly lesser in 22L compared to other strains. This result provides a direct evidence of the conformational heterogeneity of PrP(Sc) within the prion strains. Conformation-specific probes, like these mAbs, have the potential to be powerful tools for investigating conformational variations in PrP(Sc).

  18. Optimal humanization of 1B4, an anti-CD18 murine monoclonal antibody, is achieved by correct choice of human V-region framework sequences. (United States)

    Singer, I I; Kawka, D W; DeMartino, J A; Daugherty, B L; Elliston, K O; Alves, K; Bush, B L; Cameron, P M; Cuca, G C; Davies, P


    The murine anti-CD18 mAb 1B4 has been humanized using CDR grafting. Three VH (Gal, Jon, and New) and two VL (Rei and Len) human frameworks, whose selection was based exclusively on their sequence identity with m1B4, were used to construct five human gamma 4/kappa recombinant antibodies: Gal/Rei, Gal/Len, Jon/Rei, and New/Rei, and a "hemichimeric" antibody pairing the VH of m1B4 with grafted Rei. Each of these h1B4 constructs completely inhibited the binding of m1B4 to activated human leukocytes with avidities (IC50) ranging from 1.5 to 8.0 nM, compared to 0.5 nM for m1B4. Replacement of three VH residues in the best VH framework, Gal, with the corresponding m1B4 "packing" (nonsolvent exposed) residues gave an h1B4 (mutant Gal/Rei) with the same avidity as m1B4. Avidity correlated with overall percent identity between the human and murine VH frameworks and, in particular, with conservation of "packing" residues. Rei and Len VL frameworks proved to be interchangeable. Further characterization showed that the Gal/Rei prototype was equipotent to m1B4 in blocking adhesion of polymorphonuclear leukocytes and monocytes to human vascular endothelium in vitro, and polymorphonuclear leukocyte extravasation into C5a-injected rabbit or monkey skin sites. Dual-label immunofluorescence microscopy of bone marrow cells with Gal/Rei h1B4 and m1B4 demonstrated that the fine specificity of the combining sites had not been altered by humanization. Reduced immunogenicity was demonstrated in rhesus monkeys that tolerated weekly treatment with h1B4 for 6 wk, whereas m1B4 induced profound anaphylaxis at 3 wk. Anti-1B4 titers in h1B4-treated rhesus were 50 to 66% lower and developed 1 wk later than in m1B4-treated monkeys. Crucially, the anti-h1B4 antibodies were anti-idiotypic while the anti-m1B4 antibodies were directed against constant and framework regions. We conclude that sequence identity searches are sufficient to identify suitable human frameworks for CDR-grafting of m1B4

  19. Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette--Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity. (United States)

    Newton, M R; Askeland, E J; Andresen, E D; Chehval, V A; Wang, X; Askeland, R W; O'Donnell, M A; Luo, Y


    Effective treatment of bladder cancer with bacillus Calmette-Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients.

  20. Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette–Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity (United States)

    Newton, M R; Askeland, E J; Andresen, E D; Chehval, V A; Wang, X; Askeland, R W; O'Donnell, M A; Luo, Y


    Effective treatment of bladder cancer with bacillus Calmette–Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients. PMID:24593764

  1. Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea Attainment and characterization of murine monoclonal anti-factor VIII antibodies

    Directory of Open Access Journals (Sweden)

    Rosana Rossi-Ferreira


    Full Text Available Entre os avanços da engenharia celular e biotecnologia nas últimas décadas, destaca-se a produção de anticorpos monoclonais murinos (AcMm utilizados no aprimoramento diagnóstico nas rotinas laboratoriais. A produção de fator VIII de alta pureza sempre foi o desejo e a preocupação das indústrias de hemoderivados para tratamento de pacientes portadores de hemofilia A, porém este produto inexiste no Brasil, sendo necessária sua obtenção no mercado internacional a custos elevados. O trabalho tem por objetivo a produção de AcMm anti-fator VIII humano (FVIII H através da expansão dos clones e caracterização imunoquímica do anticorpo. Camundongos Balb/c foram imunizados com FVIII H purificado como também proveniente de crioprecipitado e as células esplênicas dos animais foram fusionadas com células mielomatosas murinas segundo o método descrito por Kohler e Milstein para produção de híbridos em cultura. Foram testados 1.983 híbridos dos quais 105 foram submetidos à clonagem. Destes, 39 obtiveram monoclonalidade e 7 destes clones foram caracterizados através de técnicas de immunoblotting. Foram submetidas à purificação por cromatografia três imunoglobulinas de diferentes classes pertencentes aos clones LAMB1-10A1A4, LAMB1-17A1A1 e LAMB1-24A2A1. A imunoglobulina purificada pertencente ao clone LAMB1-10A1A4 foi adsorvida em coluna de imunoafinidade para purificação de concentrado de FVIII proveniente de crioprecipitado plasmático.Among the advances in cellular engineering and biotechnology over the last decades, the production of murine monoclonal antibodies (AcMm, used to improve laboratory diagnoses, stands out. The production of very pure factor VIII has always been a concern of suppliers of blood products to treat patients with hemophilia A and this product is still not produced in Brazil. Hence, it can only be attained on the international market at a high cost. The aim of this work was to produce AcMm anti

  2. 登革病毒EDⅢ特异性单抗的体内外中和活性观察%A murine monoclone antibody against envelope protein domain Ⅲ neutralizes dengue virus four serotypes in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    黄艳芬; 邓永强; 丘立文; 刘利东; 秦鄂德; 车小燕; 秦成峰


    目的 鉴定1株能同时识别登革病毒Ⅰ~Ⅳ型(dengue virus serotypesⅠ-Ⅳ,DENV 1~4)的EDⅢ特异性单抗2D73的体内外中和活性.方法 采用间接酶联免疫吸附实验(ELISA)和间接免疫荧光法(IFA)对单抗2D73的特性进行鉴定,并用基于酶联免疫斑点法的微中和实验(ELISPOT-MNT)和乳鼠保护实验观察单抗2D73对DENV 1~4的体内外中和活性.结果 单抗2D73对4型DENV均具有较强的中和活性,其50%抑制浓度(IC50)分别为0.28、0.16、0.18和18.82 μg/ml.乳鼠保护实验显示,该抗体对DENV 1~4同样具有较好的体内保护效果,与对照组相比,实验组小鼠发病时间明显延迟,生存率显著提高(P<0.05).结论 获得1株同时针对DENV 1~4且具有体内外中和活性的单抗,该单抗可进一步用于DENV致病和免疫机制的研究以及抗病毒药物的研制.%Objective To evaluate the neutralizing activities of a murine monoclonal antibody ( mAb) 2D73 against dengue virus serotypes Ⅰ-Ⅳ( DENV1-4) in vitro and in vivo. Methods Indirect ELISA and immuno fluorescence assay (IFA) were applied to identify specificity of mAb 2D73. Enzyme-linked immunospot micro -neutralizing test (ELISPOT-MNT) was performed to identify its neutralizing activities in vitro, and protective capacity was evaluated in a stringent in -tracranial challenge suckling mice model. Results All the four DENV serotypes were strongly neutralized by mAb 2D73 with 50% inhibitory concentrations (IC50) of 0. 28 , 0. 16 , 0. 18 and 18. 82 μg/ml to DENV1-4 , respectively. The onset of pathogenesis was delayed and the survival rate was improved obviously (P < 0. 05 ) in experimental group compared with control group. Conclusion MAb 2D73 against all the four DENV serotypes has strong neutralizing activities in vitro and protective efficacy in vivo, and might be used for the study of the pathogenic and immunologic mechanism of DENV and for the development of antiviral therapy.

  3. Therapeutic Recombinant Monoclonal Antibodies (United States)

    Bakhtiar, Ray


    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  4. Murine Typhus (United States)

    Dzul-Rosado, Karla R; Zavala Velázquez, Jorge Ernesto; Zavala-Castro, Jorge


    Rickettsia typhi: is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against Rickettsia typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of R. typhi are rats (some species belonging the Rattus Genus) and fleas (Xenopsylla cheopis) are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi. PMID:24893060

  5. Monoclonal antibody "gold rush". (United States)

    Maggon, Krishan


    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.

  6. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.;


    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  7. Apoptosis and the thymic microenvironment in murine lupus. (United States)

    Takeoka, Y; Taguchi, N; Shultz, L; Boyd, R L; Naiki, M; Ansari, A A; Gershwin, M E


    The thymus of New Zealand black (NZB) mice undergoes premature involution. In addition, cultured thymic epithelial cells from NZB mice undergo accelerated preprogrammed degeneration. NZB mice also have distinctive and well-defined abnormalities of thymic architecture involving stromal cells, defined by staining with monoclonal antibodies specific for the thymic microenvironment. We took advantage of these findings, as well as our large panel of monoclonal antibodies which recognize thymic stroma, to study the induction of apoptosis in the thymus of murine lupus and including changes of epithelial architecture. We studied NZB, MRL/lpr, BXSB/Yaa, C3H/gld mice and BALB/c and C57BL/6 as control mice. Apoptosis was studied both at basal levels and following induction with either dexamethasone or lipopolysaccharide (LPS). The apoptotic cells were primarily found in the thymic cortex, and the frequency of apoptosis in murine lupus was less than 20% of controls. Moreover, all strains of murine lupus had severe abnormalities of the cortical network. These changes were not accentuated by dexamethasone treatment in cultured thymocytes. However, the thymus in murine lupus was less susceptible to LPS-induced apoptosis than control mice. Finally we note that the number of thymic nurse cells (TNC) was lowest in NZB mice. Our findings demonstrate significant abnormalities in the induction of apoptosis and the formation of TNC-like epithelial cells in SLE mice, and suggest that the abnormalities of the thymic microenvironment have an important role in the pathogenesis of murine lupus.

  8. 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.


    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

  9. Monoclonal IgA Antibodies for Aflatoxin Immunoassays



    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible ...

  10. Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Claësson, M H


    (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...... the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native...

  11. Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

    Directory of Open Access Journals (Sweden)

    Michael H Matho


    Full Text Available Vaccinia virus A33 is an extracellular enveloped virus (EEV-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

  12. Use of AN Eosinophil Specific Monoclonal Antibody in Assessing Eosinophil Function. (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

  13. Preliminary characterisation of Toxoplasma gondii isolates from Zimbabwe, with stage-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Hove, T.; Lind, Peter; Mukaratirwa, S.


    Cell-culture-derived clones of eight Toxoplasma gondii isolates from Zimbabwe were characterised in IFAT with a panel of five monoclonal antibodies (mAb). Each clone had been established from a single murine brain cyst. The antibodies were bradyzoite-specific (4.3), tachyzoite-specific (4.25, 5...... in the IFAT in a similar way to the Danish reference strain of T. gondii, SSI-119....

  14. Mammalian tissue distribution of a large heparan sulfate proteoglycan detected by monoclonal antibodies

    DEFF Research Database (Denmark)

    Couchman, J R; Ljubimov, A V


    A panel of nine monoclonal antibodies has been characterized, all of which have reactivity with the core protein of a large heparan sulfate proteoglycan derived from the murine EHS tumor matrix. These rat monoclonal antibodies stained mouse basement membranes intensely, including those of all...... muscle, endothelia, peripheral nerve fibers and epithelia so far examined. In addition, two of the monoclonal antibodies show cross-species reactivity, staining bovine and human basement membranes, and immunoprecipitating proteoglycans from human endothelial cell cultures. These antibodies do not......, however, cross-react with avian tissues. These results show the ubiquitous distribution of a heparan sulfate proteoglycan in mammalian tissues, which will be useful in vitro and in vivo for studies on the biology of basement membrane proteoglycans and investigations of possible roles of these molecules...

  15. Detection of Campylobacter species using monoclonal antibodies (United States)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.


    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  16. Uses of monoclonal antibody 8H9 (United States)

    Cheung, Nai-Kong V.


    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  17. Partial analysis of the flagellar antigenic determinant recognized by a monoclonal antibody to Clostridium tyrobutyricum. (United States)

    Bédouet, L; Arnold, F; Robreau, G; Batina, P; Talbot, F; Malcoste, R


    In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D-glucose and N-acetyl-glucosamine in a molar ration of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D-glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The over-all stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain alpha (1-->4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.

  18. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells (United States)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.


    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  19. Comparison of the inhibition of urokinase-type plasminogen activator (u-PA) activity by monoclonal antibodies specific for u-PA as assessed by different assays

    NARCIS (Netherlands)

    Boheemen, P.A. van; Hoogen, N.M. van den; Koolwijk, N.


    Six murine monoclonal antibodies (MAbs) specific for urokinase-type plasminogen activator (u-PA) were tested for their ability to inhibit u-PA activity in three different assays with respect to amidolytic activity, plasminogen activation and fibrinolytic activity. Two of the MAbs were able to inhibi

  20. 19-DEJ-1, a hemidesmosome-anchoring filament complex-associated monoclonal antibody. Definition of a new skin basement membrane antigenic defect in junctional and dystrophic epidermolysis bullosa

    DEFF Research Database (Denmark)

    Fine, J D; Horiguchi, Y; Couchman, J R


    A murine monoclonal antibody (19-DEJ-1) was recently produced that recognizes a unique antigenic epitope of human skin basement membrane localized to the midlamina lucida exclusively in those areas bordered by overlying hemidesmosomes. To determine whether the antigen defined by 19-DEJ-1 is norma...

  1. Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody. (United States)

    Vázquez, A M; Pérez, A; Hernández, A M; Macías, A; Alfonso, M; Bombino, G; Pérez, R


    An IgM monoclonal antibody (MAb), named P3, has the characteristic to react specifically with a broad battery of N-glycolyl containing-gangliosides and with antigens expressed on breast tumors. When this MAb was administered alone in syngeneic mice, an specific IgG anti-idiotypic antibody (Ab2) response was induced, this Ab2 response was increased when P3 MAb was injected coupled to a carrier protein and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments, using the murine myeloma cell line P3-X63-Ag8.653 as fusion partner. Five Ab2 MAbs specific to P3 MAb were selected. These IgG1 Ab2 MAbs were able to block the binding of P3 MAb to GM3(NeuGc) ganglioside and to a human breast carcinoma cell line. Cross-blocking experiments demonstrated that these Ab2 MAbs are recognizing the same or very close sites on the Abl MAb. The five Ab2 MAbs were injected into syngeneic mice and four of them produced strong anti-anti-idiotypic antibody (Ab3) response. While these Ab2 MAbs were unable to generate Ab3 antibodies with the same antigenic specificity than P3 MAb, three of them induced antibodies bearing P3 MAb idiotopes (Ag-Id+ Ab3). These results demonstrated that these Ab2 MAbs are not "internal image" antibodies, but they could define "regulatory idiotopes."

  2. Monoclonal IgA Antibodies for Aflatoxin Immunoassays (United States)

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma


    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

  3. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    Directory of Open Access Journals (Sweden)

    Özlem Ertekin


    Full Text Available Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA isotype with a strong binding affinity to aflatoxin B1 (AFB1, aflatoxin B2 (AFB2, aflatoxin G1 (AFG1, aflatoxin G2 (AFG2 and aflatoxin M1 (AFM1. The antibody was effectively used in immunoaffinity column (IAC and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31. The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort.

  4. Monoclonal IgA Antibodies for Aflatoxin Immunoassays. (United States)

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma


    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2-50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort.

  5. Advances in monoclonal antibody application in myocarditis

    Institute of Scientific and Technical Information of China (English)

    Li-na HAN; Shuang HE; Yu-tang WANG; Li-ming YANG; Si-yu LIU; Ting ZHANG


    Monoclonal antibodies have become a part of daily preparation technologies in many laboratories.Attempts have been made to apply monoclonal antibodies to open a new train of thought for clinical treatments of autoimmune diseases,inflammatory diseases,cancer,and other immune-associated diseases.This paper is a prospective review to anticipate that monoclonal antibody application in the treatment of myocarditis,an inflammatory disease of the heart,could be a novel approach in the future.In order to better understand the current state of the art in monoclonal antibody techniques and advance applications in myocarditis,we,through a significant amount of literature research both domestic and abroad,developed a systematic elaboration of monoclonal antibodies,pathogenesis of myocarditis,and application of monoclonal antibodies in myocarditis.This paper presents review of the literature of some therapeutic aspects of monoclonal antibodies in myocarditis and dilated cardiomyopathy to demonstrate the advance of monoclonal antibody application in myocarditis and a strong anticipation that monoclonal antibody application may supply an effective therapeutic approach to relieve the severity of myocarditis in the future.Under conventional therapy,myocarditis is typically associated with congestive heart failure as a progressive outcome,indicating the need for alternative therapeutic strategies to improve long-term results.Reviewing some therapeutic aspects of monoclonal antibodies in myocarditis,we recently found that monoclonal antibodies with high purity and strong specificity can accurately act on target and achieve definite progress in the treatment of viral myocarditis in rat model and may meet the need above.However,several issues remain.The technology on howto make a higher homologous and weak immunogenic humanized or human source antibody and the treatment mechanism of monoclonal antibodies may provide solutions for these open issues.If we are to further stimulate

  6. Development of a novel monoclonal antibody with reactivity to a wide range of Venezuelan equine encephalitis virus strains

    Directory of Open Access Journals (Sweden)

    Phelps Amanda L


    Full Text Available Abstract Background There is currently a requirement for antiviral therapies capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV, as a licensed vaccine is not available for general human use. Monoclonal antibodies are increasingly being developed as therapeutics and are potential treatments for VEEV as they have been shown to be protective in the mouse model of disease. However, to be truly effective, the antibody should recognise multiple strains of VEEV and broadly reactive monoclonal antibodies are rarely and only coincidentally isolated using classical hybridoma technology. Results In this work, methods were developed to reliably derive broadly reactive murine antibodies. A phage library was created that expressed single chain variable fragments (scFv isolated from mice immunised with multiple strains of VEEV. A broadly reactive scFv was identified and incorporated into a murine IgG2a framework. This novel antibody retained the broad reactivity exhibited by the scFv but did not possess virus neutralising activity. However, the antibody was still able to protect mice against VEEV disease induced by strain TrD when administered 24 h prior to challenge. Conclusion A monoclonal antibody possessing reactivity to a wide range of VEEV strains may be of benefit as a generic antiviral therapy. However, humanisation of the murine antibody will be required before it can be tested in humans. Crown Copyright © 2009

  7. Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo

    Directory of Open Access Journals (Sweden)

    Laura A. Novotny


    Full Text Available The vast majority of chronic and recurrent bacterial diseases are attributed to the presence of a recalcitrant biofilm that contributes significantly to pathogenesis. As such, these diseases will require an innovative therapeutic approach. We targeted DNABII proteins, an integral component of extracellular DNA (eDNA which is universally found as part of the pathogenic biofilm matrix to develop a biofilm disrupting therapeutic. We show that a cocktail of monoclonal antibodies directed against specific epitopes of a DNABII protein is highly effective to disrupt diverse biofilms in vitro as well as resolve experimental infection in vivo, in both a chinchilla and murine model. Combining this monoclonal antibody cocktail with a traditional antibiotic to kill bacteria newly released from the biofilm due to the action of the antibody cocktail was highly effective. Our results strongly support these monoclonal antibodies as attractive candidates for lead optimization as a therapeutic for resolution of bacterial biofilm diseases.

  8. Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Schjoldager, Katrine T; Cló, Emiliano


    of the nature and timing of induction of auto-antibodies to distinct O-glycopeptide epitopes induced by cancer. The results demonstrate that truncated O-glycopeptides constitute highly distinct antibody epitopes with great potential as targets for biomarkers and immunotherapeutics.......Auto-antibodies induced by cancer represent promising sensitive biomarkers and probes to identify immunotherapeutic targets without immunological tolerance. Surprisingly few epitopes for such auto-antibodies have been identified to date. Recently, a cancer-specific syngeneic murine monoclonal...... antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity...

  9. Radioimmunoimaging of subacute infective endocarditis using a technetium-99m monoclonal granulocyte-specific antibody

    Energy Technology Data Exchange (ETDEWEB)

    Munz, D.L.; Sandrock, D.; Emrich, D. (Goettingen Univ. (Germany). Abt. fuer Nuklearmedizin); Morguet, A.J.; Heim, A.; Sold, G.; Figulla, H.R.; Kreuzer, H. (Goettingen Univ. (Germany). Abt. fuer Kardiologie und Pulmonologie)


    Immunoscintigraphy with a technetium-99m murine monoclonal IgG{sub 1} antibody directed against non-specific cross-reacting antigen (NCA-95) and carcinoembryonic antigen was performed with 20 patients with suspected subacute infective endocarditis (SIE) and 6 controls with suspected inflammatory/infectious disease elsewhere in the body. Immunoscintigraphy and echocardiography localised SIE in 11 of 15 patients in whom the disease could be confirmed. In 4 patients with validated SIE, the immunoscan was abnormal, and the echocardiogram was normal. In another 4 patients, the result was exactly the opposite. These findings suggest that the combination of immunoscintigraphy and echocardiography improves diagnostic efficacy in patients with suspected SIE. (orig.).

  10. Monoclonal antibodies in chronic lymphocytic leukemia. (United States)

    Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J


    Multiple options are now available for the treatment of chronic lymphocytic leukemia. Over the last 10 years, monoclonal antibodies have become an integral part of the management of this disease. Alemtuzumab has received approval for use in patients with fludarabine-refractory chronic lymphocytic leukemia. Rituximab has been investigated extensively in chronic lymphocytic leukemia both as a single agent and in combination with chemotherapy and other monoclonal antibodies. Epratuzumab and lumiliximab are newer monoclonal antibodies in the early phase of clinical development. This article will review the monoclonal antibodies more commonly used to treat chronic lymphocytic leukemia, the results obtained with monoclonal antibodies as single agents and in combination with chemotherapy, and other biological agents and newer compounds undergoing clinical trials.


    Directory of Open Access Journals (Sweden)

    Mahdi Fasihi-Ramandi


    Full Text Available There are many studies related to immunological and molecular methods for diagnosis of Vibrio cholera (V. cholerae. However, most assays dependent on enrichment of culture of bacteria, which need more time and involves the use of costly equipment and reagents. In this study Balb/c mice were immunized with recombinant Outer Membrane Protein (rOMPw of vibrio cholerae and splenocytes of hyper immunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using rOMPw as coating antigen. The monoclonal antibodies from ascitic fluids were purified and its reaction with rOMPw was assessed by ELISA. Polyclonal antibodies were also produced by immunization of rabbits with the above mentioned antigen. The rabbit sera was affinity purified using Hi-Trap protein G column. The result showed that monoclonal antibody specific to rOMPw has been successfully generated. The monoclonal antibody reacted with recombinant OMPw in ELISA and immunonoblat method. Rabbit polyclonal antibody was also bound to rOMPw by ELISA. The results of agglutination test with whole bacteria also showed that both mouse monoclonal and rabbit polyclonal antibodies reacted with whole vibrio cholera but not other related bacteria. The purpose of this study was to check out if anti OMPw antibodies could use as diagnostic assay for detection of V. cholerae. Our results demonstrated that anti recombinant OMPw monoclonal and polyclonal antibodies are able to diagnose whole bacteria in pure culture using agglutination test but not by home made immunochromatic strip test.

  12. Production and Screening of Monoclonal Peptide Antibodies. (United States)

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


    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.

  13. Pharmacokinetics interactions of monoclonal antibodies. (United States)

    Ferri, Nicola; Bellosta, Stefano; Baldessin, Ludovico; Boccia, Donatella; Racagni, Giorgi; Corsini, Alberto


    The clearance of therapeutic monoclonal antibodies (mAbs) typically does not involve cytochrome P450 (CYP450)-mediated metabolism or interaction with cell membrane transporters, therefore the pharmacokinetics interactions of mAbs and small molecule drugs are limited. However, a drug may affect the clearance of mAbs through the modulation of immune response (e.g., methotrexate reduces the clearance of infliximab, adalimumab, and golimumab, possibly due to methotrexate's inhibitory effect on the formation of antibodies against the mAbs). In addition, mAbs that are cytokine modulators may modify the metabolism of drugs through their effects on P450 enzymes expression. For example, cytokine modulators such as tocilizumab (anti-IL-6 receptor antibody) may reverse the "inhibitory" effect of IL-6 on CYP substrates, resulting in a "normalization" of CYP activities. Finally, a drug may alter the clearance of mAbs by either increasing or reducing the levels of expression of targets of mAbs on the cell surface. For instance, statins and fibrates induce PCSK9 expression and therefore increase cellular uptake and clearance of alirocumab and evolocumab, anti-PCSK9 antibodies. In the present review, we will provide an overview on the pharmacokinetics properties of mAbs as related to the most relevant examples of mAbs-small molecule drug interaction.

  14. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies. (United States)

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles


    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

  15. [Inhibition of invasion and multiplication of Toxoplasma gondii in human colonic epithelial cells by a monoclonal antibody against protein SAG2]. (United States)

    Osorio, J C; Sánchez, R M; Iraola, R C; Pérez, J S


    By an bromodeoxyuridine (BrdU) incorporation assay, it was proved hat an IgG 1 subclass, murine monoclonal antibody to surface protein SAG2 of Toxoplasma gondii is capable of reducing the invasion and multiplication of the parasites in highly differentiated mucine secretory HT29-18N2 line cells from a human colon adenocarcinoma. This result shows the importance of surface protein SAG2 of T.gondii in invasion and further multiplication of parasites in the host cell.

  16. Development and Evaluation of Monoclonal Antibodies for Paxilline

    Directory of Open Access Journals (Sweden)

    Chris M. Maragos


    Full Text Available Paxilline (PAX is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs the concentrations of PAX required to inhibit signal development by 50% (IC50s ranged from 1.2 to 2.5 ng/mL. One mAb (2-9 was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage.

  17. Biodistribution of [sup 125]I labeled monoclonal antibody against gamma seminoprotein in the nude mice bearing human benign prostatic hyperplasia xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Fujino, Awato (Kitasato Univ., Sagamihara, Kanagawa (Japan). School of Medicine)


    The biodistribution of [sup 125]I labeled monoclonal antibody against gamma seminoprotein ([gamma]-Sm) in the nude mice bearing human benign prostatic hyperplasia xenograft was evaluated by whole body autoradiography and by counting of radioactivity in organs. The monoclonal antibody (murine Ig G[sub 1], K) to [gamma]-Sm which was established in this institute and its F(ab')[sub 2] fragment were radioiodinated using Iodogen method. The autoradiograms demonstrated specific uptake of [sup 125]I-intact Ig G as well as [sup 125]I-F(ab')[sub 2] within the prostatic adenoma xenografts 4 days after intravenous administrations. Radioactivity in the xenograft was relatively higher than those in the liver, kidney or lung. These results suggest that radio-labeled monoclonal antibody against [gamma]-Sm might be applicable for radioimmunodetection of prostatic tumors which produce [gamma]-Sm. (author).

  18. Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TU 67). (United States)

    Herrmann, F; Griffin, J D; Sabbath, K D; Oster, W; Wernet, P; Mertelsmann, R


    Anti-TU 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TU 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TU 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TU 67 antibody.

  19. Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

    Energy Technology Data Exchange (ETDEWEB)

    Malviya, G.; Dierckx, R.A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Conti, F. [Rheumatology Unit, I Faculty of Medicine and Surgery, Sapienza University of Rome (Italy); Chianelli, M. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Unit of Nuclear Medicine, Regina apostolorum Hospital, Albano, Rome (Italy); Scopinaro, F. [Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy); Signore, A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy)


    The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-{alpha}, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with {sup 99m}Tc or {sup 111}In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for

  20. Tumoricidal activation of murine resident peritoneal macrophages on pancreatic carcinoma by interleukin-2 and monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    Qi Kui Chen; Shi Zhen Yuan; Zhi Yong Zeng; Zhi Qing Huang


    @@INTRODUCTION Macrophages play an important role in tumor lysis and growth inhibition. They can be activated to a tumoricidal state by a variety of agents such as IFNr, TNFa or IL2. The killing machanisms of activated macrophages have been extensively investigated[1,2]. Recently, it has been proved that antibody dependent cellular cytotoxicity (ADCC) is one of the potent arms to lyse tumor cells resistant to cytotoxic macrophages,and that the antitumorous effect of a macrophage activator is significantly augmented by the combined use of mAbs capable of inducing ADCC to tumor cells[3].

  1. Antibody SPC-54 provides acute in vivo blockage of the murine protein C system. (United States)

    Burnier, Laurent; Fernández, José A; Griffin, John H


    Multiple protective effects of pharmacological activated protein C (APC) are reported in several organ pathologies. To help evaluate the endogenous murine PC system, we characterized a rat monoclonal anti-mouse PC antibody, SPC-54, which inhibited the amidolytic and anticoagulant activities of murine APC by>95%. SPC-54 blocked active site titration of purified APC using the active site titrant, biotinylated FPR-chloromethylketone, showing that SPC-54 blocks access to APC's active site to inhibit all enzymatic activity. A single injection of SPC-54 (10mg/kg) neutralized circulating PC in mice for at least 7days, and immunoblotting and immuno-precipitation with protein G-agarose confirmed that SPC-54 in vivo was bound to PC in plasma. Pre-infusion of SPC-54 in tissue factor-induced murine acute thromboembolism experiments caused a major decrease in mean survival time compared to controls (7min vs. 42.5min, P=0.0016). SPC-54 decreased lung perfusion in this model by 54% when monitored by vascular perfusion methodologies using infrared fluorescence of Evans blue dye. In LD50 endotoxemia murine models, SPC-54 infused at 7hr after endotoxin administration increased mortality from 42% to 100% (PSPC-54 ablates in vitro and in vivo APC protective functions and enzymatic activity. The ability of SPC-54 to block the endogenous PC/APC system provides a powerful tool to understand better the role of the endogenous PC system in murine injury models and in cell bioassays and also to neutralize the enzymatic activities of murine APC in any assay system.

  2. Human Monoclonal Antibodies as a Countermeasure Against Botulinum Toxins (United States)


    REPORT Human monoclonal antibodies as a countermeasure against Botulinum toxins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: In this report, we...Prescribed by ANSI Std. Z39.18 - 31-Aug-2012 Human monoclonal antibodies as a countermeasure against Botulinum toxins Report Title ABSTRACT In this report...DTRA Final Report: Human monoclonal antibodies as a countermeasure against Botulinum toxins   Page 1 of 22 DTRA Final Report: Human monoclonal

  3. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S


    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia. Antibod

  4. Monoclonal antibody technologies and rapid detection assays (United States)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  5. Monoclonal Antibody Therapy for Advanced Neuroblastoma (United States)

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  6. Serotyping of Actinobacillus pleuropneumoniae serotype 5 strains using a monoclonal-based polystyrene agglutination test

    DEFF Research Database (Denmark)

    Dubreuil, J.D.; Letellier, A.; Stenbæk, Eva;


    A polystyrene agglutination test has been developed for serotyping Actinobacillus pleuropneumoniae serotype 5a and 5b strains. Protein A-coated polystyrene microparticles were sensitized with a murine monoclonal antibody recognizing an epitope on serotype 5 LPS-O chain as shown by SDS......-PAGE and Western blotting, A total of 205 A. pleuropneumoniae, strains including all 12 serotype reference strains and 13 strains representing 8 common bacterial species associated with swine or related to A, pleuropneumoniae, were tested by mixing 25 mu L of polystyrene reagent with the same volume of a dense...... suspension of bacterial cells grown for 18 h. All A, pleuropneumoniae strains had been previously serotyped using standard procedures, The polystyrene agglutination test was rapid (less than 3 min) and easy to perform. Overall a very good correlation (97.3%) with the standard techniques was found...

  7. Anti-CD20 monoclonal antibodies as novel treatments for non-Hodgkin's lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    White, C.A.; Larocca, A.; Grillo-Lopez, A.J. [IDEC Pharmaceuticals, 3030 Callan Road, San Diego, CA (United States)


    Anti-CD20 monoclonal antibodies (MAbs) offer new options for patients with non-Hodgkin's lymphoma, needed because existing therapies have many limitations. The unconjugated, chimeric anti-CD20 antibody, Rituximab (MabThera, Rituxan), has recently been approved in the USA for patients with relapsed or refractory, low-grade or follicular, B-cell non-Hodgkin's lymphoma, and in Europe for therapy of relapsed stage III/IV follicular lymphoma. In the pivotal study of Rituximab, an overall response rate of 50% was achieved with median time to progressionin responders of 13.2 months. Studies are ongoing with the {sup 90}Y-labelled murine anti-CD20 antibody, IDEC-Y2B8. The response rate in a Phase I/II study in low-grade and intermediate-grade patients was 67%. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  8. Production of monoclonal antibodies against the outer cell wall of Clostridium tyrobutyricum. (United States)

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


    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.

  9. A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells. (United States)

    Griffin, J D; Linch, D; Sabbath, K; Larcom, P; Schlossman, S F


    Anti-MY9 is an IgG2b murine monoclonal antibody selected for reactivity with immature normal human myeloid cells. The MY9 antigen is expressed by blasts, promyelocytes and myelocytes in the bone marrow, and by monocytes in the peripheral blood. Erythrocytes, lymphocytes and platelets are MY9 negative. All myeloid colony-forming cells (CFU-GM), a fraction of erythroid burst-forming cells (BFU-E) and multipotent progenitors (CFU-GEMM) are MY9 positive. This antigen is further expressed by the leukemic cells of a majority of patients with AML and myeloid CML-BC. Leukemic stem cells (leukemic colony-forming cells, L-CFC) from most patients tested were also MY9 positive. In contrast, MY9 was not detected on lymphocytic leukemias. Anti-MY9 may be a valuable reagent for the purification of hematopoietic colony-forming cells and for the diagnosis of myeloid-lineage leukemias.

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

    Directory of Open Access Journals (Sweden)

    Kh. H. Haider


    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.

  11. Generation and applications of monoclonal antibodies for livestock production. (United States)

    Van Der Lende, T


    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.

  12. Use of radiolabeled monoclonal antibodies for diagnostic imaging

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Keigo (Kyoto Univ. (Japan). Faculty of Medicine)


    Monoclonal antibodies (MoAbs) are expected to carry radionuclides selectively to target tissues and to offer antigen-specific diagnosis. Indium (In)-111 has many favorable nuclear properties and is efficiently labeled with MoAbs using DAPA as a bifunctional chelating agent. In-111 labeled MoAbs are clinically employed for the diagnosis of malignant melanoma, colorectal cancer and acute myocardial infarction in Japan. Although non-specific deposit of In-111 was seen in liver and bone-marrow, scintigraphy using In-111 labeled MoAbs was encouraging, since it detected about 80% of tumors, tumors missed by conventional diagnostic methods such as CT, and tumors in patients with normal serum CEA values, and acute myocarditis as well as acute myocardial infarction was positive with In-111 labeled Fab fraction of anti-myosin Ab. Acute or subacute toxicity was not observed. Human anti-murine antibody (HAMA) was detected in 53 of 64 (82.8%) patients who were intravenously administered with 20 to 42 mg of anti-melanoma or anti-CEA MoAbs (whole IgG). In contrast, only 5 of 406 (1.2%) patients had detectable levels of HAMA in their serum after receiving 0.5 mg of Fab fraction of MoAb. Recently mouse-human chimeric Ab has been produced by recombinant DNA techniques, which localized well in xenografted tumors and seems to be promising for clinical use. Investigations are under way to increase the tumor to non-tumor ratio by modifying chelating agents for coupling MoAbs with radionuclides. (author).

  13. Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice. (United States)

    Yang, Yilong; Qian, Mengying; Yi, Shaoqiong; Liu, Shuling; Li, Bing; Yu, Rui; Guo, Qiang; Zhang, Xiaopeng; Yu, Changming; Li, Jianmin; Xu, Junjie; Chen, Wei


    Epidemic methicillin-resistant Staphylococcus aureus (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. Staphylococcus aureus surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against S. aureus infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type S. aureus and promote opsonophagocytic killing of S. aureus. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by S. aureus strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections.

  14. Dengue Virus Envelope Dimer Epitope Monoclonal Antibodies Isolated from Dengue Patients Are Protective against Zika Virus

    Directory of Open Access Journals (Sweden)

    J. A. Swanstrom


    Full Text Available Zika virus (ZIKV is a mosquito-borne flavivirus responsible for thousands of cases of severe fetal malformations and neurological disease since its introduction to Brazil in 2013. Antibodies to flaviviruses can be protective, resulting in lifelong immunity to reinfection by homologous virus. However, cross-reactive antibodies can complicate flavivirus diagnostics and promote more severe disease, as noted after serial dengue virus (DENV infections. The endemic circulation of DENV in South America and elsewhere raises concerns that preexisting flavivirus immunity may modulate ZIKV disease and transmission potential. Here, we report on the ability of human monoclonal antibodies and immune sera derived from dengue patients to neutralize contemporary epidemic ZIKV strains. We demonstrate that a class of human monoclonal antibodies isolated from DENV patients neutralizes ZIKV in cell culture and is protective in a lethal murine model. We also tested a large panel of convalescent-phase immune sera from humans exposed to primary and repeat DENV infection. Although ZIKV is most closely related to DENV compared to other human-pathogenic flaviviruses, most DENV immune sera (73% failed to neutralize ZIKV, while others had low (50% effective concentration [EC50], 1:100 serum dilution; 9% levels of cross-neutralizing antibodies. Our results establish that ZIKV and DENV share epitopes that are targeted by neutralizing, protective human antibodies. The availability of potently neutralizing human monoclonal antibodies provides an immunotherapeutic approach to control life-threatening ZIKV infection and also points to the possibility of repurposing DENV vaccines to induce cross-protective immunity to ZIKV.

  15. Recent developments in monoclonal antibody radiolabeling techniques

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Mease, R.C.


    Monoclonal antibodies (MAbs) have shown the potential to serve as selective carriers of radionuclides to specific in vivo antigens. Accordingly, there has been an intense surge of research activity in an effort to develop and evaluate MAb-based radiopharmaceuticals for tumor imaging (radioimmunoscintigraphy) and therapy (radioimmunotherapy), as well as for diagnosing nonmalignant diseases. A number of problems have recently been identified, related to the MAbs themselves and to radiolabeling techniques, that comprise both the selectivity and the specificity of the in vivo distribution of radiolabeled MAbs. This paper will address some of these issues and primarily discuss recent developments in the techniques for radiolabeling monoclonal antibodies that may help resolve problems related to the poor in vivo stability of the radiolabel and may thus produce improved biodistribution. Even though many issues are identical with therapeutic radionuclides, the discussion will focus mainly on radioimmunoscintigraphic labels. 78 refs., 6 tabs.

  16. Monoclonal Antibodies to Plant Growth Regulators (United States)

    Eberle, Joachim; Arnscheidt, Angelika; Klix, Dieter; Weiler, Elmar W.


    Four high affinity monoclonal antibodies, which recognize two plant growth regulators from the cytokinin group, namely trans-zeatin riboside and dihydrozeatin riboside and their derivatives are reported. Six hybridomas were produced from three independent fusions of Balb/c spleen cells with P3-NS1-Ag 4-1 (abbreviated NS1) or X63-Ag 8.653 (X63) myeloma cells. The mice had been hyperimmunized with zeatin riboside-bovine serum albumin conjugate or dihydrozeatin riboside-bovine serum albumin conjugate for 3 months. The hybridomas secrete antibodies of the IgG 1 or IgG 2b subclass and allow the detection of femtomole amounts of the free cytokinins, their ribosides, and ribotides in plant extracts. The use of these monoclonals in radio- and enzyme-linked immunosorbent assay is also discussed. PMID:16664848

  17. Murine gamma interferon fails to inhibit Toxoplasma gondii growth in murine fibroblasts. (United States)

    Schwartzman, J D; Gonias, S L; Pfefferkorn, E R


    Although treatment of human macrophages or fibroblasts with human gamma interferon results in the inhibition of intracellular Toxoplasma gondii, murine gamma interferon stimulated only murine macrophages, not murine fibroblasts, to inhibit T. gondii. This species difference may be important in understanding the control of acute and chronic toxoplasmosis. PMID:2106497

  18. Polyclonal and monoclonal antibodies in clinic. (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses


    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.

  19. Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. (United States)

    Zeitlin, Larry; Pettitt, James; Scully, Corinne; Bohorova, Natasha; Kim, Do; Pauly, Michael; Hiatt, Andrew; Ngo, Long; Steinkellner, Herta; Whaley, Kevin J; Olinger, Gene G


    No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED(50) = 33 μg). A version with typical heterogenous mammalian glycoforms (ED(50) = 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED(50) = 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics.

  20. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C


    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...


    Institute of Scientific and Technical Information of China (English)


    Human cervical cancer is one of the most com-mon malignant neoplas m in women with a highdeath rate.It is characterized by a lot of factors andits pathological process is very complicated.In spiteof progress in the diagnosis and therapy of humancervical cancer,ti mely and accurate methods are ur-gently needed[1].Application of murine monoclonal antibodies(McAb)for the study of diagnosis and treat mentsfor human tumors is li mited by a number of fac-tors.Due to the progress in gene engineering andphage displ...

  2. The Role of Monoclonal Antibodies in the Management of Leukemia (United States)

    Al-Ameri, Ali; Cherry, Mohamad; Al-Kali, Aref; Ferrajoli, Alessandra


    This article will review the monoclonal antibodies more commonly used in leukemias. In the last three decades, scientists have made considerable progress understanding the structure and the functions of various surface antigens, such as CD20, CD33. The introduction of rituximab, an anti CD20 monoclonal antibody, had a great impact in the treatment of lymphoproliferative disorders. Gemtuzumab, an anti CD 33 conjugated monoclonal antibody has activity in acute mylegenous leukemia (AML). As this field is undergoing a rapid growth, the years will see an increasing use of monoclonal antibodies in hematological malignancies.

  3. The Role of Monoclonal Antibodies in the Management of Leukemia

    Directory of Open Access Journals (Sweden)

    Mohamad Cherry


    Full Text Available This article will review the monoclonal antibodies more commonly used in leukemias. In the last three decades, scientists have made considerable progress understanding the structure and the functions of various surface antigens, such as CD20, CD33. The introduction of rituximab, an anti CD20 monoclonal antibody, had a great impact in the treatment of lymphoproliferative disorders. Gemtuzumab, an anti CD 33 conjugated monoclonal antibody has activity in acute mylegenous leukemia (AML. As this field is undergoing a rapid growth, the years will see an increasing use of monoclonal antibodies in hematological malignancies.

  4. Monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. (United States)

    Kyle, Robert A; San-Miguel, Jesus F; Mateos, Maria-Victoria; Rajkumar, S Vincent


    Monoclonal gammopathy of undetermined significance (MGUS) is characterized by an M spike less than 3 g/dL and a bone marrow containing fewer than 10% plasma cells without evidence of CRAB (hypercalcemia, renal insufficiency, anemia, or bone lesions). Light chain MGUS has an abnormal free light chain (FLC) ratio, increased level of the involved FLC, no monoclonal heavy chain, and fewer than 10% monoclonal plasma cells in the bone marrow. Smoldering multiple myeloma has an M protein of at least 3 g/dL and/or at least 10% monoclonal plasma cells in the bone marrow without CRAB features.


    Directory of Open Access Journals (Sweden)

    Michael Sela


    Full Text Available History of developing synergy between monoclonal antibodies, anti-tumor activity of monoclonal antibodies against tyrosine-kinases receptors EGFR/ErbB-1 and HER2/ErbB-2 as well as growth factor VEGF in various combinations are considered in the article. There were proposed hypotheses about potential molecular mechanisms underlay synergy between monoclonal antibodies (for homo- and hetero combinations of antibodies appropriately specific for antigenic determinants on the same or different receptors. Future trends in researches necessary to deeper understanding causes of this phenomenon and perspectives for practical application of monoclonal antibodies acted synergistically as immunotherapeutic drugs for human tumors treatment are reviewed.

  6. Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies. (United States)

    Cedergren, A M; Miklasz, S D; Voss, E W


    Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

  7. Anaphylaxis to chemotherapy and monoclonal antibodies. (United States)

    Castells, Mariana C


    Hypersensitivity reactions are increasingly prevalent, although underrecognized and underreported. Platins induce immunoglobulin E-mediated sensitization; taxenes and some monoclonal antibodies can induce reactions at first exposure. Severe hypersensitivity can preclude first-line therapy. Tryptase level at the time of a reaction is a useful diagnostic tool. Skin testing provides a specific diagnosis. Newer tests are promising diagnostic tools to help identify patients at risk before first exposure. Safe management includes rapid drug desensitization. This review provides information regarding the scope of hypersensitivity and anaphylactic reactions induced by chemotherapy and biological drugs, as well as diagnosis, management, and treatment options.

  8. Monoclonal Idiotope Vaccine against Streptococcus pneumoniae Infection (United States)

    McNamara, Mary K.; Ward, Ronald E.; Kohler, Heinz


    A monoclonal anti-idiotope antibody coupled to a carrier protein was used to immunize BALB/c mice against a lethal Streptococcus pneumoniae infection. Vaccinated mice developed a high titer of antibody to phosphorylcholine, which is known to protect against infection with Streptococcus pneumoniae. Measurement of the median lethal dose of the bacteria indicated that anti-idiotope immunization significantly increased the resistance of BALB/c mice to the bacterial challenge. Antibody to an idiotope can thus be used as an antigen substitute for the induction of protective immunity.

  9. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries. (United States)

    Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G


    Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur.

  10. Immunochemical Characterization of Anti-Acetylcholinesterase Inhibitory Monoclonal Antibodies (United States)


    formation. This conformation was first proposed using studies with monoclonal antibodies against a synthetic peptide mimicking the sequence of the...distinct antigenic determinants on dengue -2 virus using monoclonal antibodies, Am. J. Trop. Med. Hyg., 31 (1982) 548-555. 7 D. De la Hoz, B.P. Doctor

  11. Polyneuropathy associated with monoclonal gammopathy, cause and consequence

    NARCIS (Netherlands)

    Eurelings, Marijke


    The relation between monoclonal antibodies and polyneuropathy is best supported for polyneuropathy associated with IgM monoclonal anti-myelin associated glycoprotein (anti-MAG) antibodies. These anti-MAG antibodies are reactive against peripheral nerve autoantigen, thereby causing an autoimmune medi

  12. Flow cytometry of murine spermatocytes. (United States)

    Gaysinskaya, Valeriya; Bortvin, Alex


    Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells. Included are procedures that guide data acquisition, display, gating, and back-gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis-defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis.

  13. Monoclonal antibodies directed against protoplasts of soybean cells : Generation of hybridomas and characterization of a monoclonal antibody reactive with the cell surface. (United States)

    Villanueva, M A; Metcalf, T N; Wang, J L


    Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of (125)I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (Mr) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (Mr 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.

  14. Treatment with anti-interferon-δ monoclonal antibodies modifies experimental autoimmune encephalomyelitis in interferon-δ receptor knockout mice

    DEFF Research Database (Denmark)

    Espejo, C.; Penkowa, Milena; Saez-Torres, I.


    Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis......Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis...

  15. Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)


    textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these an

  16. 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.


    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.

  17. Fine specificity and cross-reactions of monoclonal antibodies to group B streptococcal capsular polysaccharide type III

    DEFF Research Database (Denmark)

    Pincus, Seth H; Moran, Emily; Maresh, Grace


    ) is considered the dominant "protective" immune mediator. Here we study the fine specificity and potential host reactivity of a panel of well-characterized murine monoclonal Abs against the type III CPS by examining the binding of the Abs to intact and neuraminidase-digested GBS, purified CPS, synthetic...... carbohydrate structures, and cells. The results showed marked differences in the fine specificity among these mAbs to a single carbohydrate structure. Cross-reactions with synthetic GD3 and GT3 carbohydrates, representing structures found on surfaces of neural and developing cells, were demonstrated using...... serendipitously demonstrated cross-reactions of some anti-CPS(III) mAbs with antigens, likely carbohydrates, found on human leukocytes. These studies suggest caution in the development of a maternal vaccine to prevent infection by this important human pathogen....

  18. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

    Directory of Open Access Journals (Sweden)

    Xiaodong Xiao

    Full Text Available Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252 and two anti-V-specific human mAb (m253, m254 by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

  19. A cross-reactive monoclonal antibody to nematode haemoglobin enhances protective immune responses to Nippostrongylus brasiliensis.

    Directory of Open Access Journals (Sweden)

    Natalie E Nieuwenhuizen

    Full Text Available BACKGROUND: Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection. METHODOLOGY/PRINCIPAL FINDINGS: Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four -HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens. CONCLUSION: The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity

  20. Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    Full Text Available Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9 variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D of 1.63 nM, comparable to its parental murine D9 (2.55 nM. In a mouse model, intraperitoneal (i.p. administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

  1. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM


    Full Text Available Andrea M Eisenbeis, Stefan J GrauDepartment of Neurosurgery, University Hospital of Cologne, Cologne, GermanyAbstract: Advances in biotechnology, better understanding of pathophysiological processes, as well as the identification of an increasing number of molecular markers have facilitated the use of monoclonal antibodies and Fc fragments in various fields in medicine. In this context, a rapidly growing number of these substances have also emerged in the field of oncology. This review will summarize the currently approved monoclonal antibodies used for the treatment of solid tumors with a focus on their clinical application, biological background, and currently ongoing trials.Keywords: targeted therapy, monoclonal antibodies, cancer, biological therapy

  2. Affinity Maturation of Monoclonal Antibody 1E11 by Targeted Randomization in CDR3 Regions Optimizes Therapeutic Antibody Targeting of HER2-Positive Gastric Cancer. (United States)

    Ko, Bong-Kook; Choi, Soyoung; Cui, Lei Guang; Lee, Young-Ha; Hwang, In-Sik; Kim, Kyu-Tae; Shim, Hyunbo; Lee, Jong-Seo


    Anti-HER2 murine monoclonal antibody 1E11 has strong and synergistic anti-tumor activity in HER2-overexpressing gastric cancer cells when used in combination with trastuzumab. We presently optimized this antibody for human therapeutics. First, the complementarity determining regions (CDRs) of the murine antibody were grafted onto human germline immunoglobulin variable genes. No difference in affinity and biological activity was observed between chimeric 1E11 (ch1E11) and humanized 1E11 (hz1E11). Next, affinity maturation of hz1E11 was performed by the randomization of CDR-L3 and H3 residues followed by stringent biopanning selection. Milder selection pressure favored the selection of more diverse clones, whereas higher selection stringency resulted in the convergence of the panning output to a smaller number of clones with improved affinity. Clone 1A12 had four amino acid substitutions in CDR-L3, and showed a 10-fold increase in affinity compared to the parental clone and increased potency in an in vitro anti-proliferative activity assay with HER2-overepxressing gastric cancer cells. Clone 1A12 inhibited tumor growth of NCI-N87 xenograft model with similar efficacy to trastuzumab alone, and the combination treatment of 1A12 and trastuzumab completely removed the established tumors. These results suggest that humanized and affinity matured monoclonal antibody 1A12 is a highly optimized molecule for future therapeutic development against HER2-positive tumors.

  3. Complementary innate (anti-A-specific) IgM emerging from ontogenic O-GalNAc-transferase depletion: (Innate IgM complementarity residing in ancestral antigen completeness). (United States)

    Arend, Peter


    The murine and the human genome have global properties in common. So the murine anti-A-specific complementary IgM and related human innate isoagglutinin represent developmental, 2-mercaptoethanol-sensitive, complement-binding glycoproteins, which do not arise from any measurable environmentally-induced or auto- immune response. The murine anti-A certainly originates from a cell surface- or cell adhesion molecule, which in the course of germ cell development becomes devoid of O-GalNAc-transferase and is released into the circulation. In human sera the enzyme occurs exclusively in those of blood group A- and AB subjects, while in group O(H) an identically encoded protein lets expect an opposite function and appears in conjunction with a complementary anti-A reactive glycoprotein. Since O-glycosylations rule the carbohydrate metabolism in growth and reproduction processes, we propose that the ancestral histo-(blood)-group A molecule arises in the course of O-GalNAc-glycosylations of glycolipids and protein envelops at progenitor cell surfaces. Germ cell development postulates embryonic stem cell fidelity, which is characterised by persistent production of α-linked O-GalNAc-glycans. They are determined by the A-allele within the human, "complete" histo (blood) group AB(O) structure that in early ontogeny is hypothesised to be synthesised independently from the final phenotype. The structure either passes "completely" through the germline, in transferase-secreting mature tissues becoming the "complete" phenotype AB, or disappears in exhaustive glycotransferase depletion from the differentiating cell surfaces and leaves behind the "incomplete" blood group O-phenotype, which has released a transferase- and O-glycan-depleted, complementary glycoprotein (IgM) into the circulation. The process implies, that in humans the different blood phenotypes evolve from a "complete" AB(O) molecular complex in a distinct enzymatic and/or complement cascade suggesting O

  4. Apoptosis in irradiated murine tumors. (United States)

    Stephens, L C; Ang, K K; Schultheiss, T E; Milas, L; Meyn, R E


    Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors.

  5. Development and characterization of a panel of cross-reactive monoclonal antibodies generated using H1N1 influenza virus. (United States)

    Guo, Chun-yan; Tang, Yi-gui; Qi, Zong-li; Liu, Yang; Zhao, Xiang-rong; Huo, Xue-ping; Li, Yan; Feng, Qing; Zhao, Peng-hua; Wang, Xin; Li, Yuan; Wang, Hai-fang; Hu, Jun; Zhang, Xin-jian


    To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines.

  6. Generation of monoclonal antibodies to native active human glycosyltransferases

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene Bech; Bennett, Eric Paul; Clausen, Henrik;


    using monoclonal antibodies therefore provides an excellent strategy to analyze the glycosylation process in cells. A major drawback has been difficulties in generating antibodies to glycosyltransferases and validating their specificities. Here we describe a simple strategy for generating...

  7. Generation and characterization of monoclonal antibodies specific to Coenzyme A

    Directory of Open Access Journals (Sweden)

    Malanchuk O. M.


    Full Text Available Aim. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this study, we describe for the first time the production and characterization of monoclonal antibodies against CoA. The monoclonal antibody 1F10 was shown to recognize specifically CoA in Western blotting, ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease.

  8. Monoclonal Antibodies Against Xenopus Greatwall Kinase (United States)

    Wang, Ling; Fisher, Laura A.; Wahl, James K.


    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL. PMID:22008075

  9. Drug Development of Therapeutic Monoclonal Antibodies. (United States)

    Mould, Diane R; Meibohm, Bernd


    Monoclonal antibodies (MAbs) have become a substantial part of many pharmaceutical company portfolios. However, the development process of MAbs for clinical use is quite different than for small-molecule drugs. MAb development programs require careful interdisciplinary evaluations to ensure the pharmacology of both the MAb and the target antigen are well-understood. Selection of appropriate preclinical species must be carefully considered and the potential development of anti-drug antibodies (ADA) during these early studies can limit the value and complicate the performance and possible duration of preclinical studies. In human studies, many of the typical pharmacology studies such as renal or hepatic impairment evaluations may not be needed but the pharmacokinetics and pharmacodynamics of these agents is complex, often necessitating more comprehensive evaluation of clinical data and more complex bioanalytical assays than might be used for small molecules. This paper outlines concerns and strategies for development of MAbs from the early in vitro assessments needed through preclinical and clinical development. This review focuses on how to develop, submit, and comply with regulatory requirements for MAb therapeutics.

  10. Monoclonal antibodies based on hybridoma technology. (United States)

    Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro


    Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs.

  11. Monoclonal antibody disulfide reduction during manufacturing (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.


    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. PMID:23751615

  12. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley. (United States)

    Rampitsch, Christof; Ames, Nancy; Storsley, Joanne; Marien, Lindsay


    A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.

  13. T10B9 monoclonal antibody: A short-acting nonstimulating monoclonal antibody that spares γδ T-cells and treats and prevents cellular rejection

    Directory of Open Access Journals (Sweden)

    Thomas H Waid


    Full Text Available Thomas H Waid1, John S Thompson1, Maria Siemionow2, Stephen A Brown1 1Department of Internal Medicine, University of Kentucky, Lexington, Kentucky, USA; 2Cleveland Clinic, Cleveland, Ohio, USAAbstract: T10B9.1A-31/MEDI-500 is a nonmitogenic immunoglobulin M kappa murine monoclonal antibody (mAb directed against the alpha-beta (αβ heterodimer of the T-lymphocyte receptor complex. The hybridoma was first produced by fusing spleen cells from BALB/C mice immunized with human peripheral blood T-lymphocytes with SP2/O-Ag14 mutant myeloma cells. The mAb is produced and purified using multistep ion exchange and molecular sieve chromatography protocols. T10B9 has been used successfully to treat acute cellular rejection in renal transplantation and as an immunosuppression induction agent in heart and simultaneous kidney-pancreas transplantation. Because T10B9 is nonmitogenic and causes minimal cytokine release, both treatment of rejection and induction of immunosuppression were accomplished with significantly fewer and milder untoward effects (cytokine release syndrome than its comparator OKT3. Since T10B9 is directed against the αβ heterodimer of the CD3 epitope, it spares the gamma delta (γδ region. These gamma delta (γδ T cells have a unique role in the immune response controlling many serious human diseases and perhaps facilitating the development of immunologic tolerance. T10B9 has a relatively short duration of action, depleting T cells for only 10 to 14 days, unlike the protracted depletion seen with thymoglobulin and Campath-1H. There is no B-lymphocyte depletion with T10B9 as there is with both of the aforementioned reagents. The lack of prolonged lymphocyte depletion may account for less infection observed with T10B9 treatment.Keywords: T10B9.1A-31, γδ T-cell, monoclonal antibody, Campath-1H, thymoglobulin, OKT3

  14. Generation of Monoclonal Antibodies against Immunoglobulin Proteins of the Domestic Ferret (Mustela putorius furo) (United States)


    The domestic ferret (Mustela putorius furo) serves as an animal model for the study of several viruses that cause human disease, most notably influenza. Despite the importance of this animal model, characterization of the immune response by flow cytometry (FCM) is severely hampered due to the limited number of commercially available reagents. To begin to address this unmet need and to facilitate more in-depth study of ferret B cells including the identification of antibody-secreting cells, eight unique murine monoclonal antibodies (mAb) with specificity for ferret immunoglobulin (Ig) were generated using conventional B cell hybridoma technology. These mAb were screened for reactivity against ferret peripheral blood mononuclear cells by FCM and demonstrate specificity for CD79β+ B cells. Several of these mAb are specific for the light chain of surface B cell receptor (BCR) and enable segregation of kappa and lambda B cells. Additionally, a mAb that yielded surface staining of nearly all surface BCR positive cells (i.e., pan ferret Ig) was generated. Collectively, these MαF-Ig mAb offer advancement compared to the existing portfolio of polyclonal anti-ferret Ig detection reagents and should be applicable to a wide array of immunologic assays including the identification of antibody-secreting cells by FCM. PMID:28286781

  15. Study of effects of anti-IL-10 monoclonal antibody on systemic Candidiasis

    Institute of Scientific and Technical Information of China (English)

    Hongfen Ge; Xingping Chen


    Objective: To investigate the effects of anti-interleukin- 10 monoclonal antibody( anti-IL- 10MAb) on systemic candidiasis. Methods: Control group(only candidiasis injection), and disposal group( candidiasis infection accompanying with anti-IL-10MAb infection) of cyclophosphamide-induced immuno-suppressed murine systemic candidiasis model were set in this study. Colony Forming Units (CFUs) of infected kidneys and spleens were determined using plating dilution method. The histological studies for infected livers, spleens,and kidneys were applied. Levels of interferon gamma(IFN-γ) in spleen tissue homogenare were also measured by enzyme-linked immunosorbent assay. Results: In kidneys, the numbers of CFU of the disposal group were much lower than that of the control group; the numbers of CFU in spleens were similar to the control group. The histopathological scores of the disposal group were much better than that of the control group in kindneys with significant differences( P < 0.01 ). In spleens,the histopathological scores of disposal group were also better than that of control group,but no statistic significant differences were observed ( P > 0.05). And the spleen IFN-γ level of the disposal group was significant higher than that of the control group( P < 0.01). Conclusion: Anti-IL- 10MAb effects on systemic candidiasis was concluded.

  16. Monoclonal antibodies against peptidorhamnomannans of Scedosporium apiospermum enhance the pathogenicity of the fungus.

    Directory of Open Access Journals (Sweden)

    Livia C L Lopes

    Full Text Available Scedosporium apiospermum is part of the Pseudallescheria-Scedosporium complex. Peptidorhamnomannans (PRMs are cell wall glycopeptides present in some fungi, and their structures have been characterized in S. apiospermum, S. prolificans and Sporothrix schenckii. Prior work shows that PRMs can interact with host cells and that the glycopeptides are antigenic. In the present study, three monoclonal antibodies (mAbs, IgG1 to S. apiospermum derived PRM were generated and their effects on S. apiospermum were examined in vitro and in vivo. The mAbs recognized a carbohydrate epitope on PRM. In culture, addition of the PRM mAbs increased S. apiospermum conidia germination and reduced conidial phagocytosis by J774.16 macrophages. In a murine infection model, mice treated with antibodies to PRM died prior to control animals. Thus, PRM is involved in morphogenesis and the binding of this glycopeptide by mAbs enhanced the virulence of the fungus. Further insights into the effects of these glycopeptides on the pathobiology of S. apiospermum may lead to new avenues for preventing and treating scedosporiosis.

  17. 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))


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

  18. Human C-C chemokine receptor 3 monoclonal antibody inhibits pulmonary inflammation in allergic mice

    Institute of Scientific and Technical Information of China (English)

    Kai WANG; Hua-hao SHEN; Wen LI; Hua-qiong HUANG


    Aim:To evaluate the effect of C-C chemokine receptor 3 (CCR3) blockade on pulmonary inflammation and mucus production in allergic mice. Methods:We used the synthetic peptide of the CCR3 NH2-terminal as the immunizing antigen and generated murine monoclonal antibody against the human CCR3. In addition,the generated antibody was administered to mice sensitized and challenged with ovalbumin. The inflammatory cells in bronchoalveolar lavage,cytokine levels,pulmonary histopathology,and mucus secretion were examined. Results:The Western blotting analysis indicated that the generated antibody bound to CCR3 specifically. The allergic mice treated with the antihuman CCR3 antibody exhibited a significant reduction of pulmonary inflammation accompanied with the alteration of cytokine. Conclusion:The antibody we generated was specific to CCR3. The inhibition of airway inflammation and mucus overproduction by the antibody suggested that the blockade of CCR3 is an appealing therapeutical target for asthma. The present research may provide an experimental basis for the further study of this agent.

  19. 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.

  20. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    Energy Technology Data Exchange (ETDEWEB)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Oshita, Masatoshi; Ideno, Shoji [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Yunoki, Mikihiro [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Kuhara, Motoki [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano 396-0002 (Japan); Yamamoto, Naomasa [Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611 (Japan); Okuno, Yoshinobu [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa 768-0061 (Japan); Ikuta, Kazuyoshi, E-mail: [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)


    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  1. Diffuse plane xanthomatosis associated with monoclonal gammopathy Xantomatose plana difusa associada a gamopatia monoclonal

    Directory of Open Access Journals (Sweden)

    Aristóteles Rosmaninho


    Full Text Available Diffuse plane normolipemic xanthomatosis (DPNX is a rare, non-inherited disease that is often associated with systemic diseases, mainly malignant hematological (especially multiple myeloma or lymph proliferative disorders. The DPNX can precede the appearance of such conditions by several years, so careful follow-up and periodic laboratory examinations are recommended even for patients that seemed to have no underlying disease. We describe a case associated with monoclonal gammopathy. This case shows that dermatological lesions can be the first manifestation of important hematological diseases and so physicians should be familiarized with this entityA xantomatose plana difusa normolipêmica (XPDN é uma dermatose adquirida rara, muitas vezes associada a doenças sistêmicas, nomeadamente neoplasias hematológicas(sobretudo o mieloma múltiplo ou a processos linfoproliferativos. A XPDN pode preceder o aparecimento dessas doenças em vários anos, sendo por isso recomendada uma vigilância clínica e laboratorial periódica, mesmo para os doentes que aparentemente não apresentam uma doença associada. Descrevemos um caso associado à gamopatia monoclonal. Este caso demonstra a importância das manifestações cutâneas como primeira manifestação de doenças hematológicas importantes e por isso os clínicos devem estar familiarizados com esta entidade

  2. Development of syngeneic monoclonal anti-idiotype antibodies to mouse monoclonal anti-asialoglycoprotein receptor antibody.

    Directory of Open Access Journals (Sweden)

    Hirai M


    Full Text Available Anti-idiotype antibodies (Ab2 play an important role in the homeostasis of immune responses and are related to the development and the disease activity of certain autoimmune diseases. The asialoglycoprotein receptor (ASGPR is considered one of the target antigens in the pathogenesis of autoimmune chronic active hepatitis (AIH. We previously developed a mouse monoclonal antibody (clone 8D7 which recognizes rat and human ASGPR. In this study, to help investigate the anti-ASGPR antibody-anti-idiotype antibody network in patients with AIH, we developed a syngeneic mouse monoclonal Ab2 to the 8D7 anti-ASGPR antibody (Ab1. One clone, designated as 3C8, tested positive for specific reactivity to 8D7-Ab1 and did not bind to other irrelevant immunoglobulins. By competitive inhibition assays, the binding of 8D7-Ab1 to liver membrane extracts, i.e., the crude antigen preparation, was inhibited by 3C8-Ab2 in a dose-dependent manner, and the binding of 8D7-Ab1 to 3C8-Ab2 was inhibited by the liver membrane extracts. In the immunohistochemical analysis, 3C8-Ab2 blocked the specific staining of sinusoidal margins of rat hepatocytes by 8D7-Ab1. These results suggest that 3C8 anti-idiotype antibody recognizes the specific idiotypic determinants within the antigen-binding site of 8D7-Ab1.

  3. [New immunological weapons for medicine in the 21st Century: biological therapy based on the use of the latest generation monoclonal antibodies]. (United States)

    Aguillón, Juan C; Contreras, Juan; Dotte, Andrés; Cruzat, Andrea; Catalán, Diego; Salazar, Lorena; Molina, María Carmen; Guerrero, Julia; López, Mercedes; Soto, Lilian; Salazar-Onfray, Flavio; Cuchacovich, Miguel


    The fusion of a murine B cell and a myeloma cell generates a hybridoma that produces monoclonal antibody (mAb). These murine mAb induce the HAMA (human anti-mouse antibodies) response. Murine mAb have been modified by genetic engineering, producing molecules with a higher proportion of human protein. At present, chimeric, humanized and fully human mAb are available. mAb block interactions between target molecules and their ligands or trigger the lyses of mAb-coated tumor cells. Numerous mAb have been developed using the recombinant DNA technology and several are available in the market. Trastuzumab, against HER2/neu, is useful in breast cancer; rituximab, against CD20 in B lymphocytes is useful in lymphoma; alemtuzumah, against CD52 is used in lymphoma and leukemia; daclizumab and basiliximab block the IL-2 receptor interaction and reduce acute rejection in kidney transplantation; abciximab, an antagonist of GPIIb/IIIa platelet receptor, is used in patients undergoing acute coronary syndromes. In autoimmunity diseases, blocking tumor necrosis factor by infliximab and adalimumab has demonstrated excellent results. Thus, infliximab is useful in the treatment of rheumatoid arthritis (RA), Crohn's disease and ulcerative colitis while adalimumab is the first fully human mAb available for RA. Infliximab and adalimumab reduce signs and symptoms in RA and they also interfere with progression of joint damage. Finally, the direct benefits of antagonist treatment can occur at the expense of a major adverse effect in some other biological function.

  4. Monoclonal antibodies against naturally occurring bioactive compounds. (United States)

    Shoyama, Y; Tanaka, H; Fukuda, N


    The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 mug/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 mug/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-ginsenoside Rb1 MAbs were produced. New western blotting method of determination for ginsenosides was established. Ginsenosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO(4) solution followed by BSA, resulting in a ginsenoside-BSA conjugate. Immunostaining of ginsenosides was more sensitive compared to other staining. Immunostaining of ginsenosides in the fresh ginseng root was succeeded using anti-ginsenoside Rb1 (GRb1) MAb after blotting to PVDF membrane.


    Institute of Scientific and Technical Information of China (English)

    FENGShu-Lin; BENKun-Long; LIANGZhi-Guo


    LDH--C4 is a sperm specific lactic dehydrogcnase in mammals and human, and is consid ered as a model molecule for contraceptive vaccine research. Significant contraceptive effects were observed in female mice, rabbits and baboons immunized with purified

  6. Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF. (United States)

    Weiser, W Y; Remold, H G; David, J R


    Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.

  7. Cloning and expression of murine immune interferon cDNA.



    The murine immune interferon (IFN-gamma) gene was cloned and expressed under control of the simian virus 40 early promoter in the monkey COS-1 cell line. A protein is secreted from these cells having the biological, antigenic, and biochemical characteristics of natural murine IFN-gamma. Cloned murine IFN-gamma cDNAs were obtained by using RNA from both mitogen-induced murine spleens and the transfected COS cells, and both code for identical proteins. The mature murine IFN-gamma encoded is 136...

  8. Structure of the murine Thy-1 gene

    NARCIS (Netherlands)

    V. Giguere; K-I. Isobe; F.G. Grosveld (Frank)


    textabstractWe have cloned the murine Thy-1.1 (AKR) and Thy-1.2 (Balb/c) genes. The complete exon/intron structure and the nucleotide sequence of the Thy-1.2 gene was determined. The gene contains four exons and three intervening sequences. The complete transcriptional unit gives rise to a tissue an

  9. Reemergence of Murine Typhus in the US

    Centers for Disease Control (CDC) Podcasts


    Dr. Lucas Blanton discusses the Reemergence of Murine Typhus in Galveston Texas in 2013.  Created: 4/21/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 4/27/2015.

  10. Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Bruckheimer


    Full Text Available EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID mice (which have functional NK cells and monocytes and SCID nonobese diabetic (NOD mice (which largely lack functional NK cells and monocytes. Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

  11. Unusual Manifestations of Monoclonal Gammopathy: I. Ocular Disease

    Directory of Open Access Journals (Sweden)

    Sophia R. Balderman


    Full Text Available Essential monoclonal gammopathy is usually an asymptomatic condition, the characteristics of which have been defined over approximately 70 years of study. It has a known population-attributable risk of undergoing clonal evolution to a progressive, symptomatic B-cell neoplasm. In a very small fraction of patients, the monoclonal immunoglobulin has biophysical characteristics that can lead to tissue deposition syndrome (e.g. Fanconi renal syndrome or, by chance, have characteristics of an autoantibody that may inactivate critical proteins (e.g. acquired von Willebrand disease. In this report, we describe the very uncommon forms of ocular injury that may accompany essential monoclonal gammopathy, which include crystalline keratopathy, crystal-storing histiocytosis, hypercupremic keratopathy, and maculopathy. The first three syndromes result from uncommon physicochemical alterations of the monoclonal immunoglobulin that favor crystallization or exaggerated copper binding. The last-mentioned syndrome is of uncertain pathogenesis. These syndromes may result in decreased visual acuity. These ocular findings may lead, also, to the diagnosis of monoclonal gammopathy.

  12. A Strategy for Screening Monoclonal Antibodies for Arabidopsis Flowers (United States)

    Shi, Qian; Zhou, Lian; Wang, Yingxiang; Ma, Hong


    The flower is one of the most complex structures of angiosperms and is essential for sexual reproduction. Current studies using molecular genetic tools have made great advances in understanding flower development. Due to the lack of available antibodies, studies investigating the localization of proteins required for flower development have been restricted to use commercial antibodies against known antigens such as GFP, YFP, and FLAG. Thus, knowledge about cellular structures in the floral organs is limited due to the scarcity of antibodies that can label cellular components. To generate monoclonal antibodies that can facilitate molecular studies of the flower, we constructed a library of monoclonal antibodies against antigenic proteins from Arabidopsis inflorescences and identified 61 monoclonal antibodies. Twenty-four of these monoclonal antibodies displayed a unique band in a western blot assay in at least one of the examined tissues. Distinct cellular distribution patterns of epitopes were detected by these 24 antibodies by immunofluorescence microscopy in a flower section. Subsequently, a combination of immunoprecipitation and mass spectrometry analysis identified potential targets for three of these antibodies. These results provide evidence for the generation of an antibody library using the total plant proteins as antigens. Using this method, the present study identified 61 monoclonal antibodies and 24 of them were efficiently detecting epitopes in both western blot experiments and immunofluorescence microscopy. These antibodies can be applied as informative cellular markers to study the biological mechanisms underlying floral development in plants. PMID:28293248

  13. Preparation and Identification of Anti-rabies Virus Monoclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    Wen-juan Wang; Xiong Li; Li-hua Wang; Hu Shan; Lei Cao; Peng-cheng Yu; Qing Tang; Guo-dong Liang


    To provide a foundation for the development of rapid and specific methods for the diagnosis of rabies virus infection,anti-rabies virus monoclonal antibodies were prepared and rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as immunogens to immunize 6-8 week old female BALB/c mice.Spleen cells and SP2/0 myeloma cells were fused according to conventional methods:the monoclonal cell strains obtained were selected using the indirect immunofluorescence test; this was followed by preparation of monoclonal antibody ascitic fluid; and finally,systematic identification of subclass,specificity and sensitivity was carried out.Two high potency and specific monoclonal antibodies against rabies virus were obtained and named 3B12 and 4A12,with ascitic fluid titers of 1∶8000 and 1∶10000,respectively.Both belonged to the IgG2a subclass.These strains secrete potent,stable and specific anti-rabies virus monoclonal antibodies,which makes them well suited for the development of rabies diagnosis reagents.

  14. ELISA Detection of Francisella tularensis using Polyclonaland Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Miroslav Pohanka


    Full Text Available The mouse monoclonal and polyclonal antibodies were produced for the detection of intracellular pathogenand potential warfare agent Francisella tularensis. Antibody titers obtained were 1:640 for polyclonal antibodiesand 1:320 for monoclonal antibodies. Both antibodies were used in the indirect enzyme-linked immunosorbentassay (ELISA found to detect F. tularensis whole cells. The limit of detection was 5.4×106 CFU/ml for polyclonalantibodies and 6.9×106 CFU/ml for monoclonal antibodies. The value sample could  be distinguished from anyconcentration of another gram-negative bacterium: Escherichia coli.Defence Science Journal, 2008, 58(5, pp.698-702, DOI:

  15. Immunoblotting with monoclonal antibodies: importance of the blocking solution. (United States)

    Hauri, H P; Bucher, K


    Four commonly used blocking agents, i.e., fetal calf serum, mammalian gelatin-Nonidet-P40, fish gelatin-Nonidet-P40, and defatted powdered milk were compared with respect to their efficiency to block the nonspecific background and to promote maximal immunoreactivity of monoclonal antibodies against human intestinal sucrase-isomaltase during immunoblotting. Two of five monoclonal antibodies were found to react with the electroblotted enzyme. However, one of the reacting antibodies gave optimal results with fish gelatin-Nonidet-P40 and the other with defatted powdered milk, while fetal calf serum lead to unacceptably high backgrounds. The results suggest that some of the difficulties encountered with monoclonal antibodies in immunoblotting may be due to inappropriate blocking conditions.

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

    DEFF Research Database (Denmark)

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


    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...

  17. Monoclonal gammopathy in hereditary spherocytosis: Possible pathogenetic relation

    Energy Technology Data Exchange (ETDEWEB)

    Schafer, A.I. (Univ. of Chicago); Miller, J.B.; Lester, E.P.; Bowers, T.K.; Jacob, H.S.


    Two cases of monoclonal gammopathy in patients with hereditary spherocytosis led us to consider the possible pathogenetic relation between these two disorders. Twelve adult patients with hereditary spherocytosis had significant hypergammaglobulinemia in comparison to normal subjects. Retrospective analysis of previous illness in 140 patients with multiple myeloma showed a significant association between IgA myeloma and previous gallbladder disease. We propose that the chronic reticuloendothelial stimulation due to extravascular hemolysis, possibly potentiated by the inflammation associated with cholelithiasis and cholecystitis, may foster neoplastic transformation of immunocytes in patients with hereditary spherocytosis, ultimately leading to the development of monoclonal gammopathy.

  18. Therapeutic monoclonal antibodies in human breast milk: a case study. (United States)

    Ross, Elle; Robinson, Steven E; Amato, Carol; McMillan, Colette; Westcott, Jay; Wolf, Tiffany; Robinson, William A


    Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

  19. Radiolabelled peptides and monoclonal antibodies for therapy decision making in inflammatory diseases

    NARCIS (Netherlands)

    Malviya, G.; Signore, A.; Lagana, B.; Dierckx, R. A.


    Radiolabelled peptides and monoclonal antibodies are an emerging class of radiopharmaceuticals for imaging inflammation with clinical implications for several chronic inflammatory disorders for diagnosis, therapy decision making and follow up. In the last decades, a number of novel monoclonal antibo

  20. Uptake of 111In-labeled fully human monoclonal antibody TSP-A18 reflects transferrin receptor expression in normal organs and tissues of mice. (United States)

    Sugyo, Aya; Tsuji, Atsushi B; Sudo, Hitomi; Nomura, Fumiko; Satoh, Hirokazu; Koizumi, Mitsuru; Kurosawa, Gene; Kurosawa, Yoshikazu; Saga, Tsuneo


    Transferrin receptor (TfR) is an attractive molecule for targeted therapy of cancer. Various TfR-targeted therapeutic agents such as anti-TfR antibodies conjugated with anticancer agents have been developed. An antibody that recognizes both human and murine TfR is needed to predict the toxicity of antibody-based agents before clinical trials, there is no such antibody to date. In this study, a new fully human monoclonal antibody TSP-A18 that recognizes both human and murine TfR was developed and the correlation analysis of the radiolabeled antibody uptake and TfR expression in two murine strains was conducted. TSP-A18 was selected using extracellular portions of human and murine TfR from a human antibody library. The cross-reactivity of TSP-A18 with human and murine cells was confirmed by flow cytometry. Cell binding and competitive inhibition assays with [111In]TSP-A18 showed that TSP-A18 bound highly to TfR-expressing MIAPaCa-2 cells with high affinity. Biodistribution studies of [111In]TSP-A18 and [67Ga]citrate (a transferrin-mediated imaging probe) were conducted in C57BL/6J and BALB/c-nu/nu mice. [111In]TSP-A18 was accumulated highly in the spleen and bone containing marrow component of both strains, whereas high [67Ga]citrate uptake was only observed in bone containing marrow component and not in the spleen. Western blotting indicated the spleen showed the strongest TfR expression compared with other organs in both strains. There was significant correlation between [111In]TSP-A18 uptake and TfR protein expression in both strains, whereas there was significant correlation of [67Ga]citrate uptake with TfR expression only in C57BL/6J. These findings suggest that the difference in TfR expression between murine strains should be carefully considered when testing for the toxicity of anti-TfR antibody in mice and the uptake of anti-TfR antibody could reflect tissue TfR expression more accurately compared with that of transferrin-mediated imaging probe such as [67Ga]citrate.

  1. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough. (United States)

    Nguyen, Annalee W; Wagner, Ellen K; Laber, Joshua R; Goodfield, Laura L; Smallridge, William E; Harvill, Eric T; Papin, James F; Wolf, Roman F; Padlan, Eduardo A; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A


    Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

  2. Murine Typhus: Clinical and epidemiological aspects

    Directory of Open Access Journals (Sweden)

    Gaspar Peniche Lara


    Full Text Available Rickettsia typhi is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against R. typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of Rickettsia typhi are rats (some species belonging the Rattus Genus and fleas (Xenopsylla cheopis are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi.

  3. Advances in Murine Models of Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Li-li Kong


    Full Text Available Diabetic nephropathy (DN is one of the microvascular complications of both type 1 and type 2 diabetes, which is also associated with a poor life expectancy of diabetic patients. However, the pathogenesis of DN is still unclear. Thus, it is of great use to establish appropriate animal models of DN for doing research on pathogenesis and developing novel therapeutic strategies. Although a large number of murine models of DN including artificially induced, spontaneous, and genetically engineered (knockout and transgenic animal models have been developed, none of them develops renal changes sufficiently reflecting those seen in humans. Here we review the identified murine models of DN from the aspects of genetic background, type of diabetes, method of induction, gene deficiency, animal age and gender, kidney histopathology, and phenotypic alterations in the hope of enhancing our comprehension of genetic susceptibility and molecular mechanisms responsible for this disease and providing new clues as to how to choose appropriate animal models of DN.

  4. Immunodetection of Murine Lymphotoxins in Eukaryotic Cells. (United States)

    Boitchenko, Veronika E.; Korobko, Vyacheslav G.; Prassolov, Vladimir S.; Kravchenko, Vladimir V.; Kuimov, Alexander N.; Turetskaya, Regina L.; Kuprash, Dmitry V.; Nedospasov, Sergei A.


    Lymphotoxins alpha and beta (LTalpha and LTbeta) are members of tumor necrosis factor superfamily. LT heterotrimers exist on the surface of lymphocytes and signal through LTbeta receptor while soluble LTalpha homotrimer can signal through TNF receptors p55 and p75. LT-, as well as TNF-mediated signaling are important for the organogenesis and maintenance of microarchitecture of secondary lymphoid organs in mice and has been implicated in the mechanism of certain inflammatory syndromes in humans. In this study we describe the generation of eukaryotic expression plasmids encoding murine LTalpha and LTbeta genes and a prokaryotic expression construct for murine LTalpha. Using recombinant proteins expressed by these vectors as tools for antisera selection, we produced and characterized several polyclonal antibodies capable of detecting LT proteins in eukaryotic cells.

  5. Murine models of human wound healing. (United States)

    Chen, Jerry S; Longaker, Michael T; Gurtner, Geoffrey C


    In vivo wound healing experiments remain the most predictive models for studying human wound healing, allowing an accurate representation of the complete wound healing environment including various cell types, environmental cues, and paracrine interactions. Small animals are economical, easy to maintain, and allow researchers to take advantage of the numerous transgenic strains that have been developed to investigate the specific mechanisms involved in wound healing and regeneration. Here we describe three reproducible murine wound healing models that recapitulate the human wound healing process.

  6. The clinical relevance and management of monoclonal gammopathy of undetermined significance and related disorders

    DEFF Research Database (Denmark)

    van de Donk, Niels W C J; Palumbo, Antonio; Johnsen, Hans Erik


    Monoclonal gammopathy of undetermined significance is one of the most common pre-malignant disorders. IgG and IgA monoclonal gammopathy of undetermined significance are precursor conditions of multiple myeloma; light-chain monoclonal gammopathy of undetermined significance of light-chain multiple...

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

    NARCIS (Netherlands)

    Booman, P.


    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

  8. Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Bo Wang


    Full Text Available Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS, has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa. Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

  9. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

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


    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...


    Directory of Open Access Journals (Sweden)

    Duarte Keila M.R.


    Full Text Available Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV isolated in Brazil. One antibody (8G7G2 isotyped as IgG2b (kappa light chain showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV. It can be used in identification of tomato mosaic virus (ToMV.

  11. Development of monoclonal antibodies that recognize Treponema pallidum.


    Saunders, J M; Folds, J D


    We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay.

  12. Monoclonal Antibodies to Prevent Use of Mycotoxins as Biological Weapons (United States)


    Mycotoxins as Biological Weapons PRINCIPAL INVESTIGATOR: Marta Feldmesser, M.D. CONTRACTING ORGANIZATION: Albert Einstein College of...Monoclonal Antibodies to Prevent Use of Mycotoxins as Biological Weapons 5b. GRANT NUMBER W81XWH-06-1-0085 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR

  13. Monoclonal antibodies for the control of influenza virus vaccines.

    NARCIS (Netherlands)

    H.J.M. van de Donk; M.F. van Olderen; A.D.M.E. Osterhaus (Albert); J.C. de Jong (Jan)


    textabstractHybridomas producing haemagglutination inhibiting monoclonal antibodies against influenza A/Texas/1/77 H3N2 were developed. One hybridoma producing antibodies reacting with Victoria/3/75, Texas/1/77 Bangkok/1/79 and England/496/80 was selected to determine the potency of influenza virusv

  14. 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


    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

  15. Prevention of progression in monoclonal gammopathy of undetermined significance. (United States)

    Rajkumar, S Vincent


    Monoclonal gammopathy of undetermined significance (MGUS) is a common premalignant plasma cell proliferative disorder with a lifelong risk of progression to multiple myeloma. Because myeloma is an incurable malignancy, strategies to delay or prevent progression in high-risk patients are of considerable importance.

  16. Serological comparison of tospovirus isolates using polyclonal and monoclonal antibodies.

    NARCIS (Netherlands)

    Adam, G.; Peters, D.; Goldbach, R.W.


    A test was conducted to compare tospovirus isolates using different poly- and monoclonal antibodies. All isolates and antibodies were compared under identical conditions. From 130 tospovirus isolates, which were obtained from all over the world and included well-characterized isolates from all four

  17. Monoclonal antibodies specific for the organophosphate pesticide azinphos-methyl

    NARCIS (Netherlands)

    Jones, WT; Harvey, D; Jones, SD; Ryan, GB; Wynberg, H; TenHoeve, W; Reynolds, PHS


    2-(2-Mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl,2-sulphide) methoxyacetic acid has been synthesized and used to prepare an azinphos hapten and protein conjugates. Monoclonal antibodies of high affinity against the pesticide azinphos-methyl were prepared from mice immunized with the hapten-ovalbu

  18. A mouse monoclonal antibody against Alexa Fluor 647. (United States)

    Wuethrich, Irene; Guillen, Eduardo; Ploegh, Hidde L


    Fluorophores are essential tools in molecular and cell biology. However, their application is mostly confined to the singular exploitation of their fluorescent properties. To enhance the versatility and expand the use of the fluorophore Alexa Fluor 647 (AF647), we generated a mouse monoclonal antibody against it. We demonstrate its use of AF647 for immunoblot, immunoprecipitation, and cytofluorimetry.

  19. Medullary carcinomas of the thyroid: a monoclonal origin. (United States)

    Marques, A R; Catarino, A L; Moniz, S; Cavaco, B; Roque, L; Sobrinho, L; Leite, V


    We studied the clonality of medullary thyroid carcinomas (MTC) from 16 female patients by determining X chromosome inactivation by polymerase chain reaction (PCR) amplification of a CAG repeat in exon 1 of the human androgen-receptor gene. One patient with sporadic medullary thyroid carcinoma (MTC) was homozygous for this microsatellite and was not considered for the assessment of clonality. Sixteen tumor samples from the informative 15 patients were studied: 11 were from sporadic cases and 5 were from familial cases (3 cases of multiple endocrine neoplasia type 2A [MEN 2A]; 1 case of familial medullary thyroid carcinoma [FMTC]). Fourteen tumor samples (10/11 sporadic, 3/4 MEN 2A and 1/1 FMTC) were clearly monoclonal with allelic cleavage ratios between 2.5 and 49.1. Sixty-four percent of these cases (9/14) had the preferential amplification of the shorter allele while 36 percent (5/14) had the preferential amplification of the longer allele. Two frozen tumor samples (1 sporadic and 1 MEN 2A) were polyclonal. However, the corresponding tumor embedded in paraffin from the sporadic case was monoclonal. The other polyclonal tumor was found in the right thyroid lobe of a patient with MEN 2A who had a monoclonal tumor in the left lobe. Our results clearly demonstrate that MTC have a monoclonal origin in the majority of the cases.

  20. Monoclonal Gammopathy of Undetermined Significance Disguised as Chronic Neutrophilic Leukemia

    Directory of Open Access Journals (Sweden)

    Monique A Hartley-Brown


    Full Text Available A 60-year-old woman with a medical history of diabetes mellitus, osteoporosis, peripheral vascular disease, and hypertension who was otherwise asymptomatic but continued showing elevated neutrophil levels sought a second opinion at our facility. Serum protein immunoelectrophoresis with immunofixation revealed an immunoglobulin A (IgA-κ monoclonal gammopathy concentration of 1305 mg/dL (normal 80-350 mg/dL but relatively normal concentrations of IgG of 840 mg/dL (620-1400 mg/dL and IgM of 36 mg/dL (45-250 mg/dL. Clonal analysis revealed a polyclonal expression pattern in all cell types analyzed. We concluded that our patient’s neutrophilia may have been due to the underlying monoclonal gammopathy. This is the first case in the literature of a patient with monoclonal gammopathy of undetermined significance presenting with neutrophilia, suggestive of chronic neutrophilic leukemia (CNL.  Patients with CNL have a poor prognosis; therefore, it is important to distinguish diagnostically between CNL and the less severe prognosis of monoclonal gammopathy of undetermined significance.

  1. Monoclonal Gammopathy of Undetermined Significance Disguised as Chronic Neutrophilic Leukemia

    Directory of Open Access Journals (Sweden)

    Monique A Hartley-Brown


    Full Text Available A 60-year-old woman with a medical history of diabetes mellitus, osteoporosis, peripheral vascular disease, and hypertension who was otherwise asymptomatic but continued showing elevated neutrophil levels sought a second opinion at our facility. Serum protein immunoelectrophoresis with immunofixation revealed an immunoglobulin A (IgA-κ monoclonal gammopathy concentration of 1305 mg/dL (normal 80-350 mg/dL but relatively normal concentrations of IgG of 840 mg/dL (620-1400 mg/dL and IgM of 36 mg/dL (45-250 mg/dL. Clonal analysis revealed a polyclonal expression pattern in all cell types analyzed. We concluded that our patient’s neutrophilia may have been due to the underlying monoclonal gammopathy. This is the first case in the literature of a patient with monoclonal gammopathy of undetermined significance presenting with neutrophilia, suggestive of chronic neutrophilic leukemia (CNL.  Patients with CNL have a poor prognosis; therefore, it is important to distinguish diagnostically between CNL and the less severe prognosis of monoclonal gammopathy of undetermined significance.

  2. A monoclonal antibody interferes with TIMP-2 binding and incapacitates the MMP-2-activating function of multifunctional, pro-tumorigenic MMP-14/MT1-MMP

    DEFF Research Database (Denmark)

    Shiryaev, S A; Remacle, A G; Golubkov, V S;


    Matrix metalloproteinases (MMPs) and, especially membrane type 1 (MT1)-MMP/MMP-14, are promising drug targets in malignancies. In contrast with multiple small-molecule and protein pan-inhibitors of MT1-MMP cleavage activity, the murine 9E8 monoclonal antibody targets the MMP-2-activating function...... tissue inhibitor of metalloproteinases-2 (TIMP-2) association with MT1-MMP. As a result, the 9E8 antibody incapacitates the TIMP-2-dependent MMP-2-activating function alone rather than the general enzymatic activity of human MT1-MMP. The specific function of the 9E8 antibody we determined directly...... supports an essential, albeit paradoxical, role of the protein inhibitor (TIMP-2) in MMP-2 activation via a unique membrane-tethered mechanism. In this mechanism, the formation of a tri-molecular MT1-MMPTIMP-2MMP-2 complex is required for both the capture of the soluble MMP-2 proenzyme by cells...

  3. Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein

    Directory of Open Access Journals (Sweden)

    A Malik


    Full Text Available Introduction: Emerging virulent strains of influenza virus pose a serious public health threat with potential pandemic consequences. A novel avian influenza virus, H7N9, breached the species barrier from infected domestic poultry to humans in 2013 in China. Since then, it has caused numerous infections in humans with a close contact to poultry. Materials and Methods: In this study, we describe the preliminary characterisation of five murine monoclonal antibodies (MAbs developed against recombinant haemagglutinin (rHA protein of avian H7N9 A/Anhui/1/2013 virus by their Western blot and enzyme-linked immunosorbent assay (ELISA reactivity and binding affinity. Results: Of the five MAbs, four were highly specific to H7N9 HA and did not show any cross-reactivity in ELISA with rHA protein from pandemic as well as seasonal H1N1, H2N2, H3N2, H5N1 and influenza virus B (B/Brisbane/60/2008. However, one of the MAbs, MA-24, in addition to HA protein of H7N9 also reacted strongly with HA protein of H3N2 and weakly with HA of pandemic and seasonal H1N1 and H2N2. All the five MAbs also reacted with H7N9 rHA in Western blot. The MAbs bound H7N9 rHA with an equilibrium dissociation constant (KD ranging between 0.14 and 25.20 nM, indicating their high affinity to HA. Conclusions: These antibodies may be useful in developing diagnostic tools for the detection of influenza H7N9 virus infections.

  4. Protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Lindsay Crickard

    Full Text Available Smallpox (variola virus is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs against vaccinia H3 (H3L and B5 (B5R, targeting both the mature virion (MV and extracellular enveloped virion (EV forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP studies examining severe combined immunodeficiency mice (SCID given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG. Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV. In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans.

  5. Generation and Characterization of Monoclonal Antibodies Specific to Avian Influenza H5N1 Hemagglutinin Protein. (United States)

    Malik, Ankita; Mallajosyula, V Vamsee Aditya; Mishra, Nripendra Nath; Varadarajan, Raghavan; Gupta, Satish Kumar


    Highly pathogenic avian influenza (HPAI) H5N1 virus has in the past breached the species barrier from infected domestic poultry to humans in close contact. Although human-to-human transmission has previously not been reported, HPAI H5N1 virus has pandemic potential owing to gain of function mutation(s) and/or genetic reassortment with human influenza A viruses. Monoclonal antibodies (MAbs) have been used for diagnosis as well as specific therapeutic candidates in several disease conditions including viral infections in humans. In this study, we describe the preliminary characterization of four murine MAbs developed against recombinant hemagglutinin (rHA) protein of avian H5N1 A/turkey/Turkey/1/2005 virus that are either highly specific or broadly reactive against HA from other H5N1 subtype viruses, such as A/Hong Kong/213/03, A/Common magpie/Hong Kong/2256/2006, and A/Barheaded goose/Quinghai/14/2008. The antibody binding is specific to H5N1 HAs, as none of the antibodies bound H1N1, H2N2, H3N2, or B/Brisbane/60/2008 HAs. Out of the four MAbs, one of them (MA-7) also reacted weakly with the rHA protein of H7N9 A/Anhui/1/2013. All four MAbs bound H5 HA (A/turkey/Turkey/1/2005) with high affinity with an equilibrium dissociation constant (KD) ranging between 0.05 and 10.30 nM. One of the MAbs (MA-1) also showed hemagglutination inhibition activity (HI titer; 31.25 μg/mL) against the homologous A/turkey/Turkey/1/2005 H5N1 virus. These antibodies may be useful in developing diagnostic tools for detection of influenza H5N1 virus infection.

  6. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. (United States)

    Roehrig, J T; Bolin, R A; Kelly, R G


    Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-borne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independent regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDa tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-borne encephalitis virus, determination of domain C was more problematic; however, at least four epitopes had biochemical characteristics consistent with C-domain epitopes.

  7. Variation of uptake of anti-CEA monoclonal antibody with tumor type and mass

    Energy Technology Data Exchange (ETDEWEB)

    Williams, L.E.; Philben, V.J.; Jakowatz, J.G.; Beatty, B.G.; Vlahos, W.G.; Paxton, R.J.; Shively, J.E.; Beatty, J.D.


    A nude mouse model xenografted with 3 human tumor (T) was studied with an anti-carcinoembryonic (..cap alpha..-CEA) monoclonal antibody (MoAb). The MoAb was labeled with In-111 using a bi-functional chelation technique. In vitro cross-reactivity with human blood (B) and liver (L) cells was minimal. Human colon tumors were WIDR, SW403 an LS174T. The murine carcinoma EMT6 was used as a control. In all cases only 62.5 ngm of ..cap alpha..-CEA charged at 10 was given to each animal. The corresponding value in humans, 200, is probably subimmunogenic. Organ distribution in percent injected dose/gm (% ID/gm) and images were obtained at 48 h post-injection of the MoAb. CEA levels (mgm/gm of T) were measured for each tumor using the same MoAb (T 84.66). Variation of % ID/gm with LS174T mass (m) was also determined. Uptake by EMT6 was 2.4 +- 0.2 % ID/gm. LS174T uptake varied approximately as the inverse of tumor mass. The authors conclude that tumor accumulation of ..cap alpha..-CEA MoAb is not directly correlated with the amount of CEA in the lesion. The best uptake, T/B and T/L values occurred with LS174T; this was also borne out by the 48 h images. Because of the smaller average SW403 mass, this result cannot readily be explained as a tumor size effect.

  8. Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

    Directory of Open Access Journals (Sweden)

    Qian Yajuan


    Full Text Available Abstract Background Cucumber green mottle mosaic virus (CGMMV, a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA, triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA, Dot-immunobinding assay (DBIA, direct tissue blot immunoassay (DTBIA and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR were described for detection and diagnosis of CGMMV. Results Using the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA. Conclusions The established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.

  9. Mechanism of quinine-dependent monoclonal antibody binding to platelet glycoprotein IIb/IIIa. (United States)

    Bougie, Daniel W; Peterson, Julie; Rasmussen, Mark; Aster, Richard H


    Drug-dependent antibodies (DDAbs) that cause acute thrombocytopenia upon drug exposure are nonreactive in the absence of the drug but bind tightly to a platelet membrane glycoprotein, usually α(IIb)/β3 integrin (GPIIb/IIIa) when the drug is present. How a drug promotes binding of antibody to its target is unknown and is difficult to study with human DDAbs, which are poly-specific and in limited supply. We addressed this question using quinine-dependent murine monoclonal antibodies (mAbs), which, in vitro and in vivo, closely mimic antibodies that cause thrombocytopenia in patients sensitive to quinine. Using surface plasmon resonance (SPR) analysis, we found that quinine binds with very high affinity (K(D) ≈ 10⁻⁹ mol/L) to these mAbs at a molar ratio of ≈ 2:1 but does not bind detectably to an irrelevant mAb. Also using SPR analysis, GPIIb/IIIa was found to bind monovalently to immobilized mAb with low affinity in the absence of quinine and with fivefold greater affinity (K(D) ≈ 2.2 × 10⁻⁶) when quinine was present. Measurements of quinine-dependent binding of intact mAb and fragment antigen-binding (Fab) fragments to platelets showed that affinity is increased 10 000- to 100 000-fold by bivalent interaction between antibody and its target. Together, the findings indicate that the first step in drug-dependent binding of a DDAb is the interaction of the drug with antibody, rather than with antigen, as has been widely thought, where it induces structural changes that enhance the affinity/specificity of antibody for its target epitope. Bivalent binding may be essential for a DDAb to cause thrombocytopenia.

  10. Mapping of epitopes on Poa p I and Lol p I allergens with monoclonal antibodies. (United States)

    Lin, Z W; Ekramoddoullah, A K; Jaggi, K S; Dzuba-Fischer, J; Rector, E; Kisil, F T


    Allergen Poa p I isolated from the dialysed aqueous extract of Kentucky blue grass pollen by affinity chromatography with an anti-Lol p I murine monoclonal antibody (MAb) 290A-167 was previously shown to consist of a 35.8-kilodalton (kD) component with a pI of 6.4, designated as Poa p Ia, and a 33-kD component with a pI of 9.1, designated as Poa p Ib. The present study reports on the comparative antigenic analyses of these two components, using MAbs produced separately against Poa p I and Lol p I. Thus, anti-Poa p I MAbs 60 and 61 and anti-Lol p I MAb 290A-167 recognized Poa p Ia and Poa p Ib whereas anti-Poa p I MAbs 62, 63 and 64 and anti-Lol p I MAb 348A-6 recognized only Poa p Ia. The specificities of the MAbs were further resolved by comparing their respective abilities to inhibit the binding of 125I-Poa p I or 125I-Lol p I to the different MAbs prepared in the form of solid phase. These studies revealed that at least 4 distinct epitopes (designated as E1, E2, E3 and E4) were shared by both Poa p I and Lol p I. All 4 epitopes were present on Poa p Ia whereas only E1 and E3 were detected on Poa p Ib. E1 was recognized by MAbs 60 and 61, E2 by MAbs 62, 63 and 64, E3 by MAb 290A-167 and E4 by MAb 348A-6.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Imaging of cutaneous T cell lymphoma (CTCL) with In-111-T101 monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Carrasquillo, J.A.; Bunn, P.A.; Keenan, A.M.; Reynolds, J.C.; Schroff, R.W.; Foon, K.A.; Ming-Hsu, S.; Gazdar, A.F.; Mulshine, J.M.; Perentesis, P.


    T101 is a murine monoclonal antibody (MoAb), IgC2a, directed against a cell surface pan T-cell antigen present in high concentration in CTCL cells. In-111 labelling was performed with a modification of the Krejcarek method (Hybritech, Inc.). I mg of DTPA conjugated T101 was labeled with 5 mCi, with a mean incorporation of 95%. Immunoreactivity was preserved, mean 88%. In vivo, less than 3.6% of the injected dose was on circulating transferrin. 11 patients (pts) received 2-6h intravenous infusion of 1 mg (5 pts), 10 mg (3 pts), 50 mg (3 pts) of In-111 T101. By 24h all pts showed avid uptake in pathologically or clinically involved nodes and erythroderma including several previously unsuspected nodal regions. Skin plaques were not visualized. In addition, there was localization in liver, spleen and bone marrow. Concentration of In-111 in biopsied nodes was 0.01, 0.02 and 0.03% of the injected dose per gram. Control studies with In-111Cl/sub 3/ or a nonspecific MoAb, 9.2.27, did not concentrate in nodes or skin disease. No dose dependent differences in tumor localization was seen although blood clearance was prolonged for doses less than or equal to 10 mgs of T101. All pts receiving less than or equal to 10 mgs developed transient itching, urticaria and chills. 1 of 8 pts tested had an antimouse immune response. Modulation of the antigen from circulating T-cells, skin and nodes was seen. This study shows the feasibility of imaging CTCL pts with In-111 T101 and suggest a potential for radioimmunotherapy.

  12. DMPD: The actions of bacterial DNA on murine macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10534106 The actions of bacterial DNA on murine macrophages. Sester DP, Stacey KJ, ... Show The actions of bacterial DNA on murine macrophages. PubmedID 10534106 Title The actions of bacterial DNA on murine macrophage

  13. Quality by design: impact of formulation variables and their interactions on quality attributes of a lyophilized monoclonal antibody. (United States)

    Awotwe-Otoo, David; Agarabi, Cyrus; Wu, Geoffrey K; Casey, Elizabeth; Read, Erik; Lute, Scott; Brorson, Kurt A; Khan, Mansoor A; Shah, Rakhi B


    The purpose of this study was to use QbD approaches to evaluate the effect of several variables and their interactions on quality of a challenging model murine IgG3κ monoclonal antibody (mAb), and then to obtain an optimized formulation with predefined quality target product profile. This antibody was chosen because it has a propensity to precipitate and thus represents a challenge condition for formulation development. Preliminary experiments were conducted to rule out incompatible buffer systems for the mAb product quality. A fractional factorial experimental design was then applied to screen the effects of buffer type, pH and excipients such as sucrose, sodium chloride (NaCl), lactic acid and Polysorbate 20 on glass transition temperature ( [Formula: see text] ), monoclonal antibody concentration (A(280)), presence of aggregation, unfolding transition temperature (T(m)) of the lyophilized product, and particle size of the reconstituted product. A Box-Behnken experimental design was subsequently applied to study the main, interaction, and quadratic effects of these variables on the responses. Pareto ranking analyses showed that the three most important factors affecting the selected responses for this particular antibody were pH, NaCl, and Polysorbate 20. The presence of curvature in the variables' effects on responses indicated interactions. Based on the constraints set on the responses, a design space was identified for this mAb and confirmed with experiments at three different levels of the variables within the design space. The model indicated a combination of high pH (8) and NaCl (50mM) levels, and a low Polysorbate 20 (0.008 mM) level at which an optimal formulation of the mAb could be achieved. Moisture contents and other analytical procedures such as size exclusion chromatography, protein A analysis and SDS-PAGE of the pre-lyophilized and final reconstituted lyophilized products indicated an intact protein structure with minimal aggregation after

  14. A monoclonal autoantibody that promotes central nervous system remyelination in a model of multiple sclerosis is a natural autoantibody encoded by germline immunoglobulin genes

    Energy Technology Data Exchange (ETDEWEB)

    Miller, D.J.; Rodriguez, M. [Mayo Clinic and Foundation, Rochester, MN (United States)


    Antibodies directed against self-Ags are frequently considered detrimental, and have been shown to play a pathogenic role in certain autoimmune diseases. However, the presence of autoreactive Abs in normal individuals suggests that some autoantibodies could participate in normal physiology. Our previous studies demonstrated that monoclonal autoantibodies SCH94.03 and SCH94.32, generated from the splenocytes of uninfected SJL/J mice injected with normal homogenized spinal cord, promote central nervous system remyelination when passively transferred into syngeneic mice chronically infected with Theiler`s murine encephalomyelitis virus, an established experimental model of multiple sclerosis. In this study we show that these two monoclonal autoantibodies are identical, and have phenotypic characteristics of natural autoantibodies. By using a solid phase assay system, SCH94.03 and SCH94.32 showed reactivity toward several protein Ags and chemical haptens, with prominent reactivity toward spectrin, (4-hydroxy-3-nitrophenyl) acetyl, and fluorescein. Sequence analysis showed that both SCH94.03 and SCH94.32 were encoded by identical germline Ig light chain V{sub K}10/J{sub K}l and heavy chain V23/DFL16.1/J{sub H}2 genes, with no definitive somatic mutations. These results indicate that a natural autoantibody participates in a beneficial physiologic response to central nervous system injury. 60 refs., 7 figs.

  15. Characterization of a proapoptotic antiganglioside GM2 monoclonal antibody and evaluation of its therapeutic effect on melanoma and small cell lung carcinoma xenografts. (United States)

    Retter, Marc W; Johnson, Jeffrey C; Peckham, David W; Bannink, Jeannette E; Bangur, Chaitanya S; Dresser, Karen; Cai, Feng; Foy, Teresa M; Fanger, Neil A; Fanger, Gary R; Woda, Bruce; Rock, Kenneth L


    Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.

  16. Coarse grained modeling of transport properties in monoclonal antibody solution (United States)

    Swan, James; Wang, Gang

    Monoclonal antibodies and their derivatives represent the fastest growing segment of the bio pharmaceutical industry. For many applications such as novel cancer therapies, high concentration, sub-cutaneous injections of these protein solutions are desired. However, depending on the peptide sequence within the antibody, such high concentration formulations can be too viscous to inject via human derived force alone. Understanding how heterogenous charge distribution and hydrophobicity within the antibodies leads to high viscosities is crucial to their future application. In this talk, we explore a coarse grained computational model of therapeutically relevant monoclonal antibodies that accounts for electrostatic, dispersion and hydrodynamic interactions between suspended antibodies to predict assembly and transport properties in concentrated antibody solutions. We explain the high viscosities observed in many experimental studies of the same biologics.

  17. Murine erythrocytes contain high levels of lysophospholipase activity

    NARCIS (Netherlands)

    Kamp, J.A.F. op den; Roelofsen, B.; Sanderink, G.; Middelkoop, E.; Hamer, R.


    Murine erythrocytes were found to be unique in the high levels of lysophospholipase activity in the cytosol of these cells. The specific activity of the enzyme in the cytosol of the murine cells is 10-times higher than in the cytosol of rabbit erythrocytes and approximately three orders of magnitude

  18. Strategies for Treating Autoimmune Disease With Monoclonal Antibodies


    Wofsy, David


    There is no safe and reliable therapy for most serious autoimmune diseases, such as systemic lupus erythematosus. Severe cases usually require treatment with corticosteroids or cytotoxic drugs or both, which frequently provide inadequate disease control and can cause serious complications. These therapies are not restricted in their effects to cells of the immune system, but rather have a broad range of toxic effects on cells throughout the body. The development of monoclonal antibodies has l...

  19. Monoclonal antibody to native P39 protein from Borrelia burgdorferi.


    Sullivan, T J; Hechemy, K E; Harris, H L; Rudofsky, U H; Samsonoff, W A; Peterson, A J; Evans, B. D.; Balaban, S L


    We have produced, by using a sonicate of Borrelia burgdorferi, a monoclonal antibody (MAb), NYSP39H, that is specific for the P39 protein band. This MAb reacted with 13 isolates of B. burgdorferi but not with eight different spirochetes (four borrelias, two leptospiras, and two treponemas). Surface labeling of B. burgdorferi with biotin and subsequent treatment with Nonidet P-40 showed that P39 was not biotinylated but was extracted with Nonidet P-40, indicating that it is present within the ...

  20. A monoclonal antibody for G protein-coupled receptor crystallography

    DEFF Research Database (Denmark)

    Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles


    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural inf...... information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals....

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

    Directory of Open Access Journals (Sweden)



    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. A monoclonal antibody for G protein-coupled receptor crystallography. (United States)

    Day, Peter W; Rasmussen, Søren G F; Parnot, Charles; Fung, Juan José; Masood, Asna; Kobilka, Tong Sun; Yao, Xiao-Jie; Choi, Hee-Jung; Weis, William I; Rohrer, Daniel K; Kobilka, Brian K


    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

  3. Quantification of Moraxella bovis haemagglutinating adhesins with monoclonal antibodies. (United States)

    Gil-Turnes, C; Aleixo, J A


    Six monoclonal antibodies (MAbs) against Moraxella bovis GF 9 were used to quantify haemagglutinating adhesins of 16 strains of this organism. The amount of each MAb necessary to inhibit one haemagglutinating unit of each strain varied between 4 and 0.007 times that required by strain GF 9. Five strains reacted with six MAbs, one with five, two with four, one with three, two with two and three with none. The procedures used enabled to detect dominant strains candidates for vaccines.

  4. Super-Genotype: Global Monoclonality Defies the Odds of Nature


    Johannes J Le Roux; Wieczorek, Ania M.; Wright, Mark G.; Carol T Tran


    The ability to respond to natural selection under novel conditions is critical for the establishment and persistence of introduced alien species and their ability to become invasive. Here we correlated neutral and quantitative genetic diversity of the weed Pennisetum setaceum Forsk. Chiov. (Poaceae) with differing global (North American and African) patterns of invasiveness and compared this diversity to native range populations. Numerous molecular markers indicate complete monoclonality with...

  5. Recent Progress toward Engineering HIV-1-Specific Neutralizing Monoclonal Antibodies


    Ming Sun; Yue Li; Huiwen Zheng; Yiming Shao


    The recent discoveries of broadly potent neutralizing human monoclonal antibodies represent a new generation of antiretrovirals for the treatment and prophylaxis. Antibodies are generally considered more effective and safer and have been proved to provide passive protection against mucosal challenge in humanized mice and macaques. Several neutralizing Abs could protect animals against HIV-1 but are not effective when used in an established infected model for therapy. In order to overcome the ...

  6. Prevalence of monoclonal gammopathy of undetermined significance in Thailand. (United States)

    Watanaboonyongcharoen, Phandee; Nakorn, Thanyaphong Na; Rojnuckarin, Ponlapat; Lawasut, Panisinee; Intragumtornchai, Tanin


    Individuals with monoclonal gammopathy of undetermined significance (MGUS) develop multiple myeloma and related malignancies at the rate of 1% per year. Given differences in ethnicity, data on prevalence and risk factors of MGUS in Thai population will be helpful in understanding the pathogenesis of plasma cell disorders and designing an early cancer detection strategy. Subjects of 50 years or older were included. Demographic data and suspected risk factors were collected. Monoclonal proteins were detected using serum protein electrophoresis. Serum was obtained from 3,260 participants; 1,104 males (33.9%) and 2,156 females (66.1%). The median age was 57 years (range 50-93 years). Monoclonal proteins were detectable in 2.3% (95% confidence interval [CI] 1.8-2.8). M spikes were found in gamma- and beta-globulin regions in 50 (1.5%) and 25 (0.8%) subjects, respectively. The prevalence of MGUS in subjects 50-59, 60-69, and 70 years or older was 2.0% (41/1,975), 2.6% (22/851), and 2.8% (12/434), respectively. By multivariate analysis, MGUS was associated with living outside Bangkok (odds ratio 2.25, 95% CI 1.11-4.58). The overall prevalence of MGUS in the Thai population was 2.3%, which was lower than that in Western countries, but comparable to that in Japan.

  7. Heterogeneity of monoclonal antibodies revealed by charge-sensitive methods. (United States)

    Vlasak, J; Ionescu, R


    The expanding field of monoclonal antibody-based pharmaceuticals has triggered increased interest in analytical characterization of these large proteins and in understanding of their heterogeneity and degradation pathways. As a result, a large number of enzymatic modifications as well as chemical and physical degradations have been reported in monoclonal antibodies in recent years. Most heterogeneity is related to changes in the surface charge of the antibody, either directly, as a change in the number of charged residues, or indirectly as a chemical or physical alteration that changes surface-charge distribution. This review presents an overview of the sources of charge-related heterogeneity in monoclonal antibodies and the methods used for their detection. A detailed section is dedicated to deamidation of asparagine and isomerization of aspartic acid residues, two ubiquitous degradation pathways detected in antibodies and other proteins as well. Finally, kinetic modeling of the accumulation of antibody variants is presented as a tool to determine the expected fraction of molecules that have undergone one or more degradation reactions.

  8. The Use of Monoclonal Antibodies in Human Prion Disease (United States)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

  9. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ]. (United States)

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P


    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  10. Library of monoclonal antibodies against brush border membrane epithelial antigens

    Energy Technology Data Exchange (ETDEWEB)

    Behar, M.; Katz, A.; Silverman, M.


    A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A/sub 1/, C/sub 7/, D/sub 3/, D/sub 7/ and H/sub 4/. As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D/sub 3/ exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK/sub 1/ cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells.

  11. The murine gammaherpesvirus-68 gp150 acts as an immunogenic decoy to limit virion neutralization.

    Directory of Open Access Journals (Sweden)

    Laurent Gillet

    Full Text Available Herpesviruses maintain long-term infectivity without marked antigenic variation. They must therefore evade neutralization by other means. Immune sera block murine gammaherpesvirus-68 (MHV-68 infection of fibroblasts, but fail to block and even enhance its infection of IgG Fc receptor-bearing cells, suggesting that the antibody response to infection is actually poor at ablating virion infectivity completely. Here we analyzed this effect further by quantitating the glycoprotein-specific antibody response of MHV-68 carrier mice. Gp150 was much the commonest glycoprotein target and played a predominant role in driving Fc receptor-dependent infection: when gp150-specific antibodies were boosted, Fc receptor-dependent infection increased; and when gp150-specific antibodies were removed, Fc receptor-dependent infection was largely lost. Neither gp150-specific monoclonal antibodies nor gp150-specific polyclonal sera gave significant virion neutralization. Gp150 therefore acts as an immunogenic decoy, distorting the MHV-68-specific antibody response to promote Fc receptor-dependent infection and so compromise virion neutralization. This immune evasion mechanism may be common to many non-essential herpesvirus glycoproteins.

  12. The Role of NK Cell in T Cell Recruitment in Murine Liver Infected with Adenovirus

    Institute of Scientific and Technical Information of China (English)

    游上游; 艾洪武; 黄巍; 张楚瑜


    To study the role of natural killer (NK) cells in T cell recruitment in murine liver infected with virus, mice wereintravenously injected daily with anti-NK1.1+ antibody to deplete NK cells. Lymphocytes in the liver tissue of mice infectedwith type 5 adenovirus depleted in the E1 and E3 regions were assessed by fluorometric activated cell sorting (FACS). Ex-pression of chemokine IP-10 and its receptor CXCR3 mRNA in the liver, hepatic lymphocytes and spleen tissue were examined by reverse transcription polymerase chain reaction (RT-PCR). Serum almfine aminotransferase (ALT) was measured asan indicator of liver injury. It was found that infection of adenovims and anfi-Fas monoclonal antibody (mAb) into mice caused liver injury and high expression of interfemn-γ inducible protein-10 (IP-10) mRNA in the liver. Anfi-NK1.1+ mAb, which was intraperitoneally injected into the mice infected with adenovirus, suppresses T cell recruitment and expression of IP-10 mRNA in the hver. Slighter hver injury was also observed. After vires infection, expression of CXCR3 mRNAin spleen and hver tissue was observed at different time. The results suggested that T cell recruitment was initiated by NKcell dependent chemokine IP-10, which induced activated T cells priming in the spleen to the hver of the mouse. NK cells played a key role in T cell recruitment in the liver of mouse infected with adenovims.

  13. Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies. (United States)

    Prechl, J; Tchorbanov, A; Horváth, A; Baiu, D C; Hazenbos, W; Rajnavölgyi, E; Kurucz, I; Capel, P J; Erdei, A


    Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

  14. The detection of axillary lymph node metastases from breast cancer by radiolabelled monoclonal antibodies: a prospective study

    Energy Technology Data Exchange (ETDEWEB)

    Tjandra, J.J. (Melbourne Univ., Parkville (Australia) Royal Melbourne Hospital, Parkville (Australia)); Sacks, N.P.M.; Thompson, C.H. (Melbourne Univ., Parkville (Australia)) (and others)


    Two murine monoclonal antibodies that react with human breast cancer (3E1.2 and RCC-1) were labelled with {sup 131}iodine, and the radiolabelled antibody was injected into 40 patients, 36 of whom had breast cancer and the remaining four of whom had fibroadenoma (the normal, contralateral axilla was used as a control). Immunoscintigraphy had an overall sensitivity of 33% (23% with {sup 131}I-3E1.2 and 5% with {sup 131}I-RCC-1) for the detection of lymph node metastases and a specificity of 63% (67% with {sup 131}I-3E1.2 and 60% with {sup 131}I-RCC-1) with problems of non-specific uptake by presumably normal lymph nodes. The results of immunoscintigraphy obtained with {sup 131}I-RCC-1 (IgG) were superior to {sup 131}I-3E1.2 (IgM) although the accuracy of immunoscintigraphy using {sup 131}I-RCC-1 (56%) was not much better than preoperative clinical assessment (50%). However, there were cases when immunoscintigraphy using radiolabelled antibody (IgM or IgG) detected axillary lymph node metastases not suspected by clinical examination. Thus it appears that while immunoscintigraphy may be a useful adjunct to preoperative clinical assessment and is simple and safe, a major improvement in its accuracy is needed before it can replace axillary dissection and histological examination in the accurate staging of axilla in breast cancer. (author).

  15. Generation and characterization of an anti-idiotype monoclonal antibody related to GM3(NeuGc) ganglioside. (United States)

    Rodríguez, Mabel; Llanes, Leticia; Pérez, Alexis; Pérez, Rolando; Vázquez, Ana María


    The 14F7 monoclonal antibody (MAb), IgG1 isotype, which reacts specifically to GM3(NeuGc) ganglioside induced a specific IgG anti-idiotypic antibody (Ab2) response in syngeneic mice when it was administered coupled with KLH and in the presence of Freund's adjuvant. Spleen cells from these mice were used in somatic-cell hybridization experiments using the murine myeloma cell line P3-X63-Ag8 653 as fusion partner. An IgG1 Ab2 MAb was selected. This Ab2 MAb, called 4G9, was able to block the binding of 14F7 MAb to GM3(NeuGc) ganglioside and developed a strong IgG anti-anti-idiotypic antibody (Ab3) response, when injected into syngeneic mice. These Ab3 antibodies were characterized to bear 14F7 MAb idiotopes, but did not have the same specificity as 14F7 MAb. In the other hand, a very specific anti-NeuGc-containing ganglioside response was generated in chickens immunized with this Ab2 MAb, thus behaving, in this species as an "internal image" antibody.

  16. A Novel PET Imaging Using 64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kazuko Kobayashi


    Full Text Available Mesothelin (MSLN is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

  17. Inhibition of Spontaneous Breast Cancer Metastasis by Anti—Thomsen-Friedenreich Antigen Monoclonal Antibody JAA-F11

    Directory of Open Access Journals (Sweden)

    Jamie Heimburg


    Full Text Available Thomsen-Friedenreich antigen (TF-Ag is expressed in many carcinomas, including those of the breast, colon, bladder, prostate. TF-Ag is important in adhesion and metastasis and as a potential immunotherapy target. We hypothesized that passive transfer of JAAF11, an anti -TF-Ag monoclonal antibody, may create a survival advantage for patients with TIF-Ag -expressing tumors by cytotoxicity, blocking of tumor cell adhesion, inhibition of metastasis. This was tested using in vitro models of tumor cell growth; cytotoxicity assays; in vitro, ex vivo, in vivo models of cancer metastasis; and, finally, in vivo effects in mice with metastatic breast cancer. Unlike some anti-TF-Ag antibodies, JAA-F11 did not enhance breast carcinoma cell growth. JAA-F11 did not induce the killing of 4T1 tumor cells through complement-dependent cytotoxicity or apoptotic mechanisms. However, JAA-F11 blocked the stages of metastasis that involve the adhesion of human breast carcinoma cells to human endothelial cells (human umbilical vein endothelial cells and human bone marrow endothelial cells 60 in in vitro static adhesion models, in a perfused ex vivo model, in murine lung vasculature in an in vivo metastatic deposit formation assay. JAA-F11 significantly extended the median survival time of animals bearing metastatic 4T1 breast tumors and caused a > 50% inhibition of lung metastasis.

  18. A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein.

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Deng

    Full Text Available Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV, yellow fever (YFV, West Nile (WNV, and Japanese encephalitis (JEV viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98DRXW(101 motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.

  19. Tracking Neospora caninum parasites using chimera monoclonal antibodies against its surface antigen-related sequences (rNcSRS2). (United States)

    Dong, Jinhua; Otsuki, Takahiro; Kato, Tatsuya; Park, Enoch Y


    Neosporosis, an infectious disease of cattle and dogs, causes an abortion in cattle, which has a major damage on the dairy industry worldwide. Tracking of Neospora caninum parasite that is responsible for neosporosis is required for the prevention of this infectious disease. We developed three chimera monoclonal antibodies consist of variable regions of murine antibody and constant regions of human antibody against N. caninum. Recombinant surface antigen-related sequence 2 (rNcSRS2) of N. caninum was expressed in silkworm larvae, and immunized in mice to obtain phage displaying antibody library. Through three rounds of selection, three antibodies, A6, E1 and H3, were isolated and bound to rNcSRS2 with nanomolar to micromolar affinity. In immunofluorescent staining assays, A6 and E1 bound to N. caninum strain Nc-Liv, demonstrating a successful tracking of the parasite. H3 clone bound to rNcSRS2 but not to a truncated protein without glycosylphosphatidylinositol (GPI) anchor domain in the carboxyl terminal. Amino acid sequences of A6 and E1 were similar, but that of H3 differed in the CDR-H1 region, which might be the reason of their difference of affinity. These antibodies are thought to be useful for prevention of cattle from neosporosis.

  20. Regulation of levels of serum antibodies to ryegrass pollen allergen Lol pIV by an internal image anti-idiotypic monoclonal antibody. (United States)

    Zhou, E M; Kisil, F T


    A murine monoclonal anti-idiotypic antibody (anti-Id), designated B1/1, was produced against an idiotope of a murine antibody (mAb91), which recognizes the epitope, site A, of allergen Lol pIV, one of the major groups of allergens in ryegrass (Lolium perenne) pollen. The ability of B1/1 to modulate the antibody responses to Lol pIV was investigated in murine model systems. In the first system, B1/1-keyhole limpet haemocyanin (KLH) conjugate was administered to treat three different strains of mice (C57BL/6, BALB/c and C3H). In the second and third model systems, a solution of B1/1 in phosphate-buffered saline (PBS) was used to treat syngeneic BALB/c mice at various doses and time intervals, respectively. The treatment with either form of B1/1, administered at doses ranging from 100 ng to 100 micrograms mouse, resulted in a reduction of the levels of the antibodies to Lol pIV. In particular, the level of IgE antibodies to Lol pIV was greatly reduced. The administration of a single intravenous (i.v.) injection of a solution of B1/1 8 weeks prior to the challenge with Lol pIV was still effective in reducing the level of antibodies to the allergen. Moreover, the level of antibodies to Lol pIV that expressed the idiotope mAb91 was also markedly decreased. By contrast, it was observed that the level of antibodies to Lol pIV in mice pretreated with B1/1 in PBS at a dose of 10 ng/mouse increased (albeit slightly) compared to that in mice treated with control mAb. These experimental models lend themselves for investigating the mechanism(s) by which an anti-Id modulates antibody responses to a grass pollen allergen.

  1. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid (United States)

    Davis, Christopher G.; Eisner, Eric; McGlynn, Margaret; Shelton, John M.; Richardson, James


    Objective. To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. Design. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Results. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Conclusions. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury. PMID:26069679

  2. Thermal resistance in a spontaneous murine tumour. (United States)

    Maher, J; Urano, M; Rice, L; Suit, H D


    Resistance to subsequent hyperthermia as a result of prior heating was investigated using a spontaneous murine tumour implanted into the feet of C3H/Sed mice. Tumours were treated by immersing the tumour-bearing foot into a constant-temperature hot water bath set at 45.5 degrees C and were given single and split doses of heat. Response was assessed using a tumour-growth time assay. Three aspects of thermally-induced resistance were particularly considered: the time course of development and decay; the importance of the magnitude of the priming dose and the influence of the size of the tumour at the time of treatment. Substantial resistance was induced in this tumour by short priming doses at 45.5 degrees C, rising rapidly 1-2 days after the first treatment and then starting to decay. There was no significant difference in the kinetics of thermal resistance induced in tumours treated at 4mm and those treated at 8 mm in size, although the large tumours were more sensitive to single doses of heat. Increasing the magnitude of the priming dose of heat resulted in an increase in the magnitude of resistance to the second dose. The results of this study are compared with results of similar studies in this and other laboratories using murine normal tissues and cells in culture. Possible clinical implications are considered.

  3. Splenectomy normalizes hematocrit in murine polycythemia vera.

    Directory of Open Access Journals (Sweden)

    Jan-Rung Mo

    Full Text Available Splenic enlargement (splenomegaly develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV, an activating mutation in Janus kinase 2 (JAK2(V617 induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH or splenectomy (SPL surgeries in a murine model of JAK2(V617F-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2(V617F transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2(V617 is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2(V617F-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen.

  4. Murine Typhus: Clinical and epidemiological aspects

    Directory of Open Access Journals (Sweden)

    Gaspar Peniche Lara


    Full Text Available 14.00 Normal 0 21 false false false ES-CO X-NONE X-NONE Rickettsia typhi is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against R. typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of Rickettsia typhi are rats (some species belonging the Rattus Genus and fleas (Xenopsylla cheopis are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi.

  5. Benzaldehyde suppresses murine allergic asthma and rhinitis. (United States)

    Jang, Tae Young; Park, Chang-Shin; Kim, Kyu-Sung; Heo, Min-Jeong; Kim, Young Hyo


    To evaluate the antiallergic effects of oral benzaldehyde in a murine model of allergic asthma and rhinitis, we divided 20 female BALB/c mice aged 8-10 weeks into nonallergic (intraperitoneally sensitized and intranasally challenged to normal saline), allergic (intraperitoneally sensitized and intranasally challenged to ovalbumin), and 200- and 400-mg/kg benzaldehyde (allergic but treated) groups. The number of nose-scratching events in 10 min, levels of total and ovalbumin-specific IgE in serum, differential counts of inflammatory cells in bronchoalveolar lavage (BAL) fluid, titers of Th2 cytokines (IL-4, IL-5, IL-13) in BAL fluid, histopathologic findings of lung and nasal tissues, and expressions of proteins involved in apoptosis (Bcl-2, Bax, caspase-3), inflammation (COX-2), antioxidation (extracellular SOD, HO-1), and hypoxia (HIF-1α, VEGF) in lung tissue were evaluated. The treated mice had significantly fewer nose-scratching events, less inflammatory cell infiltration in lung and nasal tissues, and lower HIF-1α and VEGF expressions in lung tissue than the allergic group. The number of eosinophils and neutrophils and Th2 cytokine titers in BAL fluid significantly decreased after the treatment (Pbenzaldehyde exerts antiallergic effects in murine allergic asthma and rhinitis, possibly through inhibition of HIF-1α and VEGF.

  6. Mechanistic insights into the neutralization of cytotoxic abrin by the monoclonal antibody D6F10.

    Directory of Open Access Journals (Sweden)

    Shradha Bagaria

    Full Text Available Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.

  7. Unarmed, tumor-specific monoclonal antibody effectively treats brain tumors (United States)

    Sampson, John H.; Crotty, Laura E.; Lee, Samson; Archer, Gary E.; Ashley, David M.; Wikstrand, Carol J.; Hale, Laura P.; Small, Clayton; Dranoff, Glenn; Friedman, Allan H.; Friedman, Henry S.; Bigner, Darell D.


    The epidermal growth factor receptor (EGFR) is often amplified and rearranged structurally in tumors of the brain, breast, lung, and ovary. The most common mutation, EGFRvIII, is characterized by an in-frame deletion of 801 base pairs, resulting in the generation of a novel tumor-specific epitope at the fusion junction. A murine homologue of the human EGFRvIII mutation was created, and an IgG2a murine mAb, Y10, was generated that recognizes the human and murine equivalents of this tumor-specific antigen. In vitro, Y10 was found to inhibit DNA synthesis and cellular proliferation and to induce autonomous, complement-mediated, and antibodydependent cell-mediated cytotoxicity. Systemic treatment with i.p. Y10 of s.c. B16 melanomas transfected to express stably the murine EGFRvIII led to long-term survival in all mice treated (n = 20; P < 0.001). Similar therapy with i.p. Y10 failed to increase median survival of mice with EGFRvIII-expressing B16 melanomas in the brain; however, treatment with a single intratumoral injection of Y10 increased median survival by an average 286%, with 26% long-term survivors (n = 117; P < 0.001). The mechanism of action of Y10 in vivo was shown to be independent of complement, granulocytes, natural killer cells, and T lymphocytes through in vivo complement and cell subset depletions. Treatment with Y10 in Fc receptor knockout mice demonstrated the mechanism of Y10 to be Fc receptor-dependent. These data indicate that an unarmed, tumor-specific mAb may be an effective immunotherapy against human tumors and potentially other pathologic processes in the “immunologically privileged” central nervous system. PMID:10852962

  8. [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies]. (United States)

    Aydın, Cevahir; Ataoğlu, Haluk


    . Sandwich ELISA and immunofluorescence (IF) methods have been used to detect the experimental reactions. In our study, highly specific class IgM murine monoclonal antibodies (mAb-2B7) against C.albicans yeast cell wall were obtained from clone 2B7. These antibodies cross-reacted with S.choleraesuis 211 and S.infantis bacteria sharing similar cell wall structure of C.albicans. The existence of mannan β-1,2 bonds on the surface of C.albicans yeast form was confirmed with a commercial monoclonal antibody (mAb-ACMK-1; Matriks Biotek(®), Turkey) specific for those bonds. Besides, mAb-ACMK-1 interacted with C.albicans yeast form and gave intense fluorescence (high positive reaction) in IF method, but no fluorescence (negative) was detected with hyphal form. This data, obtained for the first time with this study, indicates that the mannan β-1,2 bonds are either found infrequently or none in the fungal hyphal wall. Although both monoclonal antibodies recognize the mannan antigen, mAb-2B7 reacted with S.choleraesuis 211, while mAb-ACMK-1 did not, due to the difference of epitope specificity. In conclusion, monoclonal antibodies may facilitate the characterization of antigenic structures of Candida, which will lead for the identification of new determinants that may increase the sensitivity and specificity of commercial tests used for mannan detection in serum.

  9. High-efficiency screening of monoclonal antibodies for membrane protein crystallography.

    Directory of Open Access Journals (Sweden)

    Hyun-Ho Lim

    Full Text Available Determination of crystal structures of membrane proteins is often limited by difficulties obtaining crystals diffracting to high resolution. Co-crystallization with Fab fragments of monoclonal antibodies has been reported to improve diffraction of membrane proteins crystals. However, it is not simple to generate useful monoclonal antibodies for membrane protein crystallography. In this report, we present an optimized process for efficient screening from immunization to final validation of monoclonal antibody for membrane protein crystallography.

  10. Improved detection of Pneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

    DEFF Research Database (Denmark)

    Orholm, M; Holten-Andersen, W; Lundgren, Jens Dilling


    To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detecting Pneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens...... and 34 induced sputum specimens were examined. All specimens were stained by five techniques: immunofluorescence using a combination of three monoclonal antibodies (from the National Institutes of Health, USA), immunofluorescence using a single monoclonal antibody (from Dakopatts), Giemsa, methenamine...

  11. 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


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

  12. Isolation and culture of murine macrophages. (United States)

    Davies, John Q; Gordon, Siamon


    The two most convenient sources of primary murine macrophages are the bone marrow and the peritoneal cavity. Resident peritoneal macrophages can readily be harvested from mice and purified by adherence to tissue culture plastic. The injection of Bio-Gel polyacrylamide beads or thioglycollate broth into the peritoneal cavity produces an inflammatory response allowing the purification of large numbers of elicited macrophages. The production of an activated macrophage population can be achieved by using Bacillus-Calmette-Guerin as the inflammatory stimulus. Resident bone marrow macrophages can be isolated following enzymatic separation of cells from bone marrow plugs and enrichment on 30% fetal calf serum containing medium or Ficoll-Hypaque gradients. Bone marrow-derived macrophages can be produced by differentiating nonadherent macrophage precursors with medium containing macrophage colony-stimulating factor.

  13. Extracellular proteolysis in the adult murine brain. (United States)

    Sappino, A P; Madani, R; Huarte, J; Belin, D; Kiss, J Z; Wohlwend, A; Vassalli, J D


    Plasminogen activators are important mediators of extracellular metabolism. In the nervous system, plasminogen activators are thought to be involved in the remodeling events required for cell migration during development and regeneration. We have now explored the expression of the plasminogen activator/plasmin system in the adult murine central nervous system. Tissue-type plasminogen activator is synthesized by neurons of most brain regions, while prominent tissue-type plasminogen activator-catalyzed proteolysis is restricted to discrete areas, in particular within the hippocampus and hypothalamus. Our observations indicate that tissue-type plasminogen activator-catalyzed proteolysis in neural tissues is not limited to ontogeny, but may also contribute to adult central nervous system physiology, for instance by influencing neuronal plasticity and synaptic reorganization. The identification of an extracellular proteolytic system active in the adult central nervous system may also help gain insights into the pathogeny of neurodegenerative disorders associated with extracellular protein deposition.

  14. The use of combinations of monoclonal antibodies in clinical oncology. (United States)

    Henricks, Linda M; Schellens, Jan H M; Huitema, Alwin D R; Beijnen, Jos H


    Treatment with monoclonal antibodies is becoming increasingly important in clinical oncology. These antibodies specifically inhibit signaling pathways in tumor growth and/or induce immunological responses against tumor cells. By combining monoclonal antibodies several pathways may be targeted simultaneously, potentially leading to additive or synergistic effects. Theoretically, antibodies are very suitable for use in combination therapy, because of limited overlapping toxicity and lack of pharmacokinetic interactions. In this article an overview is given of preclinical and clinical data on twenty-five different combinations of antibodies in oncology. Some of these combinations have proven clinical benefit, for example the combination of trastuzumab and pertuzumab in HER2-positive breast cancer, which exemplifies an additive or synergistic effect on antitumor activity in clinical studies and the combination of nivolumab and ipilimumab, which results in significant increases in progression-free and overall survival in patients with advanced melanoma. However, other combinations may lead to unfavorable results, such as bevacizumab with cetuximab or panitumumab in advanced colorectal cancer. These combinations result in shorter progression-free survival and increased toxicity compared to therapy with a single antibody. In summary, the different published studies showed widely varying results, depending on the combination of antibodies, indication and patient population. More preclinical and clinical studies are necessary to unravel the mechanisms behind synergistic or antagonistic effects of combining monoclonal antibodies. Most research on combination therapies is still in an early stage, but it is expected that for several tumor types the use of combination therapy of antibodies will become standard of care in the near future.

  15. Monitoring monoclonal antibody delivery in oncology: the example of bevacizumab.

    Directory of Open Access Journals (Sweden)

    Guillaume Nugue

    Full Text Available Developing therapeutic monoclonal antibodies paves the way for new strategies in oncology using targeted therapy which should improve specificity. However, due to a lack of biomarkers, a personalized therapy scheme cannot always be applied with monoclonal antibodies. As a consequence, the efficacy or side effects associated with this type of treatment often appear to be sporadic. Bevacizumab is a therapeutic monoclonal antibody targeting Vascular Endothelial Growth Factor (VEGF. It is used to limit tumor vascularization. No prognosis or response biomarker is associated with this antibody, we therefore assessed whether the administration protocol could be a possible cause of heterogeneous responses (or variable efficacy. To do this, we developed a bevacizumab assay with a broad sensitivity range to measure blood bevacizumab concentrations. We then analyzed bevacizumab concentrations in 17 patients throughout the first quarter of treatment. In line with previously published data, average blood concentrations were 88+/-27 mg/L following the first dose administered, and 213+/-105 mg/L after the last (6(th dose administered. However, the individual values were scattered, with a mean 4-fold difference between the lowest and the highest concentration for each dose administered. We demonstrated that the bevacizumab administration schedule results in a high inter-individual variability in terms of blood concentrations. Comparison of assay data with clinical data indicates that blood concentrations above the median are associated with side effects, whereas values below the median favor inefficacy. In conclusion, bevacizumab-based therapy could benefit from a personalized administration schedule including follow-up and adjustment of circulating bevacizumab concentrations.

  16. Investigations into the route of uptake and pharmacokinetics of intraperitoneally-administered monoclonal antibodies: I. Transdiaphragmatic blockade of the terminal lymphatics in the rat. (United States)

    Barrett, J S; Wahl, R L; Wagner, J G; Brown, R; Fisher, S J


    Recent studies on the intraperitoneal administration of radiolabeled monoclonal antibodies indicate that the diaphragm and, in particular, the lymphatics associated with the diaphragm are more involved in the transport of such high-molecular-mass moieties than was earlier suspected. The current study examines the role of the diaphragm in the i.p. transport of an IgG2a murine monoclonal antibody, 5G6.4, by observing the effect on the absorption of the antibody produced when the diaphragm has been scarred. Normal, sham-operated, and diaphragmatically scarred (abrasions made with 600-grade sandpaper) female Sprague Dawley rats (150-250 g) were administered intraperitoneal injections of 125labeled 5G6.4 in a volume of 2.0 cm3. Approximately 5 micrograms antibody protein was administered in the individual 19-microCi injections per rat. Scarring was effective in partially blocking the amount of labeled antibody that crossed the diaphragm. Mean diaphragm levels (% injected dose/g) of 125I-labeled 5G6.4 from the scarred group were 16.8% lower than values from the sham-operated rats and 37.2% lower than those from the control rats. The blockade was effective in slowing the appearance of the labeled antibody in the systemic circulation. The half-time to absorption was significantly prolonged in the scarred group; mean t1/2 absorption values of 2.5 h for the control group, 5.3 h for the sham-operated group, and 9.6 h for the diaphragmatically blocked group were recorded. Scarring the diaphragm reduced the mean maximum blood concentration by 27.6% over the control group and 23.9% over the sham-operated group. The mean time to maximum blood concentration was lengthened by 93..0% over the control group and 35.3% over the sham-operated group due as a result of scarification. Presumably this impedence to absorption would increase the time that the radiolabeled antibody bathed the peritoneal space. The scarred group also had the largest "system mean residence time" (162.5 h

  17. Rapid analysis of small samples containing forskolin using monoclonal antibodies. (United States)

    Yanagihara, H; Sakata, R; Shoyama, Y; Murakami, H


    The effective range of the competitive ELISA test for detection of forskolin content in clonally propagated plant organs of Coleus forskohlii using monoclonal antibodies extends from 5ng to 5 micrograms. A correlation between the forskolin accumulation and the growth rate was investigated using the clonally propagated shoots. An increase of forskolin content was noted, beginning at week 6. Flowers, rachises, leaves, stems, tuberous roots, and roots were analyzed. Tuberous roots and the stem base contained higher amounts of forskolin than other organs. The forskolin content in the stem decreased gradually towards the top of the shoot.

  18. Necrobiotic xanthogranuloma associated with a benign monoclonal gammopathy. (United States)

    Burdick, Anne E; Sanchez, Jany; Elgart, George W


    Necrobiotic xanthogranuloma (NXG) is a disorder characterized by indurated, yellow-red nodules or plaques, primarily involving the face and, less frequently, the trunk and extremities. NXG may be associated with paraproteinemia, multiple myeloma, and hypertension. Histologically, xanthogranulomatous features with hyaline necrosis or necrobiosis are present. No first-line treatment has been established. This disease is a chronic process, and a patient's prognosis depends on the degree of extracutaneous involvement and the presence of visceral malignancies. We describe a patient with typical cutaneous and histologic findings of NXG with an associated monoclonal gammopathy.

  19. Monoclonal antibody therapy in the treatment of Reye's syndrome. (United States)

    Treon, S P; Broitman, S A


    A role for lipopolysaccharides (endotoxins, LPS) in 7 the pathogenesis of Reye's syndrome (RS) has previously been suggested. Impairment of hepatic LPS clearance can lead to systemic endotoxemia as previous studies by this and other laboratories have suggested for several hepatic disorders including RS. Systemic LPS may mediate many of the clinical findings associated with RS by eliciting monokines such as tumor necrosis factor-alpha, interleukin-1, interleukin-6, and interleukin-8. Monoclonal antibody therapy directed at LPS, and monokines may represent a novel approach to the treatment of RS.

  20. Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)


    A fluorescence polarization immunoassay(FPIA) for the determination of salinomycin(SAL) was developed by using anti-SAL monoclonal antibodies(mAb).Fluorescein labeled SAL(tracer) was synthesized by the N-hydroxysuccinimide active ester method and purified using thin layer chromatography(TLC).The developed FPIA for SAL had a dynamic range from 0.60 to 2193 ng/mL with an IC50 value of 33.2 ng/mL and a detection limit(LOD) of 0.08 ng/mL.No significant cross-reactivities were observed with other drugs but 67.6% with narasin.

  1. [Polyneuropathy associated with monoclonal gammopathy of undetermined significance]. (United States)

    Moth Henriksen, Marie; Kolmos, Eva Brøsted; Abildgaard, Niels; Schrøder, Henrik Daa; Sindrup, Søren


    The prevalence of polyneuropathy in patients with monoclonal gammopathy of undetermined significance (MGUS) has been reported to be 10-50%. The majority of patients have a chronic, slowly progressive, distal, symmetric and predominantly sensory polyneuropathy. A caused relationship between polyneuropathy and immunoglobulin (Ig)M MGUS is better established than the relationship between polyneuropathy and IgG/IgA MGUS because of the observed binding of IgM to myelin sheaths and widening of myelin lamellae. In randomized controlled trials plasma exchange, immunosuppressive, rituximab and intravenous Ig have been found to have a clinical meaningful effect.

  2. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Frøkiær, Hanne; Hearty, Stephen;


    The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine m......Ab-producing cell lines were cloned and their cross-reactivities characterised against a panel of airborne fungal spores representing genera commonly found in the same environment as Pst. Two specific mAbs were used to develop a competitive ELISA (Pst mAb4) and a subtractive inhibition ELISA (Pst mAb8). Standard...

  3. Monoclonal gammopathy associated with heartworm disease in a dog. (United States)

    de Caprariis, Donato; Sasanelli, Mariateresa; Paradies, Paola; Otranto, Domenico; Lia, Riccardo


    A 12-year-old, intact female, mixed Yorkshire terrier was evaluated for syncopal episodes, weakness, decreased appetite, and weight loss. Heartworm disease was diagnosed based on evidence of circulating microfilariae of Dirofilaria immitis on direct examination of blood smears and a positive SNAP heartworm antigen test. An immunoglobulin G (IgG) gammopathy, demonstrated by serum protein electrophoresis, was associated with heartworm disease in this dog. Response to treatment with both an adulticide and the microfilaricide ivermectin included remission of clinical signs and a decrease in the monoclonal gammopathy. To our knowledge, this is the first report of an IgG gammopathy associated with heartworm disease in the dog.

  4. Alemtuzumab and Natalizumab: The Monoclonal Antibody Story Continues

    Directory of Open Access Journals (Sweden)

    BL Johnston


    Full Text Available In the July/August 2006 issue of this journal, the infectious complications associated with the use of infliximab, etanercept and adalimumab were reviewed (1. These represent only three of the many monoclonal antibodies either licensed or in clinical trials for therapeutic use in cancer and autoimmune disease or to prevent rejection in both solid organ and hematopoietic stem cell transplantation. While most of these agents have not been associated with increased infection rates, alemtuzumab and natalizumab have gained particular attention related to either the frequency or type of infection seen in some individuals who have received them.

  5. Large-scale production of monoclonal antibodies in suspension culture. (United States)

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


    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.

  6. Neutralization and Binding Profile of Monoclonal Antibodies Generated Against Influenza A H1N1 Viruses. (United States)

    Shembekar, Nachiket; Mallajosyula, Vamsee V Aditya; Malik, Ankita; Saini, Ashok; Varadarajan, Raghavan; Gupta, Satish Kumar


    Monoclonal antibodies (MAbs) provide scope for the development of better therapeutics and diagnostic tools. Herein, we describe the binding and neutralization profile(s) for a panel of murine MAbs generated against influenza A H1N1 viruses elicited by immunization with pandemic H1 recombinant hemagglutinin (rHA)/whole virus or seasonal H1 rHA. Neutralizing MAbs, MA-2070 and MA-M, were obtained after pandemic A/California/07/2009 (H1N1) virus/rHA immunization(s). Both MAbs reacted specifically with rHA from A/California/07/2009 and A/England/195/2009 in ELISA. MA-2070 bound rHA of A/California/07/2009 with high affinity (KD = 51.36 ± 9.20 nM) and exhibited potent in vitro neutralization (IC50 = 2.50 μg/mL). MA-2070 bound within the stem domain of HA. MA-M exhibited both hemagglutination inhibition (HI, 1.50 μg/mL) and in vitro neutralization (IC50 = 0.66 μg/mL) activity against the pandemic A/California/07/2009 virus and showed higher binding affinity (KD = 9.80 ± 0.67 nM) than MA-2070. MAb, MA-H generated against the seasonal A/Solomon Islands/03/2006 (H1N1) rHA binds within the head domain and bound the seasonal H1N1 (A/Solomon Islands/03/2006 and A/New Caledonia/20/1990) rHAs with high affinity (KD; 0.72-8.23 nM). MA-H showed high HI (2.50 μg/mL) and in vitro neutralization (IC50 = 2.61 μg/mL) activity against the A/Solomon Islands/03/2006 virus. All 3 MAbs failed to react in ELISA with rHA from various strains of H2N2, H3N2, H5N1, H7N9, and influenza virus B, suggesting their specificity for either pandemic or seasonal H1N1 influenza virus. The MAbs reported here may be useful in developing diagnostic assays.

  7. Molecular cloning and chromosome assignment of murine N-ras.


    Ryan, J.; Hart, C P; Ruddle, F H


    The murine N-ras gene was cloned by screening an EMBL-3 recombinant phage library with a human N-ras specific probe. Hybridization of two separate unique sequence N-ras probes, isolated from the 5' and 3' flanking sequences of the murine gene, to a mouse-Chinese hamster hybrid mapping panel assigns the N-ras locus to mouse chromosome three.

  8. Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site. (United States)

    Talavera, Ariel; Mackenzie, Jenny; Garrido, Greta; Friemann, Rosmarie; López-Requena, Alejandro; Moreno, Ernesto; Krengel, Ute


    The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806.

  9. In vitro Induction of primary antibody responses to particulate and soluble protein antigens in T cell—replaced murine spleen cell cultures

    Institute of Scientific and Technical Information of China (English)



    Specific antibody responses could be induced in serumfree condition.Specific anti-SRBC or anti-SRBC ghost antibody were induced from anti-Thy treated (T-depleted) murine spleen cells in serum-free culture in the presence of Con A conditioned medium.This induction system may facilitate the study of lymphokine functions on antigen triggered B cells. In T cell-replaced cultures,the antibody responses of B cells could be successfully induced when soluble SRBC membrane proteins were used as antigens.It thus indicates that antigen together with lymphokines are sufficient to drive B cells to become antibody secreting cells in the absence of T cells.The T cell-replaced system provides a more stable way for in vitro immunization and may be applied to monoclonal antibody production when in vivo immunization is difficult to be carried out.

  10. Monoclonal antibodies directed to fucoidan preparations from brown algae. (United States)

    Torode, Thomas A; Marcus, Susan E; Jam, Murielle; Tonon, Thierry; Blackburn, Richard S; Hervé, Cécile; Knox, J Paul


    Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.

  11. Monoclonal Antibody-Based Therapeutics for Melioidosis and Glanders

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim


    Full Text Available Problem statement: Burkholderia Pseudomallei (BP and B. Mallei (BM were two closely related pathogenic gram-negative bacteria. They were the causative agents of melioidosis and glanders, respectively and are recognized by CDC as category B select agents. Significant efforts had been devoted to developing the diagnostic and therapeutic measures against these two pathogens. Monoclonal antibody-based therapeutic was a promising targeted therapy to fight against melioidosis and glanders. Valuable findings have been reported by different groups in their attempt to identify vaccine targets against these two pathogens. Approach: Our group has generated neutralizing Monoclonal Antibodies (MAbs against BP and BM and characterized them by both in vitro and in vivo experiments. We present an overview of the MAb-based therapeutic approaches against BP and BM and demonstrate some of our efforts for developing chimeric and fully human MAbs using antibody engineering. Results: Throughout conventional mouse hybridoma technique and antibody engineering (chimerization and in vitro antibody library techniques, we generated 10 chimeric MAbs (3 stable MAbs and 7 transient MAbs and one fully human MAb against BP and BM. In addition, we present the reactive antigen profiles of these MAbs. Our approaches had potentials to accelerate the development of therapeutics for melioidosis and glanders in humans. Conclusion: Our experience and findings presented here will be valuable for choosing the best antigenic targets and ultimately for the production of effective vaccines for these two pathogens.

  12. Characterization of oxidative carbonylation on recombinant monoclonal antibodies. (United States)

    Yang, Yi; Stella, Cinzia; Wang, Weiru; Schöneich, Christian; Gennaro, Lynn


    In the biotechnology industry, oxidative carbonylation as a post-translational modification of protein pharmaceuticals has not been studied in detail. Using Quality by Design (QbD) principles, understanding the impact of oxidative carbonylation on product quality of protein pharmaceuticals, particularly from a site-specific perspective, is critical. However, comprehensive identification of carbonylation sites has so far remained a very difficult analytical challenge for the industry. In this paper, we report for the first time the identification of specific carbonylation sites on recombinant monoclonal antibodies with a new analytical approach via derivatization with Girard's Reagent T (GRT) and subsequent peptide mapping with high-resolution mass spectrometry. Enhanced ionization efficiency and high quality MS(2) data resulted from GRT derivatization were observed as key benefits of this approach, which enabled direct identification of carbonylation sites without any fractionation or affinity enrichment steps. A simple data filtering process was also incorporated to significantly reduce false positive assignments. Sensitivity and efficiency of this approach were demonstrated by identification of carbonylation sites on both unstressed and oxidized antibody bulk drug substances. The applicability of this approach was further demonstrated by identification of 14 common carbonylation sites on three highly similar IgG1s. Our approach represents a significant improvement to the existing analytical methodologies and facilitates extended characterization of oxidative carbonylation on recombinant monoclonal antibodies and potentially other protein pharmaceuticals in the biotechnology industry.

  13. Generation of monoclonal antibodies against highly conserved antigens.

    Directory of Open Access Journals (Sweden)

    Hongzhe Zhou

    Full Text Available BACKGROUND: Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is very crucial for the success of the drug development. However, due to immune tolerance, some proteins that are highly conserved between mice and humans are not very immunogenic in mice, making it difficult to generate antibodies using a conventional approach. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the impaired immune tolerance of NZB/W mice was exploited to generate monoclonal antibodies against highly conserved or self-antigens. Using two highly conserved human antigens (MIF and HMGB1 and one mouse self-antigen (TNF-alpha as examples, we demonstrate here that multiple clones of high affinity, highly specific antibodies with desired biological activities can be generated, using the NZB/W mouse as the immunization host and a T cell-specific tag fused to a recombinant antigen to stimulate the immune system. CONCLUSIONS/SIGNIFICANCE: We developed an efficient and universal method for generating surrogate or therapeutic antibodies against "difficult antigens" to facilitate the development of therapeutic antibodies.

  14. Monoclonal antibodies directed to fucoidan preparations from brown algae.

    Directory of Open Access Journals (Sweden)

    Thomas A Torode

    Full Text Available Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.

  15. Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Andrew Gdowski


    Full Text Available Treatments utilizing monoclonal antibody therapeutics against intracellular protein-protein interactions in cancer cells have been hampered by several factors, including poor intracellular uptake and rapid lysosomal degradation. Our current work examines the feasibility of encapsulating monoclonal antibodies within poly(lactic-co-glycolic acid (PLGA nanoparticles using a water/oil/water double emulsion solvent evaporation technique. This method can be used to prepare protective polymeric nanoparticles for transporting functional antibodies to the cytoplasmic compartment of cancer cells. Nanoparticles were formulated and then characterized using a number of physical and biological parameters. The average nanoparticle size ranged from 221 to 252 nm with a low polydispersity index. Encapsulation efficiency of 16%–22% and antibody loading of 0.3%–1.12% were observed. The antibody molecules were released from the nanoparticles in a sustained manner and upon release maintained functionality. Our studies achieved successful formulation of antibody loaded polymeric nanoparticles, thus indicating that a PLGA-based antibody nanoformulation is a promising intracellular delivery vehicle for a large number of new intracellular antibody targets in cancer cells.

  16. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens. (United States)

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S


    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  17. Monoclonal anti-elastin antibody labelled with technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Marcia B.N. de; Silva, Claudia R. da; Araujo, Adriano C. de; Bernardo Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes. Lab. de Radiofarmacia; Porto, Luis Cristovao M.S.; Gutfilen, Bianca [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Instituto de Biologia Roberto Alcantara Gomes; Souza, J.E.Q. [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil). Centro de Pesquisa Basica; Frier, Malcolm [University Hospital, Nottingham (United Kingdom). Dept. of Medical Physics


    Technetium-99m ({sup 99m} Tc) is widely employed in nuclear medicine due to its desirable physical, chemical and biological properties. Moreover, it is easily available and normally is inexpensive. A reducing agent is necessary to label cells and molecules with {sup 99m} Tc and stannous chloride (Sn C L{sub 2}) is usually employed. Elastin is the functional protein component of the elastic fiber and it is related with some diseases such as arteriosclerosis, pulmonary emphysema and others. The present study refers to the preparation of the {sup 99m} Tc labeled monoclonal anti-elastin antibody. The monoclonal antibody was incubated with an excess of 2-iminothiolane. The free thiol groups created, were capable of binding with the reduced technetium. Labeling was an exchange reaction with {sup 99m} Tc-glucoheptonate. The labeled preparation was left at 4 deg C for one hour. Then, it was passed through a Sephadex G50 column. Various fractions were collected and counted. A peak corresponding to the radiolabeled antibody was obtained. Stability studies of the labelled anti-elastin were performed at 0,3 6, 24 hours, at both 4 deg C or room temperature. The biodistribution pattern of the {sup 99m} Tc-anti-elastin was studied in healthy male Swiss mice. The immunoreactivity was also determined. An useful labeled-anti-elastin was obtained to future immunoscintigraphic investigations. (author) 4 refs., 7 figs., 6 tabs.

  18. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani


    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. Membrane adsorbers as purification tools for monoclonal antibody purification. (United States)

    Boi, Cristiana


    Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.

  20. Monoclonal Antibody Therapy and Renal Transplantation: Focus on Adverse Effects

    Directory of Open Access Journals (Sweden)

    Gianluigi Zaza


    Full Text Available A series of monoclonal antibodies (mAbs are commonly utilized in renal transplantation as induction therapy (a period of intense immunosuppression immediately before and following the implant of the allograft, to treat steroid-resistant acute rejections, to decrease the incidence and mitigate effects of delayed graft function, and to allow immunosuppressive minimization. Additionally, in the last few years, their use has been proposed for the treatment of chronic antibody-mediated rejection, a major cause of late renal allograft loss. Although the exact mechanism of immunosuppression and allograft tolerance with any of the currently used induction agents is not completely defined, the majority of these medications are targeted against specific CD proteins on the T or B cells surface (e.g., CD3, CD25, CD52. Moreover, some of them have different mechanisms of action. In particular, eculizumab, interrupting the complement pathway, is a new promising treatment tool for acute graft complications and for post-transplant hemolytic uremic syndrome. While it is clear their utility in renal transplantation, it is also unquestionable that by using these highly potent immunosuppressive agents, the body loses much of its innate ability to mount an adequate immune response, thereby increasing the risk of severe adverse effects (e.g., infections, malignancies, haematological complications. Therefore, it is extremely important for clinicians involved in renal transplantation to know the potential side effects of monoclonal antibodies in order to plan a correct therapeutic strategy minimizing/avoiding the onset and development of severe clinical complications.

  1. Ofatumumab: a novel monoclonal anti-CD20 antibody

    Directory of Open Access Journals (Sweden)

    Thomas S Lin


    Full Text Available Thomas S LinGlaxoSmithKline Oncology R&D, Collegeville, PA, USAAbstract: Ofatumumab, a novel humanized monoclonal anti-CD20 antibody, was recently approved by the FDA for the treatment of fludarabine and alemtuzumab refractory chronic lymphocytic leukemia (CLL. Ofatumumab effectively induces complement-dependent cytotoxicity (CDC in vitro, and recent studies demonstrated that ofatumumab also effectively mediates antibody-dependent cellular cytotoxicity (ADCC. Pharmacokinetic studies indicated that increased exposure to the antibody correlated with improved clinical outcome in CLL. Thus, pharmacogenomics may be important in identifying which patients are more likely to respond to ofatumumab therapy, although such studies have not yet been performed. Patients with the high-affinity FCGR3a 158 V/V polymorphism may be more likely to respond to therapy, if ADCC is the primary in vivo mechanism of action of ofatumumab. Patients with increased expression of the complement defense proteins CD55 and CD59 may be less likely to respond if ofatumumab works in vivo primarily via CDC. Patients with increased metabolism and clearance of ofatumumab may have lower exposure and be less likely to respond clinically. Thus, pharmacogenomics may determine the responsiveness of patients to ofatumumab therapy.Keywords: monoclonal antibody, CD20, CLL, NHL, lymphoma

  2. Utility of testing for monoclonal bands in serum of patients with suspected osteoporosis

    DEFF Research Database (Denmark)

    Abrahamsen, Bo; Andersen, Ivan; Christensen, Susanne S;


    of multiple myeloma diagnosed. All patients with multiple myeloma had a history of fragility fractures. If lymphoma was included as a target condition, the specificity increased to 95.3% and the positive predictive value increased to 23.5%. Monoclonal gammopathy of undetermined significance was diagnosed...... or monoclonal gammopathy of undetermined significance....

  3. Preparation of Europium Induced Conformation—specific anti—calmodulin Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    WeiGuoLI; ChaoQI; 等


    Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

  4. Preparation of Europium Induced Conformation-specific anti-calmodulin Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)


    Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.

  5. Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

    DEFF Research Database (Denmark)

    Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang;


    Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four antib...

  6. Monoclonal antibodies against a synthetic peptide from human immunodeficiency virus type 1 Nef protein

    DEFF Research Database (Denmark)

    Steinaa, L; Wulff, A M; Saermark, T


    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes fr...

  7. Immune defects in the risk of infection and response to vaccination in monoclonal gammopathy of undetermined significance and multiple myeloma

    NARCIS (Netherlands)

    Tete, Sarah M.; Bijl, Marc; Sahota, Surinder S.; Bos, Nicolaas


    The plasma cell proliferative disorders monoclonal gammopathy of undetermined significance (MGUS) and malignant multiple myeloma (MM) are characterized by an accumulation of transformed clonal plasma cells in the bone marrow and production of monoclonal immunoglobulin. They typically affect an older

  8. Anti-PrPC monoclonal antibody infusion as a novel treatment for cognitive deficits in an alzheimer's disease model mouse

    Directory of Open Access Journals (Sweden)

    Strittmatter Stephen M


    Full Text Available Abstract Background Alzheimer's Disease (AD is the most common of the conformational neurodegenerative disorders characterized by the conversion of a normal biological protein into a β-sheet-rich pathological isoform. In AD the normal soluble Aβ (sAβ forms oligomers and fibrils which assemble into neuritic plaques. The most toxic form of Aβ is thought to be oligomeric. A recent study reveals the cellular prion protein, PrPC, to be a receptor for Aβ oligomers. Aβ oligomers suppress LTP signal in murine hippocampal slices but activity remains when pretreated with the PrP monoclonal anti-PrP antibody, 6D11. We hypothesized that targeting of PrPC to prevent Aβ oligomer-related cognitive deficits is a potentially novel therapeutic approach. APP/PS1 transgenic mice aged 8 months were intraperitoneally (i.p. injected with 1 mg 6D11 for 5 days/week for 2 weeks. Two wild-type control groups were given either the same 6D11 injections or vehicle solution. Additional groups of APP/PS1 transgenic mice were given either i.p. injections of vehicle solution or the same dose of mouse IgG over the same period. The mice were then subjected to cognitive behavioral testing using a radial arm maze, over a period of 10 days. At the conclusion of behavioral testing, animals were sacrificed and brain tissue was analyzed biochemically or immunohistochemically for the levels of amyloid plaques, PrPC, synaptophysin, Aβ40/42 and Aβ oligomers. Results Behavioral testing showed a marked decrease in errors in 6D11 treated APP/PS1 Tg mice compared with the non-6D11 treated Tg groups (p C or Aβ oligomer levels. 6D11 treated APP/PS1 Tg mice had significantly greater synaptophysin immunoreactivity in the dentate gyrus molecular layer of the hippocampus compared to vehicle treated APP/PS1 Tg mice (p Conclusions Even short term treatment with monoclonal antibodies such as 6D11 or other compounds which block the binding of Aβ oligomers to PrPC can be used to treat

  9. Research on Growth Behavior of Embryos for Bovine and Murine on Primary Murine Embryos Fibroblast Cell Feeder Layer

    Institute of Scientific and Technical Information of China (English)

    AN Li-long; XIAO Mei; FENG Xiu-Liang; DOU Zhong-ying; QIU Huai; YANG Qi; LEI An-min; YANG Chun-rong; GAO Zhi-min


    The difference in growth behavior between bovine embryos and murine embryos was studied on PMEF(primary murine embryos fibroblast)feeder layer. The results showed as follows: With embryos having attached, bovine embryonic trophoblast formed a transparent membranous structure covering on inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure. Bovine embryos formed four kinds of ICM colonies with different morphology including the mass-like, the net-like, the stream-like and the mixture-like colonies. Compared with Murine ICM, the bovine ICM grew more fast. So, the bovine ICM was passaged at first after a culture of approximately 5 - 6 days in vitro, but murine ICM was passaged at first after an attachment of 3 - 4 days on PMEF feeder layer. The mixture colonies of bovine ICM differentiated very early, while the others differentiated very late. Most ICM-like mass of Bovine grew in a defined spot, but bovine ICMs like stream and ICMs like net proliferated fast and dispersed quickly. We found that the single blastomeres derived from late bovine morula and late murine morula formed sub-blastophere; moreover, the bovine ICM cell would differentiate rapidly if the trophoblast was removed.

  10. Use of commercially available rabbit monoclonal antibodies for immunofluorescence double staining

    DEFF Research Database (Denmark)

    Bzorek, M.; Stamp, I.M.; Frederiksen, L.


    Immunohistochemistry, that is, the use of polyclonal and monoclonal antibodies to detect cell and tissue antigens at a microscopical level is a powerful tool for both research and diagnostic purposes. Especially in the field of hematologic disease, there is often a need to detect several antigens...... synchronously, and we report here a fast and easy technique for demonstrating more than 1 antigen in 1 slide using immunofluorescence. We have used commercially available rabbit monoclonal antibodies (Cyclin D1, CD3, CD5, CD23, etc.) paired with mouse monoclonal antibodies (CD7, CD20, CD79a, Pax-5, etc.......) for double immunofluorescence labeling on paraffin-embedded tissue sections. Commercially available rabbit monoclonal antibodies in combination with mouse monoclonal antibodies proved useful in double immunofluorescence labeling on paraffin-embedded tissue, and all combinations used yielded excellent results...

  11. Feline lymphoplasmacytic stomatitis associated with monoclonal gammopathy and Bence-Jones proteinuria. (United States)

    Lyon, K F


    Lymphoplasmacytic stomatitis and gingivitis was diagnosed in an 8-year old female domestic shorthair. The cat had evidence of severe generalized inflammation of the oral cavity. Biopsy samples were evaluated and displayed a lichenoid, interface stomatitis which was predominantly lymphoplasmacytic. Serum protein electrophoresis confirmed a monoclonal gammopathy. Urine protein electrophoresis confirmed Bence-Jones proteinuria. Protein electrophoresis was used to diagnose monoclonal gammopathy (the production of a monoclonal immunoglobulin, or paraprotein, which is associated with a characteristic "M" protein spike on serum electrophoresis). Diseases associated with monoclonal gammopathy are similar in the dog and cat. Alkylating agent chemotherapy is used to rapidly reduce paraprotein concentrations in multiple myeloma. Multiple myeloma is the most common disorder associated with monoclonal gammopathy. This condition is less common in the cat, compared to the dog. This report examines the diagnosis and treatment of multiple myeloma in a cat presenting with severe stomatitis.

  12. Metabolic syndrome components in murine models. (United States)

    Lawson, Heather A; Cheverud, James M


    Animal models have enriched understanding of the physiological basis of metabolic disorders and advanced identification of genetic risk factors underlying the metabolic syndrome (MetS). Murine models are especially appropriate for this type of research, and are an excellent resource not only for identifying candidate genomic regions, but also for illuminating the possible molecular mechanisms or pathways affected in individual components of MetS. In this review, we briefly discuss findings from mouse models of metabolic disorders, particularly in light of issues raised by the recent flood of human genome-wide association studies (GWAS) results. We describe how mouse models are revealing that genotype interacts with environment in important ways, indicating that the underlying genetics of MetS is highly context dependant. Further we show that epistasis, imprinting and maternal effects each contribute to the genetic architecture underlying variation in metabolic traits, and mouse models provide an opportunity to dissect these aspects of the genetic architecture that are difficult if not impossible to ascertain in humans. Finally we discuss how knowledge gained from mouse models can be used in conjunction with comparative genomic methods and bioinformatic resources to inform human MetS research.

  13. Proteomic analysis of normal murine brain parts. (United States)

    Taraslia, Vasiliki K; Kouskoukis, Alexandros; Anagnostopoulos, Athanasios K; Stravopodis, Dimitrios J; Margaritis, Lukas H; Tsangaris, George Th


    Murine brain is an excellent tool for studying protein expression and brain function in mammals. Although mice are an extensively used model to recapitulate various pathological conditions, the proteome of the normal mouse brain has not been yet reported. In the present study, we identified the total proteins of different parts of the brain of CB7BL/6 mice, a widely used strain, by applying proteomic methodologies. The adult mouse brain was dissected anatomically into the following regions: frontal cortex, olfactory bulb, hippocampus, midbrain, cerebellum, hypothalamus and medulla. Total protein extracts of these regions were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry, following in-gel digestion with trypsin. Protein identification was carried out by peptide mass fingerprint. Thus, 515 different single-gene products were identified in total, 54 expressed specifically in the olfactory bulb, 62 in the hippocampus, 36 in the frontal cortex, five in the cerebellum, nine in the midbrain, eight in the hypothamamus and 10 in the medulla. The majority of the proteins were enzymes, structural proteins and transporters. Moreover, the distribution of these molecules appears to exhibit direct correlation with the function of the brain regions where they were expressed. This study leads to the complete characterization of the normal mouse brain proteome as well as the protein expression profile of the different brain regions. These results will aid in addressing unmet scientific needs regarding physiological and pathological brain functions.

  14. Amphotropic murine leukemia viruses induce spongiform encephalomyelopathy. (United States)

    Münk, C; Löhler, J; Prassolov, V; Just, U; Stockschläder, M; Stocking, C


    Recombinants of amphotropic murine leukemia virus (A-MuLV) have found widespread use in retroviral vector systems due to their ability to efficiently and stably infect cells of several different species, including human. Previous work has shown that replication-competent recombinants containing the amphotropic env gene, encoding the major SU envelope glycoprotein that determines host tropism, induce lymphomas in vivo. We show here that these viruses also induce a spongiform encephalomyelopathy in mice inoculated perinatally. This fatal central nervous system disease is characterized by noninflammatory spongiform lesions of nerve and glial cells and their processes, and is associated with moderate astro- and microgliosis. The first clinical symptoms are ataxia, tremor, and spasticity, progressing to complete tetraparesis and incontinence, and finally death of the animal. Sequences within the amphotropic env gene are necessary for disease induction. Coinfection of A-MuLV recombinants with nonneuropathogenic ecotropic or polytropic MuLV drastically increases the incidence, degree, and distribution of the neurodegenerative disorder. The consequence of these results in view of the use of A-MuLV recombinants in the clinic is discussed.

  15. Glycosaminoglycan interactions in murine gammaherpesvirus-68 infection.

    Directory of Open Access Journals (Sweden)

    Laurent Gillet

    Full Text Available Glycosaminoglycans (GAGs commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68 infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces.

  16. The clinical relevance and management of monoclonal gammopathy of undetermined significance and related disorders: Recommendations from the European Myeloma Network

    NARCIS (Netherlands)

    N.W.C.J. van de Donk (Niels); A. Palumbo (Antonio); H.E. Johnsen (Hans); M. Engelhardt (Monika); F. Gay (Francesca); P.K. Gregersen (Peter ); R. Hajek (Roman); M. Kleber (Martina); H. Ludwig (Heinz); G. Morgan (Gareth); P. Musto (Pellegrino); T. Plesner (Torben); O. Sezer; E. Terpos (Evangelos); A. Waage (Anders); S. Zweegman (Sonja); H. Einsele (Hermann); P. Sonneveld (Pieter); H.M. Lokhorst (Henk)


    textabstractMonoclonal gammopathy of undetermined significance is one of the most common pre-malignant disorders. IgG and IgA monoclonal gammopathy of undetermined significance are precursor conditions of multiple myeloma; lightchain monoclonal gammopathy of undetermined significance of light-chain

  17. Antitumor Activity and Immune Enhancement of Murine Interleukin-23 Expressed in Murine Colon Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    Baoen Shan; Jingsheng Hao; Qiaoxia Li; Masatoshi Tagawa


    Interleukin (IL)-23, a cytokine composed of p19 and the p40 subunit of IL-12, can enhance the proliferation of memory T cells and production of IFN-γ from activated T cells. It can also induce antitumor effects in murine model. To further evaluate the antitumor activity and immune enhancement of IL-23 in vivo, murine colon carcinoma cells retrovirally transduced with mIL-23 gene were injected subcutaneously (s.c.) into BALB/c mice.Survival time and tumor volume were observed. LDH release assay, [3H]-TdR incorporation assay and ELISA were used to determine CTL activity, proliferation of splenocytes and level of cytokines, respectively. Number of dendritic cells (DCs) was analyzed by flow cytometry (FCM). IL-23 secreted by Colon26/IL-23 cells suppressed the growth of tumor and prolonged the survival time of mice, enhanced proliferation of splenocytes, CTL activity, and number of DCs. IL-23 also promoted the production of Th1 cytokines such as IFN-γ, IL-12 and TNF-o. However,the level of IL-4 was not enhanced significantly. These data suggested that IL-23 secreted by tumor cells can induce antitumor activity by enhancing immune response.

  18. Characteristics of Monoclonal Antibody Against Infectious Bursal Disease Virus

    Institute of Scientific and Technical Information of China (English)

    LiYan-Fei; WangWei; 等


    Thirteen strains of monoclonal antibodies(McAbs) against infections bursal disease virus(IBDV) were obtained by using hydridoma technique and their characteristics were studied by double immunodiffusion,enzyme-linked immunosorbent assay(ELISA),virus neutralization test(VNT) and Western-blotting assay (WBA).The result showed that nine of the thirteen McAbs belonged to IgG class and four of them belonged to IgM class.No crossreactions were detected betwween the McAbs and Newscastle disease virus (NDV),infectious bronchitis virus(IBV) and Marek's disease virus(MDV).All of McAbs were positively specific reactive with IBDV and five of them can neutralize viral infectivity.Their recognized epitopes of the neutralizing McAbs were all presented on VP2 of the IBDV.

  19. Characteristics of Monoclonal Antibody Against Infectious Bursal Disease Virus

    Institute of Scientific and Technical Information of China (English)


    Thirteen strains of monoclonal antibodies (McAbs) against infectious bursal disease virus (IBDV) were obtained by using hybridoma technique and their characteristics were studied by double immunodiffusion,en- zyme- linked immunosorbent assay (ELISA), virus neutralization test (VNT) and Western- blotting assay (WBA). The result showed that nine of the thirteen McAbs belonged to IgG class and four of them belonged to IgM class. No crossreactions were detected betwween the McAbs and Newscastle disease virus (NDV) ,in- fectious bronchitis virus(IBV) and Marek's disease virus(MDV). All of McAbs were positively specific reac- tive with IBDV and five of them can neutralize viral infectivity. Their recognized epitopes of the neutralizing McAbs were all presented on VP2 of the IBDV.

  20. Preparation and Identification of Monoclonal Antibodies Against Vibrio anguillarum

    Institute of Scientific and Technical Information of China (English)

    Chen Shiyong(陈师勇); Zhang Peijun; Mo Zhaolan; Zhang Zhendong; Zou Yuxia; Xu Yongli


    Monoclonal antibodies (Mabs) against V.anguillarum strain M3 are prepared, and their isotypes are also characterized. Among them, C1C5 is the only Mab which does not crossreact with other eleven non-V.anguillarum strains. The proteinase K digestion test shows that the epitopes recognized by C1C5, C6C3 and C6C32 Mabs contained protein. The periodate oxidation test showed that the epitopes recognized by Mabs except C1C5 are glycosylated. In addition, results of additivity test indicate that the epitopes recognized by C6C3 and C6C32 Mabs are similar, and quite different from those recognized by Mab C1C5.

  1. Monoclonal antibodies against the human leukemia cell line K 562. (United States)

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B


    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  2. Effect of polyol sugars on the stabilization of monoclonal antibodies. (United States)

    Nicoud, Lucrèce; Cohrs, Nicholas; Arosio, Paolo; Norrant, Edith; Morbidelli, Massimo


    We investigate the impact of sugars and polyols on the heat-induced aggregation of a model monoclonal antibody whose monomer depletion is rate-limited by protein unfolding. We follow the kinetics of monomer consumption by size exclusion chromatography, and we interpret the results in the frame of two mechanistic schemes describing the enhanced protein stability in the presence of polyols. It is found that the stabilization effect increases with increasing polyol concentration with a comparable trend for all of the tested polyols. However, the stabilization effect at a given polyol concentration is polyol specific. In particular, the stabilization effect increases as a function of polyol size until a plateau is reached above a critical polyol size corresponding to six carbon atoms. Our results show that the stabilization by polyols does not depend solely on the volume fraction filled by the polyol molecules, but is also affected by the polyol chemistry.

  3. Moving through three-dimensional phase diagrams of monoclonal antibodies. (United States)

    Rakel, Natalie; Baum, Miriam; Hubbuch, Jürgen


    Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three-dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however.

  4. Annexin A1 as a target for managing murine pristane-induced systemic lupus erythematosus. (United States)

    Mihaylova, Nikolina; Bradyanova, Silviya; Chipinski, Petroslav; Herbáth, Melinda; Chausheva, Stela; Kyurkchiev, Dobroslav; Prechl, József; Tchorbanov, Andrey I


    Systemic lupus erythematosus (SLE) is a polygenic pathological disorder which involves multiple organs. Self-specific B cells play a main role in the lupus pathogenesis by generating autoantibodies as well as by serving as important autoantigen-presenting cells. Autoreactive T lymphocytes, on the other hand, are responsible for B cell activation and proliferation, and cytokine production. Therefore, both factors promote the idea that a down-modulation of activated self-reactive T and B cells involved in the pathogenic immune response is a reasonable approach for SLE therapy. Annexin A1 (ANX A1) is expressed by many cell types and binds to phospholipids in a Ca(2+) dependent manner. Abnormal expression of ANX A1 was found on activated B and T cells in both murine and human autoimmunity, suggesting its potential role as a therapeutic target. While its role on T lymphocytes is through formyl peptide receptor-like molecules (FPRL), and the formed ANX A1/FPRL pathway modulates T cell receptor signalling, there is still no fool-proof data available for the role of ANX A1 in B cells. We employed a lupus model of Balb/c mice with pristane-induced SLE which very closely resembles human lupus. In the present study, we investigated the possibility to modulate the autoimmune response in a pristane-induced mouse model of SLE using an anti- ANX A1 antibody. Administration of this monoclonal antibody resulted in the inhibition of T-cell activation and proliferation, suppression of IgG anti-dsDNA antibody-secreting plasma cells and of proteinuria, decreased disease activity and prolonged survival compared to control group.

  5. Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    A. Sasikalaveni


    Full Text Available Background: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA cells, which enable quicker isolation of street rabies virus with minimum passages. Objective: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. Materials and Methods: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. Results: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. Conclusion: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

  6. Phage Display Approaches for the Isolation of Monoclonal Antibodies Against Dengue Virus Envelope Domain III from Human and Mouse Derived Libraries

    Directory of Open Access Journals (Sweden)

    Subhash G. Vasudevan


    Full Text Available Domain III of the dengue virus envelope protein (EDIII, aa295-395 has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library ( > 10 billion independent clones. Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9 that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III.

  7. Fine specificity of monoclonal antibodies directed at human T cell receptor variable regions: comparison with oligonucleotide-driven amplification for evaluation of V beta expression. (United States)

    Diu, A; Romagné, F; Genevée, C; Rocher, C; Bruneau, J M; David, A; Praz, F; Hercend, T


    Seven distinct anti-human T cell receptor (TcR) V region monoclonal antibodies (mAb) were generated by immunizing mice with either human T cell lines or transfected murine cells expressing human TcR V beta genes. The specificity of these reagents was determined as follows: T cells recognized by each mAb were purified from the peripheral blood of healthy donors and TcR transcripts expressed in these cells were analyzed using oligonucleotide-driven amplification and cDNA sequencing. Four mAb were found to delineate the V beta 3, V beta 8, V beta 17 and V beta 19 subfamilies, respectively. The remaining reagents recognize subsets within the V beta 2, V beta 5 and V beta 13 subfamilies. Reactivity of the mAb with circulating T cells from 18 unrelated healthy individuals was determined. Limited variability was found from an individual to another. In four donors, mAb staining was compared to oligonucleotide-driven amplification for evaluation of V beta 3, V beta 8, V beta 17 and V beta 19 subfamily expression in the peripheral blood. Although the V gene subfamily-specific oligonucleotides used in this study belong to a carefully controlled series, our results show that this method does not give an accurate estimate of the percentage of peripheral T cells expressing a given TcR beta chain. The present data confirm the necessity to establish a complete set of well-characterized monoclonal reagents to study human T cell responses.

  8. A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease. (United States)

    Calvert, Amanda E; Dixon, Kandice L; Piper, Joseph; Bennett, Susan L; Thibodeaux, Brett A; Barrett, Alan D T; Roehrig, John T; Blair, Carol D


    The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.

  9. Clearance of a monoclonal anti-DNA antibody following administration of DNA in normal and autoimmune mice

    Energy Technology Data Exchange (ETDEWEB)

    Jones, F.S.; Pisetsky, D.S.; Kurlander, R.J.


    To study the assembly of DNA-anti-DNA complexes in vivo, we have measured the clearance from blood and organ localization of a murine IgG2a monoclonal anti-DNA antibody, called 6/0, following the infusion of DNA intravenously or intraperitoneally. Intraperitoneal DNA caused a profound acceleration of 6/0 anti-DNA clearance that was dose dependent and demonstrable after the infusion of as little as 1.9 microgram per gram of body weight of single-stranded DNA. The antibody was cleared primarily in the liver without increased deposition in the kidney. Intraperitoneal infusions of DNA also accelerated the clearance of 6/0 in autoimmune MRL-lpr/lpr mice. In contrast, intravenous DNA given in comparable doses caused only a slight increase in 6/0 antibody clearance; this accelerated clearance was seen only at low antigen doses and only during the first 10 min following DNA infusion. Using double-radiolabeling techniques, 6/0 and Cl.18, an IgG2ak myeloma protein without anti-DNA activity, were found to disappear from blood at a comparable rate in both B6D2 mice and MRL-lpr/lpr mice. These results suggest that the DNA-anti-DNA immune complexes can form in vivo but that this process is profoundly affected by the manner in which DNA enters the circulation. In addition, the results suggest that DNA-dependent clearance is not a major pathway for anti-DNA metabolism in normal or at least one strain of autoimmune mice.

  10. Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci. (United States)

    Zhang, Shaohua; Luo, Xuenong; Guo, Aijiang; Zhu, Xueliang; Cai, Xuepeng


    The gene encoding a mature 18 kDa glycoprotein of Taenia solium cysticerci (Ts18) was cloned and bacterially expressed with a His-tagged fusion protein. Monoclonal antibodies (MAbs) against the recombinant Ts18 antigen were generated in vitro by routine murine hybridoma technique of fusing splenocytes, from BALB/c mice immunized with the vesicular fluid of T. solium cysticerci (TsVF), with mouse myeloma cells (SP2/0). The reactivity and specificity of these MAbs were evaluated by indirect ELISA and immunoblotting techniques. Three stable hybridoma clones, namely 3B11, 6C5, and 6G4, were screened using His-Ts18-based ELISA, and these showed two IgG1 isotypes and one IgM isotype. All MAbs reacted with His-Ts18 at molecular weight (MW) 12.8 kDa and the native antigen at MW 18 kDa in TsVF and whole larval extracts (WLE). In a dot blotting test, MAbs 6C5 and 6G4 showed no obvious cross-reactivity with heterologous vesicular fluids from other taeniid species, including Taenia saginata (TsaVF), Taenia pisiformis (TpVF), Taenia hydatigena (ThVF), Taenia multiceps (TmVF), and Echinococcus granulosus (EgVF). Immunofluorescent assays showed that MAb 6C5 specifically reacted with the Ts18 expressed from pEGFP-N1-Ts18-transfected HeLa cells. Immunolocalization analysis, using MAb 6C5 as a probe, indicated that Ts18 was present at high concentrations in the region of the larval sucker and spiral canal. The results indicate that the Ts18 protein is an abundantly secreted parasite protein and MAbs against it might provide a step forward for improving the diagnosis of porcine cysticercosis.

  11. Generation of human antigen-specific monoclonal IgM antibodies using vaccinated "human immune system" mice.

    Directory of Open Access Journals (Sweden)

    Pablo D Becker

    Full Text Available BACKGROUND: Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. METHODOLOGY/PRINCIPAL FINDINGS: After transplantation with CD34+CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2⁻/⁻IL-2Rγc⁻/⁻ mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. "Human Immune System" mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. CONCLUSION/SIGNIFICANCE: This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.

  12. A new monoclonal antibody radiopharmaceutical for radioimmunoscintigraphy of breast cancer: direct labeling of antibody and its quality control

    Directory of Open Access Journals (Sweden)

    Mojtaba Salouti


    Full Text Available Radioimmunoscintigraphy (RIS has found widespread clinical application in tumor diagnosis. The antibody (Ab PR81 is a new murine anti-MUC1 monoclonal antibody (MAb against human breast carcinoma. In this study a very simple, rapid and efficient method for labeling of this MAb with 99mTc, particularly suitable for development of a ‘kit’is described. The reduction of Ab was performed with 2-mercaptoethanol (2-ME at a molar ratio of 2000:1 (2-ME:MAb and the reduced Ab was labeled with 99mTc via methylene diphosphonate (MDP as a transchelator. The labeling efficiency which was determined by instant thin layer chromatography (ITLC was 94.2%±2.3. Radiocolloides measured by cellulose nitrate electrophoresis were 2.5%±1.7. In vitro stability of the labeled product in human serum which was measured by gel filtration chromatography (FPLC was 70%±5.7 over 24 hr. The integrity of labeled MAb was checked by means of SDS-PAGE and no significant fragmentation was observed. The results of the cell-binding studies showed that both labeled and unlabeled PR81 were able to compete for binding to MCF 7 cells. Biodistribution studies were performed in normal BALB/c mice at 4 and 24 hrs post-injection and no important accumulation was observed in vital organs. These results show that the new radiopharmaceutical may be considered as a promising candidate for imaging of breast cancer.

  13. Characterization of the specificity by immunohistology of a monoclonal antibody to a novel epithelial antigen of ovarian carcinomas. (United States)

    Mariani-Costantini, R; Agresti, R; Colnaghi, M I; Ménard, S; Andreola, S; Rilke, F


    An immunohistological study, using the avidin-biotin-peroxidase complex method, was carried out to define the reactivity profile of a murine monoclonal antibody, MOv2, which recognizes a novel glycoprotidic antigen associated with ovarian epithelial tumors. Among the primary ovarian tumors tested, MOv2 immunostained 93% of mucinous and 75% of serous cystadenomas, 100% of mucinous, 81% of serous and 73% of endometrioid carcinomas. Undifferentiated and clear cell tumors revealed more limited reactivity with the antibody, whereas ovarian sex cord-stromal and germinal tumors were immunonegative. Positive reactions were also documented in omental metastases from primary ovarian carcinomas. No immunoreactivity was detected in normal ovarian epithelium, whereas the cells lining Walthard's nests adjacent to the fallopian tubes and a variety of normal epithelia were consistently immunolabeled. These included the lining epithelia of the gastrointestinal tract, bronchi and endocervix, and the epithelium of salivary, biliary and pancreatic ducts and sweat glands. To a lesser extent, positive reactions were detected in other surface epithelia, such as squamous and transitional epithelia. Among tumors other than ovarian, MOv2 consistently reacted with adenocarcinomas and squamous cell carcinomas from different sites, most notably breast, lung and gastrointestinal tract, and with transitional cell carcinomas. In contrast, no staining was demonstrated in non-epithelial malignancies. The antigen defined by MOv2 may be operationally useful as a marker of epithelial lineage in tumor histopathology. Its pattern of immunohistochemical distribution indicates that an antigenic phenotype shared by normal surface epithelia and non-ovarian carcinomas is strongly associated with common epithelial neoplasms of the ovaries.

  14. Murine Typhus: An Important Consideration for the Nonspecific Febrile Illness

    Directory of Open Access Journals (Sweden)

    Gurjot Basra


    Full Text Available Murine typhus is a widely distributed flea-borne infection caused by Rickettsia typhi. Symptoms of murine typhus are nonspecific and mimic a variety of other infectious diseases. We herein report a case of murine typhus in an area where the broad use of DDT in the mid-20th century has now made it a rare disease. The patient described presented with headache, fever, and a faint macular rash. Initial laboratory studies revealed a slight transaminase elevation. Further questioning revealed exposure to opossums, prompting the consideration of murine typhus as a diagnosis. Although typhus group antibodies were not present during the patient’s acute illness, empiric therapy with doxycycline was initiated, and the patient defervesced. One month after convalescence, the patient returned to clinic with serum that contained typhus group antibodies with an IgG titer of 1 : 1024. Murine typhus is an important consideration during the workup of a patient with a nonspecific febrile illness. Exposure to reservoir hosts and the flea vector place humans at risk for this disease. Clinician recognition of this entity is required for diagnosis and effective therapy.

  15. Analysis of cardiomyocyte movement in the developing murine heart

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Hisayuki [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Yuasa, Shinsuke, E-mail: [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Tabata, Hidenori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Nakajima, Kazunori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Sakaue-Sawano, Asako; Miyawaki, Atsushi [Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Fukuda, Keiichi [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan)


    The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.

  16. Isotype specific immune responses in murine experimental toxocariasis

    Directory of Open Access Journals (Sweden)

    Cuéllar C


    Full Text Available In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.

  17. [Monoclonal gammopathy of undetermined significance (MGUS) in Mexican mestizos: one institution's experience]. (United States)

    Ruiz-Delgado, Guillermo J; Gómez Rangel, J David


    Monoclonal gammopathy of undetermined significance (MGUS) is defined as presence of serum monoclonal protein at a concentration of 3 g per deciliter or less, no monoclonal protein or only moderate amounts of monoclonal light chains in urine, absence of lytic bone lesions, anemia, hypercalemia, and renal insufficiency related with monoclonal protein, and with a proportion of plasma cells in bone marrow of 10% or less. In Caucasian population, MGUS affects about 3% of individuals > 70 years of age, whereas in Mexican mestizos this figure is substantially lower (0.7%); on the other hand, MGUS represents in Mexico only 2.4% of all monoclonal gammopathies. In a total of 9081 individuals studied prospectively at the Centro de Hematología y Medicina Interna de Puebla throughout a 20-year period, 11 patients with MGUS were identified. Median age was 70 years (range 43-83 years). Patients have been followed in periods ranging from 6 to 3270 days (median, 308 days). Two patients evolved into overt multiple myeloma at 308 and 1687 days after diagnosis of MGUS. Overall median survival (SV) of the group has not been reached, whereas 3270 days overall SV is 91%. After discussing underreporting, biasing, and other confounding factors, it would seem that MGUS, like other monoclonal gammopathies, is less frequent in Mexican mestizos than in Caucasians. Routine screening studies to identify the condition should result in increased numbers of patients.

  18. Anti-CD45 radioimmunotherapy with 90Y but not 177Lu is effective treatment in a syngeneic murine leukemia model.

    Directory of Open Access Journals (Sweden)

    Johnnie J Orozco

    Full Text Available Radioimmunotherapy (RIT for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab labeled with 131I or 90Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing 90Y and 177Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J model. Biodistribution studies showed that both 90Y- and 177Lu-anti-murine CD45 Ab conjugates (DOTA-30F11 targeted hematologic tissues, as at 24 hours 48.8 ± 21.2 and 156 ± 14.6% injected dose per gram of tissue (% ID/g of 90Y-DOTA-30F11 and 54.2 ± 9.5 and 199 ± 11.7% ID/g of 177Lu-DOTA-30F11 accumulated in bone marrow (BM and spleen, respectively. However, 90Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi 90Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi 90Y-DOTA-30F11 had a median survival 66 days. 90Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, 177Lu- anti-CD45 RIT yielded no long-term survivors. Thus, 90Y was more effective than 177Lu for anti-CD45 RIT of AML in this murine leukemia model.

  19. Occurrence of Double Monoclonal Bands on Protein Electrophoresis: An Unusual Finding. (United States)

    Srinivasan, Vishrut K; Bhagat, Priyanka; Bansal, Frainey; Chhabra, Seema


    Various techniques of protein electrophoresis are used for detection of monoclonal proteins/paraproteins in serum and/or urine of patients with monoclonal gammopathies. These are detected as the so-called 'M' bands (monoclonal bands) on serum protein electrophoresis and/or immunofixation electrophoresis. In most cases, a single M-band is detected. However, more than one M-band can be detected in the samples of a minor proportion of patients. This condition is termed as 'double gammopathy' or 'biclonal gammopathy'. A knowledge of such an unusual occurrence is essential for recognition and appropriate interpretation of this entity.

  20. Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts. (United States)

    Collins, S P; Comis, A; Marshall, M; Hartwick, R F; Howden, M E


    1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri). 2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots. 3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

  1. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria.

    Directory of Open Access Journals (Sweden)

    Ryan W Carroll

    Full Text Available BACKGROUND: Cerebral malaria (CM is a neurological syndrome that includes coma and seizures following malaria parasite infection. The pathophysiology is not fully understood and cannot be accounted for by infection alone: patients still succumb to CM, even if the underlying parasite infection has resolved. To that effect, there is no known adjuvant therapy for CM. Current murine CM (MCM models do not allow for rapid clinical identification of affected animals following infection. An animal model that more closely mimics the clinical features of human CM would be helpful in elucidating potential mechanisms of disease pathogenesis and evaluating new adjuvant therapies. METHODOLOGY/PRINCIPAL FINDINGS: A quantitative, rapid murine coma and behavior scale (RMCBS comprised of 10 parameters was developed to assess MCM manifested in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA. Using this method a single mouse can be completely assessed within 3 minutes. The RMCBS enables the operator to follow the evolution of the clinical syndrome, validated here by correlations with intracerebral hemorrhages. It provides a tool by which subjects can be identified as symptomatic prior to the initiation of trial treatment. CONCLUSIONS/SIGNIFICANCE: Since the RMCBS enables an operator to rapidly follow the course of disease, label a subject as affected or not, and correlate the level of illness with neuropathologic injury, it can ultimately be used to guide the initiation of treatment after the onset of cerebral disease (thus emulating the situation in the field. The RMCBS is a tool by which an adjuvant therapy can be objectively assessed.

  2. Exchanging murine and human immunoglobulin constant chains affects the kinetics and thermodynamics of antigen binding and chimeric antibody autoreactivity.

    Directory of Open Access Journals (Sweden)

    Marcela Torres

    Full Text Available Mouse-human chimeric antibodies composed of murine variable (V and human (C chains are useful therapeutic reagents. Consequently, we investigated whether heterologous C-regions from mice and humans affected specificity and affinity, and determined the contribution of C(H glycosylation to antigen binding. The interaction of a 12-mer peptide mimetic with monoclonal antibody (mAb 18B7 to Cryptococcus neoformans glucuronoxylomannan, and its chimeric (ch and deglycosylated forms were studied by surface plasmon resonance. The equilibrium and rate association constants for the chAb were higher than for mAb 18B7. V region affinity was not affected by C(H region glycosylation whereas heterologous C region of the same isotype altered the Ab binding affinity and the specificity for self-antigens. Structural models displayed local differences that implied changes on the connectivity of residues. These findings suggest that V region conformational changes can be dictated by the C(H domains through an allosteric effect involving networks of highly connected amino acids.

  3. Generation and characterisation of a full-length cDNA encoding murine myotonic dystrophy protein kinase from cardiac tissue

    Energy Technology Data Exchange (ETDEWEB)

    Carey, N.; Tongeren, T. van; Winchester, C. [Charing Cross & Wesminster Medical School, London (United Kingdom)] [and others


    The mutation underlying myotonic dystrophy (DM) is a CTG trinucleotide expansion in the 3{prime} untranslated region of a putative protein kinase gene (DMPK). We report the isolation of a full-length cDNA clone of the murine (DMPK) gene from a heart cDNA library. Sequence analysis shows that the clone is a splice isoform which has only previously been identified in brain, suggesting that there may be some flexibility of the splicing pattern in some tissues. We are currently analyzing the library for the presence of other isoforms. The full-length cDNA has been cloned into a bacterial expression system and the expressed protein is being used as an immunogen to generate both polyclonal and monoclonal antisera. These reagents will allow the analysis of the intracellular targets of the DMPK. Subclones of the cDNA have been generated for use as in situ hybridization probes, allowing investigation of the normal patterns of expression of the gene and the differential expression of the protein isoforms. These data will be essential for deciding on a rational use of rare patient material and will provide the necessary baseline for the analysis of transgenic and {open_quotes}knock-out{close_quotes} mice.

  4. Mutational dynamics of murine angiogenin duplicates

    Directory of Open Access Journals (Sweden)

    Fares Mario A


    Full Text Available Abstract Background Angiogenin (Ang is a protein involved in angiogenesis by inducing the formation of blood vessels. The biomedical importance of this protein has come from findings linking mutations in Ang to cancer progression and neurodegenerative diseases. These findings highlight the evolutionary constrain on Ang amino acid sequence. However, previous studies comparing human Angiogenin with homologs from other phylogenetically related organisms have led to the conclusion that Ang presents a striking variability. Whether this variability has an adaptive value per se remains elusive. Understanding why many functional Ang paralogs have been preserved in mouse and rat and identifying functional divergence mutations at these copies may explain the relationship between mutations and function. In spite of the importance of testing this hypothesis from the evolutionarily and biomedical perspectives, this remains yet unaccomplished. Here we test the main mutational dynamics driving the evolution and function of Ang paralogs in mammals. Results We analysed the phylogenetic asymmetries between the different Ang gene copies in mouse and rat in the context of vertebrate Ang phylogeny. This analysis shows strong evidence in support of accelerated evolution in some Ang murine copies (mAng. This acceleration is not due to non-functionalisation because constraints on amino acid replacements remain strong. We identify many of the amino acid sites involved in signal localization and nucleotide binding by Ang to have evolved under diversifying selection. Compensatory effects of many of the mutations at these paralogs and their key structural location in or nearby important functional regions support a possible functional shift (functional divergence in many Ang copies. Similarities between 3D-structural models for mAng copies suggest that their divergence is mainly functional. Conclusions We identify the main evolutionary dynamics shaping the variability of

  5. Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells. (United States)

    Chan, C H; Blazar, B R; Greenfield, L; Kreitman, R J; Vallera, D A


    Myeloid leukemias can express interleukin-3 receptors (IL-3R). Therefore, as an antileukemia drug, a fusion immunotoxin was synthesized consisting of the murine IL-3 (mIL-3) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mIL-3 hybrid gene was cloned into a vector under the control of an inducible promote. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was potent because it inhibited FDC-P1, an IL-3R-expressing murine myelomonocytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL). Kinetics were rapid and cell-free studies showed that DT390-mIL-3 was as toxic as native DT. DT390-mIL-3 was selective because anti-mIL-3 monoclonal antibody, but not irrelevant antibody, inhibited its ability to kill. Cell lines not expressing IL-3R were not inhibited by the fusion protein. Because the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, bone marrow (BM) progenitor assays were performed. DT390-mIL-3 selectively inhibited committed BM progenitor cells as measured by in vitro colony-forming unit-granulocyte-macrophage and in vivo colony-forming unit-spleen colony assays. To determine if this fusion protein was reactive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed. Eight million DT390-mIL-3-treated C57BL/6 Ly5.2 BM cells, but not 4 million, were able to rescue lethally irradiated congenic C57BL/6 Ly5.1 recipients, suggesting that progenitor cells might be heterogenous in their expression of IL-3R. This idea was supported in competitive repopulation experiments in which DT390-mIL-3 treated C57BL/6 Ly5.2 BM cells were mixed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57BL/6 Ly5.1 mice. A significant reduction

  6. Immunolocation of antisperm monoclonal antibody 6B10 and corresponding antigen

    Institute of Scientific and Technical Information of China (English)

    高绍荣; 胡国俊; 段崇文; 刘辉; 韩之明; 宋祥芬; 陈大元


    An antisperm monoclonal antibody 6B10 was produced by hybridoma technique of the isotype IgG. The monoclonal antibody was purified by means of ammonium sulfate precipitation and protein A-Sepharose Cl-4B affinity chromatography. SDS polyacrylamide gel electrophoresis was used to evaluate the purity of the antibody. Evaluation of the sperm acrosomal status was determined by chlortetracycline (CTC) staining. It was found that monoclonal antibody 6B10 can inhibit the sperm acrosome reaction induced by progesterone. The corresponding antigen recognized by monoclonal antibody 6B10 was located on the plasma membrane of the sperm acrosome by indirect immunofluorescent microscopy and immunoelectronmicroscopy. Sperm protein was extracted by 1% Triton X-100. The molecular weight of the antigen is 50 ku, detected by Western blot. The antigen is a key protein in the sperm acrosome reaction and may be the receptor of progesterone on the sperm acrosome. It may either be developed as a candidate contraceptive vaccine

  7. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels. (United States)

    Wilkinson, Trevor C I


    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed.


    Institute of Scientific and Technical Information of China (English)



    Three monoclonal antibodies (MAbs) were raised against both pig and rabbit zona pellucida with a dual immunization protocol employing heat soluble pig zona (HSPZ) and heat soluble rabbit zona (HSRZ), Of the 140 wells screencd, 12 wells were positive to

  9. Purification of a Mycoplasma pneumoniae adhesin by monoclonal antibody affinity chromatography.


    Leith, D K; Baseman, J B


    A 165,000-dalton surface protein of Mycoplasma pneumoniae, designated protein P1, appears to be the major attachment ligand of the pathogen. We employed monoclonal antibody affinity chromatography to obtain purified protein P1.

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


    Institute of Scientific and Technical Information of China (English)

    WANGMin-Yi; HEZhi-Ying; WANGHan-Zheng


    The purpose of this study is to investigate the mechanism by which antiprogcsterone monoclonal antibodies block early pregnancy in mice. The mechanism of passive immunization is a complex issue as indicated below:

  12. Immunoglobulin heavy chain/light chain pairs (HLC, Hevylite™) assays for diagnosing and monitoring monoclonal gammopathies. (United States)

    Kraj, Maria


    Immunofixation (IFE) is a standard method for detecting monoclonal immunoglobulins and characterizing its isotype. Recently clonality can also be determined by using immunoglobulin (Ig) heavy chain/light chain immunoassays - HLC, HevyliteTM. HLC separately measures in pairs light chain types of each intact Ig class generating ratios of monoclonal Ig/uninvolved polyclonal Ig concentrations. Studies have shown that HLC and IFE are complementary methods. HLC assays quantify monoclonal proteins and identify monoclonality. It is possible to predict prognosis in multiple myeloma and to monitor response to treatment using HLC ratio. HLC ratio may serve as a parameter for myeloma induced immunoparesis and serve as a new marker for validating remission depth and relapse probabilities.

  13. Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model

    Directory of Open Access Journals (Sweden)

    Hill Colin


    Full Text Available Abstract Background Internalin A (InlA is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26, multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlAm*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlAm* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced

  14. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes Internalin A for enhanced infectivity in the murine oral infection model

    LENUS (Irish Health Repository)

    Monk, Ian R


    Abstract Background Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA m*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA m* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human

  15. Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation


    J. F. G. Vliegenthart; Maccarrone, M.; Veldink, G.A.


    The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporat...

  16. New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system.


    Galen, F X; Devaux, C.; Atlas, S; Guyenne, T; Menard, J; Corvol, P; Simon, D.; Cazaubon, C; Richer, P; Badouaille, G


    Monoclonal antibodies directed against human renin were obtained by the fusing of myeloma cells with spleen cells from Balb/c or high-responder Biozzi mice injected with pure tumoral or highly purified renal renin. These procedures resulted in the production of seven stable monoclonal antibodies to human renin. Antibodies in the hybridoma culture medium were screened by binding to pure iodinated renin or insolubilized renin in a solid phase assay. The concentration of purified antibodies that...

  17. Laboratory Characterizations on 2007 Cases of Monoclonal Gammopathies in East China

    Institute of Scientific and Technical Information of China (English)

    Hao Wang; Chunfang Gao; Lingling Xu; Zaixing Yang; Wenjing Zhao


    Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulin in patients with or without evidence of multiple myeloma (MM), macroglobulinemia, amyloidosis (AL), or a related plasma cell proliferative disorder. This study aims to evaluate laboratory diagnostic characters of monoclonal gammopathies and investigates the correlation between monoclonal gammopathies and transforming growth factor β1 (TGFβ1). Immunofixation electrophoresis (IFE), serum protein electrophoresis (SPE), nephelometry and urine light chain ELISA were used for laboratory identification of monoclonal immunoglobulins. Plasma TGFβ1 was detected with double-antibodies ELISA. Lightcycler was used for single nucleotide polymorphism (SNP) analysis. Totally 2,007 cases of monoclonai immunogiobulin (M protein) were identified in 10,682 samples. The isotypes of M protein were IgG type 47.1%, IgA 23.0%, IgM 8.7%, IgD 5.3%, free light chain κ 6.1%, λ 9.8%. In reference to IFE, the coherency of diagnosis was serum light chain ratio (κ/λ) 94.4%, quantitation of lgs 83%, light chain quantitation 80.9%, and urine light chain ratio (κ/λ) 58.0%. Plasma TGFβ1 was elevated significantly compared to normal control. The allelic frequency of codon 10 (C > T) was neither associated with the existence of the M protein nor with the M protein isotype. Monoclonal gammopathies can be identified with the combination of IFE, SPE, Igs quantitaion and urine light chain determination. Although TGFβ1, an important cytokine in immune regulation, was elevated in monoclonal gammopathies, the SNPs in coding region of TGFβ1 gene did not confer susceptibility to the development of monoclonal gammopathies in this study. Cellular & Molecular Immunology. 2008;5(4): 293-298.

  18. Does My Patient with a Serum Monoclonal Spike have Multiple Myeloma?


    Bianchi, Giada; Ghobrial, Irene M.


    A monoclonal spike (M spike or paraprotein) on serum protein electrophoresis (SPEP) is a frequent finding in the general population and typically is pathognomonic of an asymptomatic, premalignant condition called monoclonal gammopathy of undetermined significance (MGUS). MGUS occurs in around 3% of people older than 50 and is associated with a lifelong, low, yet non negligible, risk of progression to multiple myeloma (MM) or a related plasma cell dyscrasia. It is generally an incidental diagn...

  19. Preparation and Biological Evaluation of 188Re Labeled Monoclonal Antibody TGLA

    Institute of Scientific and Technical Information of China (English)

    WEN; Kai; ZHANG; Jun-li; CHEN; Bao-jun; CUI; Hai-ping


    <正>Monoclonal antibody TGLA is a specific targeting CD20 chimeric antibody. It can kill tumor cells and inhibit tumor cells’ growth effectively, which has been applied to clinical therapy of lymphoma cell B. 188 Re is easy to get, and emits both β and γ rays. 188Re labeled monoclonal antibody TGLA can be used for the study of lymphoma therapy and imaging. This work got the product 188Re-TGLA by direct labeling

  20. Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay.


    Hamel, J.; Brodeur, B R; Belmaaza, A; Montplaisir, S; Musser, J M; Selander, R K


    A coagglutination assay using monoclonal antibody is described for the identification of Haemophilus influenzae type b. An immunoglobulin G2a monoclonal antibody, Hb-2, directed against a serotype-specific outer membrane protein of H. influenzae type b was adsorbed to Staphylococcus aureus Cowan 1 cells. In a dot enzyme immunoassay, Hb-2 reacted with 453 of 455 H. influenzae type b isolates and did not react with H. influenzae of other serotypes, untypeable H. influenzae strains, or other bac...

  1. Efecto de un anticuerpo monoclonal anti CD20 (Rituximab) en trombocitopenia inmune.


    Untama, José; Médico, Departamento de Hematología, Hospital Nacional Edgardo Rebagliati Martins – EsSalud. Lima.; Del Carpio, Daniel; Médico, Departamento de Hematología, Hospital Nacional Edgardo Rebagliati Martins – EsSalud. Lima.


    Objetivo: Describir la respuesta terapéutica con un anticuerpo monoclonal anti CD20 (Rituximab), en pacientes con Trombocitopenia Inmune (PTI). Material y métodos: Estudio retrospectivo, descriptivo y observacional tipo serie de casos. Se revisaron las historias clínicas de pacientes adultos con PTI que recibieron el anticuerpo monoclonal anti CD20 (Rituximab), desde diciembre 2005 hasta diciembre 2010. Se definió respuesta: conteo plaquetario >30 mil, por lo menos duplicar el conteo plaqu...

  2. Studies of monoclonal antibodies IOR-CEA-1 and IOR-EGF/R3 labelled with {sup 99m}Tc; Estudo de marcacao dos anticorpos monoclonais IOR-CEA-1 e IOR-EGF/R3 com {sup 99m}Tc

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Carla Roberta de Barros Rodrigues


    Nuclear Medicine is a speciality that uses radioisotopes for the diagnosis or treatment of diseases and it is considered one of the best tools among the diagnostic modalities for detection of cancer. {sup 99m}Tc is one of the main isotopes for labelling antibodies and in Nuclear Medicine in general, due to its adequate physical properties, availability and low cost. Labelled monoclonal antibodies have shown promising results for diagnosis and therapy of cancer and their use has brought great experimental and clinical advances in the field of oncology. The main clinical applications of immunoscintigraphy with monoclonal antibodies are staging and evaluation of tumoral reappearance. The antibodies employed in this work were: OIR-CEA-1, a murine monoclonal antibody that acts directly against CEA expressed in several neoplasia in particular those from the gastrointestinal tract (colorectal cancer) and IOR-EGF/R3, a murine monoclonal antibody that binds to the external domain of EGF-R and it has been used in the diagnosis of tumors of epithelial origin. The objectives of this work were the development and optimization of the reduction and purification processes, the radiolabelling techniques and quality control procedures (radiochemical, immunoreactivity and cystein challenge) and imaging studies of monoclonal antibodies OIR-CEA-1 and IOR-EGF/R3, using the simple, fast and efficient method of direct labelling of the antibody with {sup 99m}Tc. The final results was the definition of the best conditions for the preparation of lyophilized reactive kits of OIR-CEA-1 and IOR- EGF/R3 for an efficient diagnostic application in Nuclear Medicine. The most adequate conditions for the labelling of the antibodies were: 1.0 mg Ab, 29 {mu}L MDP, 3.0 {mu}g Sn{sup 2+}, 1 mL of {sup 99m}Tc and 30 min. reaction time. With these conditions the labelling yield was always higher than 95% and the maximum activity of {sup 99m}Tc was about 2220 MBq (60 mCi). The evidences of the efficiency and

  3. Monoclonal antibodies: pharmacokinetics as a basis for new dosage regimens? (United States)

    Azanza, J-R; Sádaba, B; Gómez-Guiu, A


    Complete monoclonal IgG antibodies which are in use in clinical practice share some pharmacological properties resulting in high concentrations in plasma. This fact is reflected in their low volumes of distribution, which can also be correlated with a high molecular weight and water solubility. This feature allows a novel approach to be applied to the dosing schedule for this group of drugs with fixed doses being used instead of the initially developed weight- or body surface-adjusted dosing schedules. In addition, the development of a new formulation containing hyaluronidase allows a subcutaneous route of administration to be used, because hyaluronidase creates a space in the subcutaneous tissue that helps antibody absorption. This method requires higher doses, but has allowed testing the feasibility of administering a fixed dose, with no individual dose adjustments based on weight or body surface. Moreover, loading doses are not needed, because the first dose results, within 3 weeks, in minimum concentrations that are higher than effective concentrations.

  4. Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab. (United States)

    Brand, Toni M; Iida, Mari; Wheeler, Deric L


    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance.

  5. Anti-bacterial monoclonal antibodies: back to the future? (United States)

    Oleksiewicz, Martin B; Nagy, Gábor; Nagy, Eszter


    Today's medicine has to deal with the emergence of multi-drug resistant bacteria, and is beginning to be confronted with pan-resistant microbes. This worsening inadequacy of the antibiotics concept, which has ruled infectious medicine in the last six decades creates an increasing unmet medical need that can be addressed by passive immunization. While past experience from the pre-antibiotic era with serum therapy was in many cases encouraging, antibacterial monoclonal antibodies have so far suffered high attrition rates in the clinic, generally from lack of efficacy. Yet, we believe that recent developments in a number of areas such as infectious disease pathogenesis research, translational medicine, mAb engineering, mAb manufacturing and rapid bedside diagnostics are converging to make the medium-term future permissive for antibacterial mAb development. Here, we review antibacterial mAb-based approaches that are or were in clinical development, and may potentially act as paradigms with regards to molecular targets, antibody formats and mode-of-action, pre-clinical validation and selection of most relevant patient populations, in order to increase the likelihood of successful product development in this field.

  6. Novel neutralizing monoclonal antibodies protect rodents against lethal filovirus challenges

    Directory of Open Access Journals (Sweden)

    Caleb D. Marceau


    Full Text Available Filoviruses are the causative agents of lethal hemorrhagic fever in human and non-human primates (NHP. The family of Filoviridae is composed of three genera, Ebolavirus, Marburgvirus and Cuevavirus. There are currently no approved vaccines or antiviral therapeutics for the treatment of filovirus infections in humans. Passive transfer of neutralizing antibodies targeting the Ebola virus (EBOV glycoprotein (GP has proven effective in protecting mice, guinea pigs and NHP from lethal challenges with EBOV. In this study, we generated two neutralizing monoclonal antibodies (MAbs, termed S9 and M4 that recognize the GP of EBOV or multiple strains of Marburg virus (MARV, respectively. We characterized the putative binding site of S9 as a linear epitope on the glycan cap of the GP1 subunit of the EBOV-GP. The M4 antibody recognizes an unknown conformational epitope on MARV-GP. Additionally, we demonstrated the post-exposure protection potential of these antibodies in both the mouse and guinea pig models of filovirus infection. These data indicate that MAbs S9 and M4 would be good candidates for inclusion in an antibody cocktail for the treatment of filovirus infections.

  7. Plasmid copy number noise in monoclonal populations of bacteria (United States)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy


    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  8. An update on newer monoclonal antibodies in lymphoma therapy

    Directory of Open Access Journals (Sweden)

    Subhashini Archana Kadavakolan


    Full Text Available In 2014, an estimated 9.4% of all new cancers in the US were accounted to hematological cancers. Most of these cancers have a B-cell origin and on the cell surface express antigen CD20-known to restrict B-cells. Considering the intrinsic immune status of the patients receiving chemotherapy, monoclonal antibodies (mAbs are designed to provide active or passive immunotherapy. Clinical success of rituximab-anti-CD20 mAb in the treatment of lymphoma has led to the development of newer generations of mAb to increase the anti-tumor activity. Hence, recent advances in lymphoma therapy are being built on the conventional prototype of anti-CD20 mAb-rituximab. Our review is an update on the advances in lymphoma therapy using mAb against CD20 including the second generation-ofatumumab, veltuzumab, ocrelizumab, and the third-generation mAbs-ocaratuzumab and obinutuzumab.

  9. Monoclonal antibodies against NS1 protein of Goose parvovirus. (United States)

    Qiu, Zheng; Tian, Wei; Yu, Tianfei; Li, Li; Ma, Bo; Wang, Junwei


    In the present study, monoclonal antibodies (MAbs) against NS1 protein of Goose parvovirus (GPV) were generated. The secreted MAbs were obtained by fusing mouse myeloma cells and spleen cells of BALB/c mice, which were immunized with the plasmid pcDNA3.1-GPV-NS1 and recombinant protein of GPV-NS1. With indirect ELISA, six hybridoma cell lines against GPV-NS1 were screened. The subtypes of the two MAbs were IgG2a; the others were IgM. The light chain was κ. Western blot analysis showed that six MAbs reacted with recombinant protein GPV-NS1. GPV-NS1 was dissected into 15 overlapping epitopes, which were used to react with MAbs in Western blot. Results showed that six MAbs recognized NS1 protein linear B-cell epitopes located at the C-terminus 453-514 aa, 485-542 aa, and 533-598 aa.

  10. Monoclonal antibody-based candidate therapeutics against HIV type 1. (United States)

    Chen, Weizao; Dimitrov, Dimiter S


    Treatment of HIV-1 infection has been highly successful with small molecule drugs. However, resistance still develops. In addition, long-term use can lead to toxicity with unpredictable effects on health. Finally, current drugs do not lead to HIV-1 eradication. The presence of the virus leads to chronic inflammation, which can result in increased morbidity and mortality after prolonged periods of infection. Monoclonal antibodies (mAbs) have been highly successful during the past two decades for therapy of many diseases, primarily cancers and immune disorders. They are relatively safe, especially human mAbs that have evolved in humans at high concentrations to fight diseases and long-term use may not lead to toxicities. Several broadly neutralizing mAbs (bnmAbs) against HIV-1 can protect animals but are not effective when used for therapy of an established infection. We have hypothesized that HIV-1 has evolved strategies to effectively escape neutralization by full-size antibodies in natural infections but not by smaller antibody fragments. Therefore, a promising direction of research is to discover and exploit antibody fragments as potential candidate therapeutics against HIV-1. Here we review several bnmAbs and engineered antibody domains (eAds), their in vitro and in vivo antiviral efficacy, mechanisms used by HIV-1 to escape them, and strategies that could be effective to develop more powerful mAb-based HIV-1 therapeutics.

  11. DNA immunization as a technology platform for monoclonal antibody induction. (United States)

    Liu, Shuying; Wang, Shixia; Lu, Shan


    To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail.

  12. Analysis of viral clearance unit operations for monoclonal antibodies. (United States)

    Miesegaes, George; Lute, Scott; Brorson, Kurt


    Demonstration of viral clearance is a critical step in assuring the safety of biotechnology products. We generated a viral clearance database that contains product information, unit operation process parameters, and viral clearance data from monoclonal antibody and antibody-related regulatory submissions to FDA. Here we present a broad overview of the database and resulting analyses. We report that the diversity of model viruses tested expands as products transition to late-phase. We also present averages and ranges of viral clearance results by Protein A and ion exchange chromatography steps, low pH chemical inactivation, and virus filtration, focusing on retro- and parvoviruses. For most unit operations, an average log reduction value (LRV, a measure of clearance power) for retrovirus of >4 log(10) were measured. Cases where clearance data fell outside of the anticipated range (i.e., outliers) were rationally explained. Lastly, a historical analysis did not find evidence of any improvement trend in viral clearance over time. The data collectively suggest that many unit operations in general can reliably clear viruses.

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

    Institute of Scientific and Technical Information of China (English)



    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.


    Institute of Scientific and Technical Information of China (English)

    刘小云; 甄永苏


    Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DTT),while Fab′ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β mercaptoethanol.The conjugation between Fab′ fragment and pingyangmycin (PYM),an antitumor antibiotic,was mediated by dextran T 40.Immunoreactivity of Fab′ PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay.Antitumor effects of the Fab′ PYM conjugates were evaluated by subcutaneously transplanted tumors in mice. Results.The molecular weight of Fab′ fragment was approximately 53 kD,while the average molecular weight of Fab′ PYM conjugate was 170 kD.The Fab′ PYM conjugates showed immunoreactivity with antigen relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab′ PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb. Conclusion.Fab′ PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.

  15. Establishment of a novel monoclonal antibody against LGR5. (United States)

    Sasaki, Yuka; Kosaka, Hiromichi; Usami, Katsuaki; Toki, Hiroe; Kawai, Hironori; Shiraishi, Norihiko; Ota, Toshio; Nakamura, Kazuyasu; Furuya, Akiko; Satoh, Mitsuo; Hasegawa, Kazumasa; Masuda, Kazuhiro


    LGR5 is an orphan G-protein-coupled receptor (GPCR) that is expressed on the cell surface membrane. LGR5 is reported to be overexpressed in colon, liver, and ovary tumor compared to normal tissue. However, a specific ligand for LGR5 has not yet been determined, and the function is still not clear. An LGR5-specific monoclonal antibody (mAb) is needed as a tool for detection and analysis of LGR5 biological function and cancer therapy. To date, no mAb against LGR5 that retains high affinity and specificity has been reported. Here, we report successful establishment and characterization of a mAb (KM4056) that specifically recognizes the extracellular N-terminal domain of human LGR5, but not LGR4 or LGR6. This mAb has potent complement-dependent cytotoxicity (CDC) activity in vitro and shows strong anti-tumor activity in vivo against xenograft model by transplanting LGR5 expressing CHO transfectants into SCID mice. Thus, KM4056 can be a useful tool for detection of LGR5 positive cells and analysis of LGR5 biological function.

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


    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.

  17. Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy. (United States)

    Poppe, Leszek; Jordan, John B; Lawson, Ken; Jerums, Matthew; Apostol, Izydor; Schnier, Paul D


    Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

  18. Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Ramos Franco Antonia Maria


    Full Text Available A large number of Endotrypanum stocks (representing an heterogeneous population of strains have been screened against a panel of monoclonal antibodies (MAbs derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12 or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5 reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent, but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex

  19. Role of cosolutes in the aggregation kinetics of monoclonal antibodies. (United States)

    Nicoud, Lucrèce; Sozo, Margaux; Arosio, Paolo; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo


    We propose a general strategy based on kinetic analysis to investigate how cosolutes affect the aggregation behavior of therapeutic proteins. We apply this approach to study the impact of NaCl and sorbitol on the aggregation kinetics of two monoclonal antibodies, an IgG1 and an IgG2. By using a combination of size exclusion chromatography and light scattering techniques, we study the impact of the cosolutes on the monomer depletion, as well as on the formation of dimers, trimers, and larger aggregates. We analyze these macroscopic effects in the frame of a kinetic model based on Smoluchowski's population balance equations modified to account for nucleation events. By comparing experimental data with model simulations, we discriminate the effect of cosolutes on the elementary steps which contribute to the global aggregation process. In the case of the IgG1, it is found that NaCl accelerates the kinetics of aggregation by promoting specifically aggregation events, while sorbitol delays the kinetics of aggregation by specifically inhibiting protein unfolding. In the case of the IgG2, whose monomer depletion kinetics is limited by dimer formation, NaCl and sorbitol are found respectively to accelerate and inhibit conformational changes and aggregation events to the same extent.

  20. Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions. (United States)

    Nicoud, Lucrèce; Jagielski, Jakub; Pfister, David; Lazzari, Stefano; Massant, Jan; Lattuada, Marco; Morbidelli, Massimo


    Gaining understanding on the aggregation behavior of proteins under concentrated conditions is of both fundamental and industrial relevance. Here, we study the aggregation kinetics of a model monoclonal antibody (mAb) under thermal stress over a wide range of protein concentrations in various buffer solutions. We follow experimentally the monomer depletion and the aggregate growth by size exclusion chromatography with inline light scattering. We describe the experimental results in the frame of a kinetic model based on population balance equations, which allows one to discriminate the contributions of the conformational and of the colloidal stabilities to the global aggregation rate. Finally, we propose an expression for the aggregation rate constant, which accounts for solution viscosity, protein-protein interactions, as well as aggregate compactness. All these effects can be quantified by light scattering techniques. It is found that the model describes well the experimental data under dilute conditions. Under concentrated conditions, good model predictions are obtained when the solution pH is far below the isoelectric point (pI) of the mAb. However, peculiar effects arise when the solution pH is increased toward the mAb pI, and possible explanations are discussed.

  1. Monoclonal antibody probe for assessing beer foam stabilizing proteins. (United States)

    Onishi, A; Proudlove, M O; Dickie, K; Mills, E N; Kauffman, J A; Morgan, M R


    A monoclonal antibody (Mab; IFRN 1625) has been produced, which is specific for the most hydrophobic polypeptides responsible for foam stabilization. The binding characteristics of the Mab suggest that it is the conformation of certain hydrophobic polypeptides which is important for foam stabilization. An enzyme-linked immunosorbent assay (ELISA) for assessing the foam-positive form of the foam-stabilizing polypeptides in beer was developed using IFRN 1625. A good correlation was obtained between ELISA determination of foam-stabilizing polypeptides and an empirical means of determining foaming, that is, the Rudin head retention values, for a collection of beers of various foam qualities. Application of the ELISA to different stages of the brewing process showed that the amounts of foam-positive polypeptides increased during barley germination. During the brewing process the proportion of foam-positive polypeptides present after fermentation increased slightly, although a large amount was lost along with other beer proteins during subsequent steps, such as filtering. The present study demonstrates that the amounts of beer polypeptide present in a foam-positive form have a direct relationship with the foaming potential of beer, that their levels are altered by processing, and that there is potential for greater quality control.

  2. Desmoids in familial adenomatous polyposis are monoclonal proliferations. (United States)

    Middleton, S B; Frayling, I M; Phillips, R K


    Desmoids are poorly-understood, locally aggressive, non-metastasizing fibromatoses that occur with disproportionate frequency in patients with familial adenomatous polyposis (FAP). Their nature is controversial with arguments for and against a neoplastic origin. Neoplastic proliferations are by definition monoclonal, whereas reactive processes originate from a polyclonal background. We examined clonality of 25 samples of desmoid tissue from 11 female FAP patients by assessing patterns of X-chromosome inactivation to calculate a clonality ratio. Polymerase chain reaction (PCR) amplification of a polymorphic CAG short tandem repeat (STR) sequence adjacent to a methylation-sensitive restriction enzyme site within the human androgen receptor (HUMARA) gene using fluorescent-labelled primers enabled analysis of PCR products by Applied Biosystems Genescan II software. Twenty-one samples from nine patients were informative for the assay. Samples from all informative cases comprised a median of 66% (range 0-75%) clonal cells but from the six patients with a clonality ratio < or =0.5 comprised a median of 71% (65-75%) clonal cells. FAP-associated desmoid tumours are true neoplasms. This may have implications in the development of improved treatment protocols for patients with these aggressive tumours.

  3. Super-genotype: global monoclonality defies the odds of nature.

    Directory of Open Access Journals (Sweden)

    Johannes J Le Roux

    Full Text Available The ability to respond to natural selection under novel conditions is critical for the establishment and persistence of introduced alien species and their ability to become invasive. Here we correlated neutral and quantitative genetic diversity of the weed Pennisetum setaceum Forsk. Chiov. (Poaceae with differing global (North American and African patterns of invasiveness and compared this diversity to native range populations. Numerous molecular markers indicate complete monoclonality within and among all of these areas (F(ST = 0.0 and is supported by extreme low quantitative trait variance (Q(ST = 0.00065-0.00952. The results support the general-purpose-genotype hypothesis that can tolerate all environmental variation. However, a single global genotype and widespread invasiveness under numerous environmental conditions suggests a super-genotype. The super-genotype described here likely evolved high levels of plasticity in response to fluctuating environmental conditions during the Early to Mid Holocene. During the Late Holocene, when environmental conditions were predominantly constant but extremely inclement, strong selection resulted in only a few surviving genotypes.

  4. Super-genotype: global monoclonality defies the odds of nature. (United States)

    Le Roux, Johannes J; Wieczorek, Ania M; Wright, Mark G; Tran, Carol T


    The ability to respond to natural selection under novel conditions is critical for the establishment and persistence of introduced alien species and their ability to become invasive. Here we correlated neutral and quantitative genetic diversity of the weed Pennisetum setaceum Forsk. Chiov. (Poaceae) with differing global (North American and African) patterns of invasiveness and compared this diversity to native range populations. Numerous molecular markers indicate complete monoclonality within and among all of these areas (F(ST) = 0.0) and is supported by extreme low quantitative trait variance (Q(ST) = 0.00065-0.00952). The results support the general-purpose-genotype hypothesis that can tolerate all environmental variation. However, a single global genotype and widespread invasiveness under numerous environmental conditions suggests a super-genotype. The super-genotype described here likely evolved high levels of plasticity in response to fluctuating environmental conditions during the Early to Mid Holocene. During the Late Holocene, when environmental conditions were predominantly constant but extremely inclement, strong selection resulted in only a few surviving genotypes.

  5. Pharmacokinetics of biotech drugs: peptides, proteins and monoclonal antibodies. (United States)

    Lin, Jiunn H


    With the advances in recombinant DNA biotechnology, molecular biology and immunology, the number of biotech drugs, including peptides, proteins and monoclonal antibodies, available for clinical use has dramatically increased in recent years. Although pharmacokinetic principles are equally applicable to the large molecule drugs and conventional small molecule drugs, the underlying mechanisms for the processes of absorption, distribution, metabolism and excretion (ADME) of large molecule drugs are often very different from that of small molecule drugs. Therefore, a good understanding of the ADME processes of large molecule drugs is essential in support of the development of therapeutic biologics. The purpose of this article is to review the current knowledge of the ADME processes that govern the pharmacokinetics of biotech drugs. The challenges encountered by orally administered peptide and protein drugs, and the nature of lymphatic absorption after subcutaneous administration will be discussed. In addition, molecular mechanisms of biodistribution, metabolism and renal excretion of biotech drugs will also be discussed. Finally, approaches used for prediction of human pharmacokinetics of protein drugs will be briefly discussed.

  6. Secondary monoclonal gammopathy of undetermined significance after allogeneic stem cell transplantation in multiple myeloma. (United States)

    Schmitz, Marian F; Otten, Henny G; Franssen, Laurens E; van Dorp, Suzanne; Strooisma, Theo; Lokhorst, Henk M; van de Donk, Niels W C J


    In the course of multiple myeloma, patients may develop a M-protein band different from the original: secondary monoclonal gammopathy of undetermined significance. In this retrospective single center analysis, we describe the occurrence and clinical relevance of secondary monoclonal gammopathy of undetermined significance after allogeneic stem cell transplantation (post-transplant monoclonal gammopathy of undetermined significance). A total of 138 patients who had undergone 139 allogeneic stem cell transplantations (39.6% in the upfront setting and 60.4% for relapsed multiple myeloma) were included in the study. Sixty-seven (48.2%) patients developed secondary monoclonal gammopathy of undetermined significance, after a median latency of 6.9 months. Secondary monoclonal gammopathy of undetermined significance occurred more often in patients who achieved at least very good partial response after allogeneic stem cell transplantation, compared to partial response or less (54.8% vs. 26.5%; P=0.005). The incidence was also higher in the upfront setting as compared to relapsed disease, or with a sibling donor compared to matched unrelated donor, but less often after T-cell depletion. Importantly, development of post-transplant monoclonal gammopathy of undetermined significance as a time-dependent variable independently predicted for superior progression-free and overall survival (median progression-free survival 37.5 vs. 6.3 months, Pundetermined significance should not be confused with relapse or progression of disease. (Trial registered with; HOVON 108: NTR 2958.).

  7. Statistical analysis of data from limiting dilution cloning to assess monoclonality in generating manufacturing cell lines. (United States)

    Quiroz, Jorge; Tsao, Yung-Shyeng


    Assurance of monoclonality of recombinant cell lines is a critical issue to gain regulatory approval in biological license application (BLA). Some of the requirements of regulatory agencies are the use of proper documentations and appropriate statistical analysis to demonstrate monoclonality. In some cases, one round may be sufficient to demonstrate monoclonality. In this article, we propose the use of confidence intervals for assessing monoclonality for limiting dilution cloning in the generation of recombinant manufacturing cell lines based on a single round. The use of confidence intervals instead of point estimates allow practitioners to account for the uncertainty present in the data when assessing whether an estimated level of monoclonality is consistent with regulatory requirements. In other cases, one round may not be sufficient and two consecutive rounds are required to assess monoclonality. When two consecutive subclonings are required, we improved the present methodology by reducing the infinite series proposed by Coller and Coller (Hybridoma 1983;2:91-96) to a simpler series. The proposed simpler series provides more accurate and reliable results. It also reduces the level of computation and can be easily implemented in any spreadsheet program like Microsoft Excel. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1061-1068, 2016.

  8. Preparation of monoclonal antibody to P53 and its clinical application

    Institute of Scientific and Technical Information of China (English)

    Wenqing Wei; Junhua Wu; Jing Liu; Yuxia Wang


    Objective:The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initial y in clinical immunoassay. Methods:Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by af inity chro-matography. Antibody titer was determined by enzyme linked immunosorbent assay (ELISA). The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results:Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by af inity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1:6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor celllines and determine the expression of P53 in tumor tis-sue. Conclusion:Three strains of monoclonal antibodies with high af inity to P53 were successful y established, which can be used for detecting the expression of P53 in tumor cells or tissue.

  9. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies (United States)

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


    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

  10. Pharmacodynamics of Doxycycline in a Murine Malaria Model▿


    Batty, Kevin T.; Law, Angela S. F.; Stirling, Verity; Moore, Brioni R.


    Doxycycline is reported to impair second-generation parasite schizogony. The effects of doxycycline alone and combined with dihydroartemisinin were investigated in a murine malaria model. Doxycycline lowered the rate of parasite growth within 2 days, with maximum effect in 6 days. Addition of dihydroartemisinin led to an additive antimalarial effect.

  11. Murine Sirt3 protein isoforms have variable half-lives (United States)

    Sirt3 is a NAD+-dependent protein deacetylase mainly localized in mitochondria. Recent studies indicate that the murine Sirt3 gene expresses different transcript variants resulting in three possible Sirt3 protein isoforms with variable lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3...

  12. Increased rejection of murine allogeneic bone marrow in presensitized recipients

    NARCIS (Netherlands)

    vanOs, R; deWitte, T; Dillingh, JH; Mauch, PM; Down, JD


    The role of presensitizing murine recipients with donor spleen cells prior to T cell-depleted or -repleted H-2 compatible allogeneic bone marrow transplantation (BMT) was investigated at two different doses of total body irradiation (TBI). Recipients that were presensitized with 2 x 10(7) irradiated

  13. Macropinocytosis is the Entry Mechanism of Amphotropic Murine Leukemia Virus

    DEFF Research Database (Denmark)

    Rasmussen, Izabela; Vilhardt, Frederik


    The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV does not internalize by caveolae or other pinocytic mechanism, but by macropinocytosis. Thus A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin...

  14. Expression of biologically active murine interleukin-18 in Lactococcus lactis. (United States)

    Feizollahzadeh, Sadegh; Khanahmad, Hossein; Rahimmanesh, Ilnaz; Ganjalikhani-Hakemi, Mazdak; Andalib, Alireza; Sanei, Mohammad Hossein; Rezaei, Abbas


    The food-grade bacterium Lactococcus lactis is increasingly used for heterologous protein expression in therapeutic and industrial applications. The ability of L. lactis to secrete biologically active cytokines may be used for the generation of therapeutic cytokines. Interleukin (IL)-18 enhances the immune response, especially on mucosal surfaces, emphasizing its therapeutic potential. However, it is produced as an inactive precursor and has to be enzymatically cleaved for maturation. We genetically manipulated L. lactis to secrete murine IL-18. The mature murine IL-18 gene was inserted downstream of a nisin promoter in pNZ8149 plasmid and the construct was used to transform L. lactis NZ3900. The transformants were selected on Elliker agar and confirmed by restriction enzyme digestion and sequencing. The expression and secretion of IL-18 protein was verified by SDS-PAGE, western blotting and ELISA. The biological activity of recombinant IL-18 was determined by its ability to induce interferon (IFN)-γ production in L. lactis co-cultured with murine splenic T cells. The amounts of IL-18 in bacterial lysates and supernatants were 3-4 μg mL(-1) and 0.6-0.7 ng mL(-1), respectively. The successfully generated L. lactis strain that expressed biologically active murine IL-18 can be used to evaluate the possible therapeutic effects of IL-18 on mucosal surfaces.

  15. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

    Directory of Open Access Journals (Sweden)

    Yang Wang


    Full Text Available The N-linked glycosylation in recombinant monoclonal antibodies (mAb occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF to analyze a recombinant mAb produced in murine myeloma (NS0 cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC high performance liquid chromatography (HPLC to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS analysis.

  16. Generation and characterization of a tetraspanin CD151/integrin α6β1-binding domain competitively binding monoclonal antibody for inhibition of tumor progression in HCC (United States)

    Cai, Jia-Bin; Huang, Xiao-Yong; Wu, Chao; Zhang, Lu; Kang, Qiang; Liu, Li-Xin; Xie, Nan; Shen, Zao-Zhuo; Hu, Mei-Yu; Cao, Ya; Qiu, Shuang-Jian; Sun, Hui-Chuan; Zhou, Jian; Fan, Jia; Shi, Guo-Ming


    Our previous studies revealed that tetraspanin CD151 plays multiple roles in the progression of hepatocellular carcinoma (HCC) by forming a functional complex with integrin α6β1. Herein, we generated a monoclonal antibody (mAb) that dissociates the CD151/integrin α6β1 complex, and we evaluated its bioactivity in HCCs. A murine mAb, tetraspanin CD151 (IgG1, called CD151 mAb 9B), was successfully generated against the CD151-integrin α6β1 binding site of CD151 extracellular domains. Co-immunoprecipitation using CD151 mAb 9B followed by Western blotting detected a 28 kDa protein. Both immunofluorescent and immunohistochemical staining showed a good reactivity of CD151 mAb 9B in the plasma membrane and cytoplasm of HCC cells, as well as in liver cells. In vitro assays demonstrated that CD151 mAb 9B could inhibit neoangiogenesis and both the mobility and the invasiveness of HCC cells. An in vivo assay showed that CD151 mAb 9B inhibited tumor growth potential and HCC cells metastasis. We successfully produced a CD151 mAb 9B targeting the CD151/integrin α6β1-binding domain, which not only can displayed good reactivity to the CD151 antigen but also prevented tumor progression in HCC. PMID:26756217

  17. Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies. (United States)

    Sasaki, Takanori; Terano, Yoshitake; Shibata, Tadayoshi; Kawamoto, Hiroyoshi; Kuzuguchi, Tsuyoshi; Kohyama, Erina; Watanabe, Toshihiro; Ohyama, Tohru; Gemba, Munekazu


    Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin-biotin sandwich ELISA (ABS-ELISA) system for SEA, SEB, and SEC1 using newly-developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78-25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantifi-cation and simplicity in detecting SEs.

  18. Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles for active targeting of breast cancer cells. (United States)

    Kouchakzadeh, Hasan; Shojaosadati, Seyed Abbas; Mohammadnejad, Javad; Paknejad, Malihe; Rasaee, Mohammad Javad


    With PR81 as a murine monoclonal antibody (mAb) that was prepared against the human breast cancer, the MUC1 receptor specific targeting is possible. In this study, PR81-conjugated bovine serum albumin (BSA) nanoparticles loaded with anticancer drug 5-fluorouracil (5-FU) were developed. Enzyme linked immunosorbant assay (ELISA) results showed high immunoreactivity of PR81 mAb conjugated to nanoparticles towards MUC1 related peptide or native cancerous MUC1 and almost no cross-reaction to non-specific proteins. In vitro experiments were performed to determine the ability of this new drug delivery system on overcoming MCF-7 breast cancer cells in comparison with four other systems. The results revealed that these cell-type specific drug loaded nanoparticles could achieve more cell death as compared to when the 5-FU was used with no carriers. Stability studies of produced drug delivery system proved high immunoreactivity of conjugated PR81 even after 11 days of storage in room temperature.

  19. Uptake of indium-111 in the liver of mice following administration of indium-111-DTPA-labeled monoclonal antibodies: Influence of labeling parameters, physiologic parameters, and antibody dose

    Energy Technology Data Exchange (ETDEWEB)

    Schuhmacher, J.; Klivenyi, G.; Matys, R.; Kirchgebner, H.; Hauser, H.; Maier-Borst, W.; Matzku, S. (Institute of Radiology and Pathophysiology, Heidelberg (Germany, F.R.))


    Liver uptake of indium-111 ({sup 111}In) in mice was investigated following administration of {sup 111}In-DTPA murine monoclonal antibodies ({sup 111}In-DTPA-MAbs) labeled by the cyclic anhydride method. Biodistribution of HPLC-purified {sup 111}In-DTPA-MAb preparations was checked with a low (0.2 micrograms) and a high (8.0 micrograms) MAb dose. Using Bio Gel P-30 for desalting the MAb-conjugates, {sup 111}In uptake in the liver amounted to 8%-9% of the injected dose (ID) and was independent from the MAb dose, the DTPA-to-MAb molar ratio, tumor growth and biologic variability (different MAbs and different strains of mice). Using Sephadex G-25 for desalting, 0.2 micrograms doses from 7 out of 26 preparations showed increased liver accumulation of {sup 111}In in non-tumor mice ranging from 15%-25% of ID. Corresponding high doses led to a normal value of 8%-9%. Increased liver uptake of the low dose could not be reduced by coadministration of the unconjugated MAb, but was normal after reinjection of in vivo filtered material. An inverse intracellular distribution of {sup 111}In activity between sediment and supernatant of liver homogenates, following the administration of the low and the high MAb dose, indicated an artifact of the labeling procedure rather than an inherent biological property of labeled MAbs.

  20. Comprehensive evaluation of two HLA-B17 monoclonal antibodies for flow cytometry-based HLA-B57/B58 screening prior to abacavir prescription. (United States)

    Stevens, R; Coates, E; Street, J; Cook, E; Darke, C


    Hypersensitivity reactions to the drug abacavir, used to treat HIV/AIDS patients, is associated with possession of HLA-B*57:01. We have carefully assessed two commercially available HLA-B57/B58 murine monoclonal antibodies [0196HA and BIH0243 (One Lambda Inc.)] in a simple flow cytometry-based assay. The evaluation involved tests on 228 reference and random samples covering 91% of all WHO recognized HLA-A, B and C specificities. These involved donors with six different HLA-B*57 alleles and included 19 examples of B*57:01. Both antibodies unambiguously detected B57, but there were small difference in their reactivity against B57-positive non-B*57:01 samples. Importantly, there was no reactivity against B57/B58-negative samples. The possible amino acid motifs involved in the reactivity of these antibodies with B57/B58 were delineated. Thus, HLA-B57/B58, normally present in <10% of patients, can be easily recognized using these two antibodies and further tested by a DNA-based typing method to identify B*57:01.

  1. Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces (United States)

    Coleman, David A.; Oh, Soon-Hwan; Zhao, Xiaomin; Zhao, Hongyuan; Hutchins, Jeff T.; Vernachio, John H.; Patti, Joseph M.; Hoyer, Lois L.


    Two monoclonal antibodies (MAbs) were raised against the Candida albicans cell-surface glycoprotein Als3 using the N-terminal domain of the protein as the immunogen. ELISA was used to demonstrate the specificity of the MAbs for the Als3 fragment, but not for the corresponding N-terminal domain fragments from other proteins in the Als family. The anti-Als3 MAbs immunolabeled the surface of germ tubes from a diverse collection of wild-type C. albicans isolates, but did not label yeast cells, an als3Δ/als3Δ deletion mutant strain, nor isolates of other Candida species associated with human disease. Als3 was visualized readily in fresh and formalin-fixed, paraffin-embedded kidney tissue from a murine model of candidiasis. The anti-Als3 MAbs were also useful for immunogold electron microscopy and Western blotting. Both MAbs blocked C. albicans adhesion to vascular endothelial cells and buccal epithelial cells. These versatile MAbs are a valuable addition to the reagents available to study C. albicans cell surface dynamics and interaction of the fungus with host cells. PMID:19427882

  2. Structure and function of the sigma-70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription. (United States)

    Strickland, M S; Thompson, N E; Burgess, R R


    Murine monoclonal antibodies reactive with the major sigma subunit (sigma-70) of Escherichia coli RNA polymerase were obtained by standard hybridoma techniques. Western blot analyses established that seven antibodies had unique specificities after various chemical and enzymatic methods were used to fragment sigma. Peptides were purified by HPLC using size-exclusion, reverse-phase, or ion-exchange chromatography. The epitopes for six of these antibodies have been localized to specific peptides. These peptides were further characterized by amino acid composition and N-terminal sequencing. Sigma, which has a molecular weight of 70.2K, runs as 83K on SDS gels in this study. This anomalous behavior has been localized to the very acidic N-terminal half of the molecule. One antibody is unable to bind to native sigma. Two others do not bind well to sigma when it is contained in holoenzyme, indicating that their epitopes are in regions of sigma which are inaccessible in the holoenzyme complex. All three of these antibodies fail to inhibit in vitro transcription by holoenzyme. The other four antibodies all can inhibit in vitro transcription.

  3. Development of porcine ficolin-alpha monoclonal and polyclonal antibodies for determining the binding capacity of multiple GlcNAc-binding proteins to bacterial danger components. (United States)

    Nahid, M Abu; Ross, Steven J; Umiker, Benjamin R; Li, Huapeng; Sugii, Sunji; Bari, Latiful


    Ficolins are a group of oligomeric defense proteins assembled from collagen-like stalks and fibrinogen-like domains that have common biochemical specificity for N-acetyl-d-glucose amine (GlcNAc) and can function as opsonins. In this report, GlcNAc-binding protein (GBP) purified from porcine nonimmune serum was biochemically characterized as ficolin-α. Ficolin-α was used as an immunogen to generate both rabbit polyclonal and murine monoclonal anti-ficolin-α antibodies, which are not yet commercially available. GBPs have been shown to be present in many animals, including humans; however, their functions are largely unknown. GBPs from chicken, dog, horse, bovine, and human sera were isolated using various chromatography methods. Interestingly, anti-ficolin-α antibody showed cross-reaction with those animal sera GBPs. Furthermore, anti-ficolin-α antibody was reactive with the GlcNAc eluate of Escherichia coli O26-bound and Salmonella-bound porcine serum proteins. Functionally, GBPs and bacteria-reactive pig serum proteins were able to bind with pathogen-associated molecular patterns such as lipopolysaccharides and lipoteichoic acids. Our studies demonstrate that ficolin-α specific antibody was reactive with GBPs from many species as well as bacteria-reactive serum proteins. These proteins may play important roles in innate immunity by sensing danger components that can lead to antibacterial activity.

  4. Extracorporeal adsorption therapy: A Method to improve targeted radiation delivered by radiometal-labeled monoclonal antibodies.

    Energy Technology Data Exchange (ETDEWEB)

    Nemecek, Eneida R.; Green, Damian J.; Fisher, Darrell R.; Pagal, John M.; Lin, Yukang; Gopal, A. K.; Durack, Lawrence D.; Rajendran, Joseph G.; Wilbur, D. S.; Nilsson, Rune; Sandberg, Bengt; Press, Oliver W.


    Many investigators have demonstrated the ability to treat hematologic malignancies with radiolabeled monoclonal antibodies targeting hematopoietic antigens such as anti-CD20 and anti-CD45. [1-5] Although the remission rates achieved with radioimmunotherapy (RIT) are relatively high, many patients subsequently relapse presumably due to suboptimal delivery of enough radiation to eradicate the malignancy. The dose-response of leukemia and lymphoma to radiation has been proven. Substantial amounts of radiation can be delivered by RIT if followed by hematopoietic cell transplantation to rescue the bone marrow from myeloablation.[ref] However, the maximum dose of RIT that can be used is still limited by toxicity to normal tissues affected by nonspecific delivery of radiation. Efforts to improve RIT focus on improving the therapeutic ratios of radiation in target versus non-target tissues by removing the fraction of radioisotope that fails to bind to target tissues and circulates freely in the bloodstream perfusing non-target tissues. Our group and others have explored several alternatives for removal of unbound circulating antibody. [refs] One such method, extracorporeal adsorption therapy (ECAT) consists of removing unbound antibody by a method similar to plasmapheresis after critical circulation time and distribution of antibody into target tissues have been achieved. Preclinical studies of ECAT in murine xenograft models demonstrated significant improvement in therapeutic ratios of radioactivity. Chen and colleagues demonstrated that a 2-hour ECAT procedure could remove 40 to 70% of the radioactivity from liver, lung and spleen. [ref] Although isotope concentration in the tumor was initially unaffected, a 50% decrease was noted approximately 36 hours after the procedure. This approach was also evaluated in a limited phase I pilot study of patients with refractory B-cell lymphoma. [ref] After radiographic confirmation of tumor localization of a test dose of anti-CD20

  5. Monoclonal antibody:the corner stone of modern biotherapeutics%Monoclonal antibody: the corner stone of modern biotherapeutics

    Institute of Scientific and Technical Information of China (English)

    XIA Zhi-nan; CAI Xue-ting; CAO Peng


    Worldwide sales of biologic drugs exceeded 100 billion USD in 2011.About 32% is from therapeutic monoclonal antibody (mAb).With many blockbuster biopharmaceutical patents expiring over the next decade,there is a great opportunity for biosimilar to enter the worldwide especially emerging market.Both European Medicines Agency (EMA) and Food and Drug Administration (FDA) have introduced regulatory frameworks for the potential approval of biosimilar mAb therapeutics.Rather than providing a highly abbreviated path,as in the case for small molecule chemical drug,approval for biosimilar mAb will require clinical trial and the details will be very much on a case-by-case basis.Since mAb is the dominant category of biologic drugs,mAb will be the focus of this review.First,the United States (US) and European Union (EU) approved mAb and those in phase 3 trials will be reviewed,then strategies on how to win biosimilar competition will be reviewed.

  6. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model.

    LENUS (Irish Health Repository)

    Monk, Ian R


    Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.

  7. Crystallization and preliminary X-ray analysis of birch-pollen allergen Bet v 1 in complex with a murine monoclonal IgG Fab' fragment

    DEFF Research Database (Denmark)

    Spangfort, M D; Mirza, Osman Asghar; Gajhede, M


    The human type I allergic response is characterized by the presence of allergen-specific serum immunoglobulin E (IgE). Allergen-mediated cross-linking of receptor-bound IgE on the surface of mast cells and circulating basophils triggers the release of mediators, resulting in the development of th......, with unit-cell parameters a = 91.65, b = 99.14, c = 108.90 A, alpha = 105.7, beta = 98.32, gamma = 97.62 degrees, and diffract to 2.9 A resolution when analyzed at 100 K using synchrotron-generated X--rays....

  8. Functional characterization and evaluation of in vitro protective efficacy of murine monoclonal antibodies BURK24 and BURK37 against Burkholderia pseudomallei.

    Directory of Open Access Journals (Sweden)

    Bhavani V Peddayelachagiri

    Full Text Available Burkholderia pseudomallei, the causative agent of melioidosis has been recognized by CDC as a category B select agent. Although substantial efforts have been made for development of vaccine molecules against the pathogen, significant hurdles still remain. With no licensed vaccines available and high relapse rate of the disease, there is a pressing need for development of alternate protection strategies. Antibody-mediated passive protection is promising in this regard and our primary interest was to unravel this frontier of specific mAbs against Burkholderia pseudomallei infections, as functional characterization of antibodies is a pre-requisite to demonstrate them as protective molecules. To achieve this, we designed our study on in vitro-based approach and assessed two mAbs, namely BURK24 and BURK37, reactive with outer membrane proteins and lipopolysaccharide of the pathogen respectively, for their ability to manifest inhibitory effects on the pathogenesis mechanisms of B. pseudomallei including biofilm formation, invasion and induction of apoptosis. The experiments were performed using B. pseudomallei standard strain NCTC 10274 and a clinical isolate, B. pseudomallei 621 recovered from a septicemia patient with diabetic ailment. The growth kinetic studies of the pathogen in presence of various concentrations of each individual mAb revealed their anti-bacterial properties. Minimal inhibitory concentration and minimal bactericidal concentration of both the mAbs were determined by using standards of Clinical and Laboratory Standards Institute (CLSI and experiments were performed using individual mAbs at their respective bacteriostatic concentration. As an outcome, both mAbs exhibited significant anti-Burkholderia pseudomallei properties. They limited the formation of biofilm by the bacterium and completely crippled its invasion into human alveolar adenocarcinoma epithelial cells. Also, the mAbs were appreciably successful in preventing the bacterium to induce apoptosis in A549 cells. The present study design revealed the protection attributes possessed by BURK24 and BURK37 that has to be further substantiated by additional in vivo studies.

  9. Belimumab: anti-BLyS human monoclonal antibody, anti-BLyS monoclonal antibody, BmAb, human monoclonal antibody to B-lymphocyte stimulator. (United States)


    Belimumab is a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of B-lymphocyte stimulator, or BLyS. Belimumab is in phase III trials for the treatment of systemic lupus erythematosus (SLE) and has completed a phase II trial in rheumatoid arthritis (RA); the product may also have potential in the treatment of other autoimmune disorders. In May 2001, Cambridge Antibody Technology (now MedImmune) completed its discovery programme and Human Genome Sciences identified belimumab as a candidate for clinical development. More than 1000 distinct human antibodies specific to BLyS were characterized by the collaboration.B-lymphocyte stimulator is a naturally occurring protein discovered by Human Genome Sciences that stimulates B-lymphocytes to develop into mature B cells. Laboratory studies have indicated that higher than normal levels of B-lymphocyte stimulator may contribute to the pathogenesis of autoimmune diseases, such as SLE and RA. Human Genome Sciences (HGS) and Cambridge Antibody Technology signed a collaborative agreement in August 1999 to study the B-lymphocyte stimulator as a human protein target. HGS is also developing other BLyS products. In March 2000, HGS and Cambridge Antibody Technology expanded their agreement into a 10-year collaboration and product development alliance, providing Human Genome Sciences with the right to use the antibody technology of Cambridge Antibody Technology to fully develop human antibodies for therapeutic and diagnostic purposes. Cambridge Antibody Technology will receive royalty payments on product sales from HGS, as well as the development and milestone payments it has already received. Belimumab will be manufactured in Human Genome Sciences' manufacturing facility, located in Rockville, MD, USA. HGS holds commercial rights to the drug. In July 2005, GlaxoSmithKline (GSK) exercised its co-development and co-promotion option to belimumab. In an agreement made in June 1996, HGS had

  10. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies. (United States)

    Nicoud, Lucrèce; Arosio, Paolo; Sozo, Margaux; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo


    We investigate by kinetic analysis the aggregation mechanism of two monoclonal antibodies belonging to the IgG1 and IgG2 subclass under thermal stress. For each IgG, we apply a combination of size exclusion chromatography and light scattering techniques to resolve the time evolution of the monomer, dimer, and trimer concentrations, as well as the average molecular weight and the average hydrodynamic radius of the aggregate distribution. By combining the detailed experimental characterization with a theoretical kinetic model based on population balance equations, we extract relevant information on the contribution of the individual elementary steps on the global aggregation process. The analysis shows that the two molecules follow different aggregation pathways under the same operating conditions. In particular, while the monomer depletion of the IgG1 is found to be rate-limited by monomeric conformational changes, bimolecular collision is identified as the rate-limiting step in the IgG2 aggregation process. The measurement of the microscopic rate constants by kinetic analysis allows the quantification of the protein-protein interaction potentials expressed in terms of the Fuchs stability ratio (W). It is found that the antibody solutions exhibit large W values, which are several orders of magnitude larger than the values computed in the frame of the DLVO theory. This indicates that, besides net electrostatic repulsion, additional effects delay the aggregation kinetics of the antibody solutions with respect to diffusion-limited conditions. These effects likely include the limited efficiency of the collision events due to the presence of a limited number of specific aggregation-prone patches on the heterogeneous protein surface, and the contribution of additional repulsive non-DLVO forces to the protein-protein interaction potential, such as hydration forces.

  11. Monoclonal antibodies against human granulocytes and myeloid differentiation antigens. (United States)

    Mannoni, P; Janowska-Wieczorek, A; Turner, A R; McGann, L; Turc, J M


    Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315-43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1, 80H.3, and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (IgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (IgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence, i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited CFU-GM growth stimulated by leukocyte feeder layers or placental conditioned media, but did not inhibit BFU-E and CFU-E. Antigens recognized by 80H.3, 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.

  12. The clinical relevance and management of monoclonal gammopathy of undetermined significance and related disorders: recommendations from the European Myeloma Network. (United States)

    van de Donk, Niels W C J; Palumbo, Antonio; Johnsen, Hans Erik; Engelhardt, Monika; Gay, Francesca; Gregersen, Henrik; Hajek, Roman; Kleber, Martina; Ludwig, Heinz; Morgan, Gareth; Musto, Pellegrino; Plesner, Torben; Sezer, Orhan; Terpos, Evangelos; Waage, Anders; Zweegman, Sonja; Einsele, Hermann; Sonneveld, Pieter; Lokhorst, Henk M


    Monoclonal gammopathy of undetermined significance is one of the most common pre-malignant disorders. IgG and IgA monoclonal gammopathy of undetermined significance are precursor conditions of multiple myeloma; light-chain monoclonal gammopathy of undetermined significance of light-chain multiple myeloma; and IgM monoclonal gammopathy of undetermined significance of Waldenström's macroglobulinemia and other lymphoproliferative disorders. Clonal burden, as determined by bone marrow plasma cell percentage or M-protein level, as well as biological characteristics, including heavy chain isotype and light chain production, are helpful in predicting risk of progression of monoclonal gammopathy of undetermined significance to symptomatic disease. Furthermore, alterations in the bone marrow microenvironment of monoclonal gammopathy of undetermined significance patients result in an increased risk of venous and arterial thrombosis, infections, osteoporosis, and bone fractures. In addition, the small clone may occasionally be responsible for severe organ damage through the production of a monoclonal protein that has autoantibody activity or deposits in tissues. These disorders are rare and often require therapy directed at eradication of the underlying plasma cell or lymphoplasmacytic clone. In this review, we provide an overview of the clinical relevance of monoclonal gammopathy of undetermined significance. We also give general recommendations of how to diagnose and manage patients with monoclonal gammopathy of undetermined significance.

  13. Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer. (United States)

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


    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.

  14. Effect on renal function of an iso-osmolar contrast agent in patients with monoclonal gammopathies

    Energy Technology Data Exchange (ETDEWEB)

    Preda, Lorenzo [Division of Radiology, European Institute of Oncology, IRCCS, Milan (Italy); Agazzi, Alberto; Martinelli, Giovanni [Division of Haematology, European Institute of Oncology, IRCCS, Milan (Italy); Raimondi, Sara [Division of Epidemiology and Biostatistics, European Institute of Oncology, IRCCS, Milan (Italy); University of Milan, Department of Occupational Medicin ' ' Clinica del Lavoro Luigi Devoto' ' Section of Medical Statistics and Biometry ' ' GA Maccacaro' ' , Milan (Italy); Lanfranchi, Carla Federica [University of Milan, IRCCS, School of Medicine, Milan (Italy); Passerini, Rita [Unit of Laboratory Medicine, European Institute of Oncology, IRCCS, Milan (Italy); Calvetta, Albania [Nephrology and Dialysis Unit, Istituto Clinico Humanitas, IRCCS, Rozzano, Milan (Italy); Bellomi, Massimo [Division of Radiology, European Institute of Oncology, IRCCS, Milan (Italy); University of Milan, IRCCS, School of Medicine, Milan (Italy)


    To assess the safety of the non-ionic iso-osmolar contrast agent iodixanol on renal function in patients with monoclonal gammopathies undergoing CT. We explored the effect of iodixanol on renal function in 30 patients with monoclonal gammopathies and 20 oncological patients with a normal electrophoretic profile (control group). The parameters used to estimate renal function were: serum creatinine, eGFR (determined 24 h before and 48 h after the administration of iodixanol), and urinary excretion of Neutrophil Gelatinase-Associated Lipocalin (NGAL) determined 2 h and 24 h after. Serum creatinine was also determined 1 month after the administration of iodixanol. No significant increase in serum creatinine values were observed in the monoclonal gammopathies group and in 19/20 patients in the control group. Only 1 patient in the control group developed a transient contrast agent-induced nephropathy. We found no statistically significant difference between the two groups regarding the percentage variation from baseline values of serum creatinine, creatinine clearance, NGAL 2 h after, and eGFR. Whereas NGAL at 24 h showed a statistically significant increase in patients with Monoclonal gammopathies. The use of iodixanol appears to be safe in patients with monoclonal gammopathies and an eGFR {>=} 60 ml/min/1.73 mq. (orig.)

  15. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Holers, V.M.; Kotzin, B.L.


    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  16. Effects of the murine skull in optoacoustic brain microscopy. (United States)

    Kneipp, Moritz; Turner, Jake; Estrada, Héctor; Rebling, Johannes; Shoham, Shy; Razansky, Daniel


    Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

  17. Lactobacillus rhamnosus GG as an Effective Probiotic for Murine Giardiasis

    Directory of Open Access Journals (Sweden)

    Nisha Goyal


    Full Text Available The gut microflora is an important constituent in the intestinal mucosal barrier and has been introduced as the concept of probiotic therapy that beneficially affects the host by improving its intestinal microbial balance. Therefore, the main objective of the study was to explore the protective potential of various lactobacilli strains for murine giardiasis. By experimentation, it was found that the probiotic supplementation of either Lactobacillus casei, L. acidophilus, L. plantarum, or L. rhamnosus GG, 7 days prior to inoculation with G. lamblia trophozoites, reduced the rate of cyst excretion compared with Giardia-infected mice. Interestingly, L. GG was found to be the most effective probiotic in reducing the duration of giardia cycle and acts as an effective prophylactic probiotic for murine giardiasis but needs to be clinically correlated due to entirely different human microflora.

  18. T Cell Integrin Overexpression as a Model of Murine Autoimmunity

    Directory of Open Access Journals (Sweden)

    Yung Raymond L.


    Full Text Available Integrin adhesion molecules have important adhesion and signaling functions. They also play a central role in the pathogenesis of many autoimmune diseases. Over the past few years we have described a T cell adoptive transfer model to investigate the role of T cell integrin adhesion molecules in the development of autoimmunity. This report summarizes the methods we used in establishing this murine model. By treating murine CD4+ T cells with DNA hypomethylating agents and by transfection we were able to test the in vitro effects of integrin overexpression on T cell autoreactive proliferation, cytotoxicity, adhesion and trafficking. Furthermore, we showed that the ability to induce in vivo autoimmunity may be unique to the integrin lymphocyte function associated antigen-1 (LFA-1.

  19. Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model.

    Directory of Open Access Journals (Sweden)

    Natalia Makarova

    Full Text Available BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC. Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP that had the size and morphology of live infectious XMRV. RESULTS: Immunization elicited Env-specific binding and neutralizing antibodies (NAb against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations. CONCLUSIONS: Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.

  20. Osteopontin Is Upregulated in Human and Murine Acute Schistosomiasis Mansoni (United States)

    Pereira, Thiago Almeida; Syn, Wing-Kin; Amâncio, Frederico Figueiredo; Cunha, Pedro Henrique Diniz; Caporali, Julia Fonseca Morais; Trindade, Guilherme Vaz de Melo; Santos, Elisângela Trindade; Souza, Márcia Maria; Andrade, Zilton Araújo; Witek, Rafal P; Secor, William Evan; Pereira, Fausto Edmundo Lima; Lambertucci, José Roberto; Diehl, Anna Mae


    Background Symptomatic acute schistosomiasis mansoni is a systemic hypersensitivity reaction against the migrating schistosomula and mature eggs after a primary infection. The mechanisms involved in the pathogenesis of acute schistosomiasis are not fully elucidated. Osteopontin has been implicated in granulomatous reactions and in acute hepatic injury. Our aims were to evaluate if osteopontin plays a role in acute Schistosoma mansoni infection in both human and experimentally infected mice and if circulating OPN levels could be a novel biomarker of this infection. Methodology/Principal Findings Serum/plasma osteopontin levels were measured by ELISA in patients with acute (n = 28), hepatointestinal (n = 26), hepatosplenic (n = 39) schistosomiasis and in uninfected controls (n = 21). Liver osteopontin was assessed by immunohistochemistry in needle biopsies of 5 patients. Sera and hepatic osteopontin were quantified in the murine model of schistosomiasis mansoni during acute (7 and 8 weeks post infection, n = 10) and chronic (30 weeks post infection, n = 8) phase. Circulating osteopontin levels are increased in patients with acute schistosomiasis (p = 0.0001). The highest levels of OPN were observed during the peak of clinical symptoms (7–11 weeks post infection), returning to baseline level once the granulomas were modulated (>12 weeks post infection). The plasma levels in acute schistosomiasis were even higher than in hepatosplenic patients. The murine model mirrored the human disease. Macrophages were the major source of OPN in human and murine acute schistosomiasis, while the ductular reaction maintains OPN production in hepatosplenic disease. Soluble egg antigens from S. mansoni induced OPN expression in primary human kupffer cells. Conclusions/Significance S. mansoni egg antigens induce the production of OPN by macrophages in the necrotic-exudative granulomas characteristic of acute schistosomiasis mansoni. Circulating OPN levels are upregulated in human and

  1. Effect of blockage of costimulatory signal on murine abortion-prone model

    Institute of Scientific and Technical Information of China (English)

    ZHAO Fu-xi; ZHANG Yuan-yuan; LIU Run-hua; LI Shuan-ming


    Background Inhibition of the key costimulatory signals results in T cell anergy, indicating the alloantigen-specific immunologic unresponsiveness. In this study, the effect of blockage of costimulatory signal CD86 on murine abortion-prone model was studied.Methods Thirty CBA/J female mice cohabitated with DBA/2 male or BALB/c male mice were investigated. CBA/J ×DBA/2 matings were used as the abortion-prone model, and CBA/J × BALB/c matings were used as the normal pregnant model. The abortion-prone models were divided into experimental and control groups, and the normal pregnant models were set as a normal group (10 mice in each group). The mice in the experimental group were treated with anti-mouse CD86 monoclonal antibody (mAb) (100 μg) on day 4.5 of gestation, while the controls received irrelevant-isotype matched rat IgG2b. As for the normal group, nothing was given to the mice. The mice were killed on day 13.5 of gestation, embryo resorption rate and the expression of transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor 1 (PAI-1), and matrix metalloproteinase 9 (MMP-9) were detected. Then the data were analyzed by Chi-square test and Fisher's exact test.Results The embryo resorption rate in the experimental (8.2%) and normal groups (7.7%) was significantly lower than that of the control (23.5%, P<0.05). No significant difference was detected between the experimental and normal groups (P>0.05). The positive expression rates of TGF-β1 and PAI-1 proteins in the experimental and normal groups were significantly higher than those in the control group (P<0.05). The positive expression rate of MMP-9 protein in the experimental and normal groups was significantly lower than that in the control group (P<0.05). No significant difference in the positive expression rates of the three proteins was detected between the experimental and normal groups (P>0.05).Conclusions Blockage of costimulatory signal CD86 at early pregnancy can treat

  2. The Monoclonal Antitoxin Antibodies (Actoxumab–Bezlotoxumab) Treatment Facilitates Normalization of the Gut Microbiota of Mice with Clostridium difficile Infection (United States)

    Džunková, Mária; D'Auria, Giuseppe; Xu, Hua; Huang, Jun; Duan, Yinghua; Moya, Andrés; Kelly, Ciarán P.; Chen, Xinhua


    Antibiotics have significant and long-lasting impacts on the intestinal microbiota and consequently reduce colonization resistance against Clostridium difficile infection (CDI). Standard therapy using antibiotics is associated with a high rate of disease recurrence, highlighting the need for novel treatment strategies that target toxins, the major virulence factors, rather than the organism itself. Human monoclonal antibodies MK-3415A (actoxumab–bezlotoxumab) to C. difficile toxin A and toxin B, as an emerging non-antibiotic approach, significantly reduced the recurrence of CDI in animal models and human clinical trials. Although the main mechanism of protection is through direct neutralization of the toxins, the impact of MK-3415A on gut microbiota and its restoration has not been examined. Using a CDI murine model, we compared the bacterial diversity of the gut microbiome of mice under different treatments including MK-3415A, vancomycin, or vancomycin combined with MK-3415A, sampled longitudinally. Here, we showed that C. difficile infection resulted in the prevalence of Enterobacter species. Sixty percent of mice in the vehicle group died after 2 days and their microbiome was almost exclusively formed by Enterobacter. MK-3415A treatment resulted in lower Enterobacter levels and restoration of Blautia, Akkermansia, and Lactobacillus which were the core components of the original microbiota. Vancomycin treatment led to significantly lower survival rate than the combo treatment of MK-3415A and vancomycin. Vancomycin treatment decreased bacterial diversity with predominant Enterobacter and Akkermansia, while Staphylococcus expanded after vancomycin treatment was terminated. In contrast, mice treated by vancomycin combined with MK-3415A also experienced decreased bacterial diversity during vancomycin treatment. However, these animals were able to recover their initial Blautia and Lactobacillus proportions, even though episodes of Staphylococcus overgrowth were

  3. The monoclonal antitoxin antibodies (actoxumab-bezlotoxumab treatment facilitates normalization of the gut microbiota of mice with Clostridium difficile infection

    Directory of Open Access Journals (Sweden)

    Mária Džunková


    Full Text Available Antibiotics have significant and long-lasting impacts on the intestinal microbiota and consequently reduce colonization resistance against Clostridium difficile infection (CDI. Standard therapy using antibiotics is associated with a high rate of disease recurrence, highlighting the need for novel treatment strategies that target toxins, the major virulence factors, rather than the organism itself. Human monoclonal antibodies MK-3415A (actoxumab-bezlotoxumab to C. difficile toxin A and toxin B, as an emerging non-antibiotic approach, significantly reduced the recurrence of CDI in animal models and human clinical trials. Although the main mechanism of protection is through direct neutralization of the toxins, the impact of MK-3415A on gut microbiota and its restoration has not been examined. Using a CDI murine model, we compared the bacterial diversity of the gut microbiome of mice under different treatments including MK-3415A, vancomycin or vancomycin combined with MK-3415A, sampled longitudinally. Here we showed that C. difficile infection resulted in the prevalence of Enterobacter species. 60% of mice in the vehicle group died after two days and their microbiome was almost exclusively formed by Enterobacter. MK-3415A treatment resulted in lower Enterobacter levels and restoration of Blautia, Akkermansia and Lactobacillus which were the core components of the original microbiota. Vancomycin treatment led to significantly lower survival rate than the combo treatment of MK-3415A and vancomycin. Vancomycin treatment decreased bacterial diversity with predominant Enterobacter and Akkermansia, while Staphylococcus expanded after vancomycin treatment was terminated. In contrast, mice treated by vancomycin combined with MK-3415A also experienced decreased bacterial diversity during vancomycin treatment. However, these animals were able to recover their initial Blautia and Lactobacillus proportions, even though episodes of Staphylococcus overgrowth were

  4. Preparation of Monoclonal Antibody and Development of Enzyme-linked Immunosorbent Assay Specific for Escherichia coli O157 in Foods

    Institute of Scientific and Technical Information of China (English)


    Objective To prepare monoclonal antibodies (Mab) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect E.coli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157:H7 were fused with murine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the Mab 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized milk were tested to confirm efficiency of the method. Results Mab 3A5 specific for E.coli O157 and O113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1×106. No cross-reactivity of the Mab was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1:1×105 with E.coli O157. The detection limit of this sandwich ELISA was 103-104 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion Mab 3A5 specific for E.coli O157 and O113:H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the Mab 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.

  5. Generation and characterization of a panel of monoclonal antibodies specific for human fibroblast growth factor receptor 4 (FGFR4). (United States)

    Chen, Chaoyuan; Patel, Sima; Corisdeo, Susanne; Liu, Xiangdong; Micolochick, Holly; Xue, Jiyang; Yang, Qifeng; Lei, Ying; Wang, Baiyang; Soltis, Daniel


    Fibroblast growth factor receptor 4 (FGFR4) is a member of the FGFR family of receptor tyrosine kinases, and plays important roles in a variety of biological functions such as cell proliferation, differentiation, migration, angiogenesis, tissue repair, and tumorigenesis. The human FGFRs share a high degree of sequence homology between themselves, as well as with their murine homologs. Consequently, it has been suggested that it may be difficult to prepare monoclonal antibodies (MAbs) that are specific for the individual receptor types. In this communication, we report on the development and characterization of a panel of anti-human FGFR4 MAbs that were generated in mice using a rapid immunization protocol. Using a modified rapid immunization at multiple sites (RIMMS) protocol with the soluble extracellular domain of human FGFR4 (FGFR4-ECD), the immunized mice developed high levels of polyclonal IgG to the immunogen within 13 days of the first immunization. The lymph node cells isolated from the immunized animals were then fused with mouse myeloma cells for hybridoma generation. Use of an efficient hybridoma cloning protocol in combination with an ELISA screening procedure allowed for early identification of stable hybridomas secreting antihuman FGFR4 IgG. Several identified MAbs specifically reacted with the FGFR4 protein without binding to the other human isoforms (FGFR1, FGFR2, and FGFR3). As evaluated by BIAcore analysis, most anti-FGFR4 MAbs displayed high affinities (8.6 x 10(8) approximately 3.9 x 10(10) M) to FGFR4. Furthermore, these MAbs were able to bind to FGFR4 expressed on human breast tumor cell lines MDA-MB-361 and MDA-MB-453. Taken together, the results demonstrate that the RIMMS strategy is an effective approach for generating class-switched, high-affinity MAbs in mice to evolutionarily conserved proteins such as human FGFR4. These MAbs may be useful tools for further investigation of the biological functions and pathological roles of human FGFR4.

  6. Administration guidelines for radioimmunotherapy of non-Hodgkin's lymphoma with (90)Y-labeled anti-CD20 monoclonal antibody. (United States)

    Wagner, Henry N; Wiseman, Gregory A; Marcus, Carol S; Nabi, Hani A; Nagle, Conrad E; Fink-Bennett, Darlene M; Lamonica, Dominick M; Conti, Peter S


    90Y-ibritumomab tiuxetan is a novel radioimmunotherapeutic agent recently approved for the treatment of relapsed or refractory low-grade, follicular, or CD20+ transformed non-Hodgkin's lymphoma (NHL). (90)Y-ibritumomab tiuxetan consists of a murine monoclonal antibody covalently attached to a metal chelator, which stably chelates (111)In for imaging and (90)Y for therapy. Both health care workers and patients receiving this therapy need to become familiar with how it differs from conventional chemotherapy and what, if any, safety precautions are necessary. Because (90)Y is a pure beta-emitter, the requisite safety precautions are not overly burdensome for health care workers or for patients and their families. (90)Y-ibritumomab tiuxetan is dosed on the basis of the patient's body weight and baseline platelet count; dosimetry is not required for determining the therapeutic dose in patients meeting eligibility criteria similar to those used in clinical trials, such as shielding during dose preparation and administration; primary lead shielding should be avoided because of the potential exposure risk from bremsstrahlung. Because there are no penetrating gamma-emissions associated with the therapy, (90)Y-ibritumomab tiuxetan is routinely administered on an outpatient basis. Furthermore, the risk of radiation exposure to patients' family members has been shown to be in the range of background radiation, even without restrictions on contact. There is therefore no need to determine activity limits or dose rate limits before patients who have been treated with (90)Y radioimmunotherapy are released, as is necessary with patients who have been treated with radiopharmaceuticals that contain (131)I. Standard universal precautions for handling body fluids are recommended for health care workers and patients and their family members after (90)Y-ibritumomab tiuxetan administration. In summary, (90)Y-ibritumomab tiuxetan introduces (90)Y into clinical practice and expands the role

  7. Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Laura Tarnawski

    Full Text Available In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

  8. Diagnostic imaging advances in murine models of colitis. (United States)

    Brückner, Markus; Lenz, Philipp; Mücke, Marcus M; Gohar, Faekah; Willeke, Peter; Domagk, Dirk; Bettenworth, Dominik


    Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are chronic-remittent inflammatory disorders of the gastrointestinal tract still evoking challenging clinical diagnostic and therapeutic situations. Murine models of experimental colitis are a vital component of research into human IBD concerning questions of its complex pathogenesis or the evaluation of potential new drugs. To monitor the course of colitis, to the present day, classical parameters like histological tissue alterations or analysis of mucosal cytokine/chemokine expression often require euthanasia of animals. Recent advances mean revolutionary non-invasive imaging techniques for in vivo murine colitis diagnostics are increasingly available. These novel and emerging imaging techniques not only allow direct visualization of intestinal inflammation, but also enable molecular imaging and targeting of specific alterations of the inflamed murine mucosa. For the first time, in vivo imaging techniques allow for longitudinal examinations and evaluation of intra-individual therapeutic response. This review discusses the latest developments in the different fields of ultrasound, molecularly targeted contrast agent ultrasound, fluorescence endoscopy, confocal laser endomicroscopy as well as tomographic imaging with magnetic resonance imaging, computed tomography and fluorescence-mediated tomography, discussing their individual limitations and potential future diagnostic applications in the management of human patients with IBD.

  9. Diagnosis and follow-up of monoclonal gammopathies of undetermined significance; information for referring physicians. (United States)

    Caers, Jo; Vekemans, Marie-Christiane; Bries, Greet; Beel, Karolien; Delrieu, Vanessa; Deweweire, Anne; Demuynck, Hilde; De Prijck, Bernard; De Samblanx, Hadewijch; Kentos, Alain; Meuleman, Nathalie; Mineur, Philippe; Offner, Fritz; Vande Broek, Isabelle; Van Droogenbroeck, Jan; Vande Velde, Ann; Wu, Ka Lung; Delforge, Michel; Schots, Rik; Doyen, Chantal


    The prevalence of monoclonal gammopathy of undetermined significance (MGUS) is generally estimated at 3.4% in the general population over 50 years, and its incidence increases with age. MGUS represents a preneoplastic entity that can transform into multiple myeloma or other lymphoproliferative disorders. The risk of malignant transformation is estimated at 1% per year and persists over time. Predictors of malignant transformation have been identified such as the heavy chain isotype, The level of monoclonal proteins, increasing levels of the monoclonal component during the first years off follow-up, the percentage of bone marrow plasmocytosis, the dosage of serum free light chains, the presence of immunophenotypically abnormal plasma cells, aneuploidy, and the presence of circulating plasma cells. Prognostic scores that combine certain of these factors have been proposed and allow the identification of high-risk patients. Their use could assist in tailoring the care for each patient, based on his/her risk profile.

  10. Monoclonal gammopathy of undetermined significance and risk of infections: a population-based study. (United States)

    Kristinsson, Sigurdur Y; Tang, Min; Pfeiffer, Ruth M; Björkholm, Magnus; Goldin, Lynn R; Blimark, Cecilie; Mellqvist, Ulf-Henrik; Wahlin, Anders; Turesson, Ingemar; Landgren, Ola


    No comprehensive evaluation has been made to assess the risk of viral and bacterial infections among patients with monoclonal gammopathy of undetermined significance. Using population-based data from Sweden, we estimated risk of infections among 5,326 monoclonal gammopathy of undetermined significance patients compared to 20,161 matched controls. Patients with monoclonal gammopathy of undetermined significance had a 2-fold increased risk (Pundetermined significance had an increased risk (Pundetermined significance with M-protein concentrations over 2.5 g/dL at diagnosis had highest risks of infections. However, the risk was also increased (Pundetermined significance who developed infections had no excess risk of developing multiple myeloma, Waldenström macroglobulinemia or related malignancy. Our findings provide novel insights into the mechanisms behind infections in patients with plasma cell dyscrasias, and may have clinical implications.

  11. Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization. (United States)

    Liu, Na; Zhao, Zhiyong; Tan, Yanglan; Lu, Lei; Wang, Lin; Liao, Yucai; Beloglazova, Natalia; De Saeger, Sarah; Zheng, Xiaodong; Wu, Aibo


    Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.

  12. Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis. (United States)

    Monzó, César; Urbaneja, Alberto; Ximénez-Embún, Miguel; García-Fernández, Julia; García, José Luis; Castañera, Pedro


    Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

  13. 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


    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.

  14. Non-secretory immunoglobulin E myeloma associated with immunoglobulin G monoclonal gammopathy of undetermined significance

    Directory of Open Access Journals (Sweden)

    Masako Yasuyama


    Full Text Available A 68-year old woman came to our hospital with a severe case of anemia. Serum immunoelectropheresis identified a monoclonal immunoglobulin (Ig G and κ protein. The serum IgE level was within the nomal range and the amounts of remaining immuno - globlins were low. On bone marrow aspirate, plasma cells made up 55.5% of nucleated cells and the plasma cells showed positive readings for IgE κ and IgG by immunohistochemistry. Serum immunofixation did not reveal the IgE monoclonal band. She was diagnosed as having non-secretory IgE myeloma with IgG monoclonal gammopathy of undetermined significance. The nature of this rare myeloma will be discussed.

  15. Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

    Energy Technology Data Exchange (ETDEWEB)

    Srivastava, S.C.; Buraggi, G.L.


    This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

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


    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.

  17. Intradermal Infection Model for Pathogenesis and Vaccine Studies of Murine Visceral Leishmaniasis



    The levels of protection found in vaccine studies of murine visceral leishmaniasis are significantly lower than for cutaneous leishmaniasis; whether this is due to the high-challenge murine model employed and/or is a consequence of differences required in tissue-specific local immune responses is not understood. Consequently, an intradermal murine model of visceral leishmaniasis has been explored. Intradermal inoculation established a chronic infection in susceptible mice which was associated...

  18. Limited cross-reactivity of mouse monoclonal antibodies against Dengue virus capsid protein among four serotypes

    Directory of Open Access Journals (Sweden)

    Noda M


    Full Text Available Megumi Noda,1 Promsin Masrinoul,1 Chaweewan Punkum,1 Chonlatip Pipattanaboon,2,3 Pongrama Ramasoota,2,4 Chayanee Setthapramote,2,3 Tadahiro Sasaki,6 Mikiko Sasayama,1 Akifumi Yamashita,1,5 Takeshi Kurosu,6 Kazuyoshi Ikuta,6 Tamaki Okabayashi11Mahidol-Osaka Center for Infectious Diseases, 2Center of Excellence for Antibody Research, 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand; 5Graduate School of Life Science, Tohoku University, Sendai, Miyagi, 6Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanBackground: Dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. Four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4 are classified in the Flavivirus genus of the family Flaviviridae. We prepared monoclonal antibodies against DENV capsid protein from mice immunized with DENV-2 and determined the cross-reactivity with each serotype of DENV and Japanese encephalitis virus.Methods and results: To clarify the relationship between the cross-reactivity of monoclonal antibodies and the diversity of these viruses, we examined the situations of flaviviruses by analyses of phylogenetic trees. Among a total of 60 prepared monoclonal antibodies specific for DENV, five monoclonal antibodies stained the nuclei of infected cells and were found to be specific to the capsid protein. Three were specific to DENV-2, while the other two were cross-reactive with DENV-2 and DENV-4. No monoclonal antibodies were cross-reactive with all four serotypes. Phylogenetic analysis of DENV amino acid sequences of the capsid protein revealed that DENV-2 and DENV-4 were clustered in the same branch, while DENV-1 and DENV-3 were clustered in the other branch. However, these classifications of the capsid protein were different from those of the


    Institute of Scientific and Technical Information of China (English)

    LI Zhen-fu; ZHANG Hong; NIU Yong-ge


    Monoclonal antibody (McAb) 3H11 against gastric cancer was grown in the mouse ascites system. To acquire a clinical grade product for cancer radioimmuno-imaging was purified by two step high performance liquid chromatography (HPLC) protocol using protein A and high-performance hydroxylapatite (HPHT). An analysis of data reported shows the two step HPLC method to be the best purification procedure. This protocol satisfies purity and immunoreactivity requirement, and provides an sample sterility,free-pyrogens, free-mycoplasma and non-specific IgG contamination. This procedure described was capable of generating large amounts of clinical grade monoclonal antibody.

  20. Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies. (United States)

    Lewis, A P; Parry, N; Peakman, T C; Crowe, J S


    Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

  1. Invasion of erythrocytes in vitro by Plasmodium falciparum can be inhibited by monoclonal antibody directed against an S antigen. (United States)

    Saul, A; Cooper, J; Ingram, L; Anders, R F; Brown, G V


    A monoclonal antibody has been produced which binds to the heat stable S antigen present in the FCQ-27/PNG isolate of Plasmodium falciparum. This monoclonal antibody also inhibits the invasion in vitro of erythrocytes by malarial merozoites thus demonstrating that the S antigens of Plasmodium falciparum may be a target of protective immune responses.

  2. Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response

    NARCIS (Netherlands)

    Tete, Sarah M.; Kipling, David; Westra, Johanna; de Haan, Aalzen; Bijl, Marc; Dunn-Walters, Deborah K.; Sahota, Surinder S.; Bos, Nicolaas A.


    Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examin

  3. Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains. (United States)

    Schlapschy, Martin; Fogarasi, Marton; Gruber, Helga; Gresch, Oliver; Schäfer, Claudia; Aguib, Yasmine; Skerra, Arne


    An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

  4. Detection of auto-anti-idiotypic antibodies to Lol p I (rye I) IgE antibodies in human sera by the use of murine idiotypes: levels in atopic and non-atopic subjects and effects of immunotherapy. (United States)

    Hébert, J; Bernier, D; Mourad, W


    Anti-idiotypic antibodies (anti-Id Abs) are involved in the regulation of a number of immune responses including the IgE antibody production. In atopic patients, the increased synthesis of IgE antibodies could be related to a defective production of regulatory anti-Id Abs. In the present study, we first developed a sensitive assay for measuring the levels of anti-Id Abs directed against antibodies specific for Lol p I, the major allergenic determinant of Lolium perenne (rye grass). In this assay, we used previously described murine monoclonal anti-Lol p I antibodies that were shown to share epitopic specificities with human anti-Lol p I IgE and IgG antibodies, thus short-cutting the need for purification of F(ab')2 fragments of human IgG Abs and insuring optimal specificity and sensitivity. Levels of anti-Id Abs against two anti-Lol p I monoclonal antibodies (290A-167, 348A-6) were higher in normal volunteers than in untreated atopic patients. Specific immunotherapy increased the levels of anti-Id Abs to those of normal volunteers. These observations suggest a role for the Id-anti-Id network in the regulation of IgE antibody production.

  5. Inhibition of the alternative complement activation pathway in traumatic brain injury by a monoclonal anti-factor B antibody: a randomized placebo-controlled study in mice

    Directory of Open Access Journals (Sweden)

    Holers V Michael


    Full Text Available Abstract Background The posttraumatic response to traumatic brain injury (TBI is characterized, in part, by activation of the innate immune response, including the complement system. We have recently shown that mice devoid of a functional alternative pathway of complement activation (factor B-/- mice are protected from complement-mediated neuroinflammation and neuropathology after TBI. In the present study, we extrapolated this knowledge from studies in genetically engineered mice to a pharmacological approach using a monoclonal anti-factor B antibody. This neutralizing antibody represents a specific and potent inhibitor of the alternative complement pathway in mice. Methods A focal trauma was applied to the left hemisphere of C57BL/6 mice (n = 89 using a standardized electric weight-drop model. Animals were randomly assigned to two treatment groups: (1 Systemic injection of 1 mg monoclonal anti-factor B antibody (mAb 1379 in 400 μl phosphate-buffered saline (PBS at 1 hour and 24 hours after trauma; (2 Systemic injection of vehicle only (400 μl PBS, as placebo control, at identical time-points after trauma. Sham-operated and untreated mice served as additional negative controls. Evaluation of neurological scores and analysis of brain tissue specimens and serum samples was performed at defined time-points for up to 1 week. Complement activation in serum was assessed by zymosan assay and by murine C5a ELISA. Brain samples were analyzed by immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL histochemistry, and real-time RT-PCR. Results The mAb 1379 leads to a significant inhibition of alternative pathway complement activity and to significantly attenuated C5a levels in serum, as compared to head-injured placebo-treated control mice. TBI induced histomorphological signs of neuroinflammation and neuronal apoptosis in the injured brain hemisphere of placebo-treated control mice for up to 7 days. In contrast, the

  6. Experimental studies for immunoscintigraphy of adenocarcinoma of the prostate with [sup 125]I-labeled monoclonal antibody to [gamma]-seminoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Fujino, Awato; Suyama, Kazuho; Shidara, Toshiya; Ao, Teruaki; Ishibashi, Akira; Koshiba, Ken (Kitasato Univ., Sagamihara, Kanagawa (Japan). School of Medicine)


    We established LNCaP tumor in male nude mice after inoculation of LNCaP cells. The tumor produces the organ-specific glycoproteins in the human prostate, [gamma]-seminoprotein ([gamma]-Sm). The LNCaP tumor is composed of fluid and solid portions. The fluid portion contains much more [gamma]-Sm than the solid part. Monoclonal antibody against [gamma]-Sm (murine IgG[sub 1], K) was radiolabeled with iodine-125 by lactoperoxidase method followed by passage over column chromatography. Finally the conjugates with specific activity of 49.8-56.6 KBq/[mu]g were obtained. Biodistribution of [sup 125]I- labeled monoclonal antibody to [gamma]-Sm ([sup 125]I-[gamma]MAb) was evaluated by whole-body autoradiography (ARG) and by determination of label uptake (DLU) by the LNCaP tumor using the well type gamma counter. The ARG were obtained 1-9 days after intravenous administration of 32-38 [mu]g of [sup 125]I-[gamma]MAb to 12 nude mice bearing LNCaP tumor. The DLU were evaluated 3, 4 and 7 days after intravenous administration of 18-25 [mu]g of [sup 125]I- [gamma]MAb to 9 nude mice bearing LNCaP tumor. The DLU by organs (fluid portion, solid portion, liver, kidney, spleen, lung, etc.) were expressed as F-values (%) which were calculated by the following formula: F=radioactivity per gram of tissue (dpm/g) / injected dose per gram of animal (dpm/g)x100 and tissue-to-blood ratios (T/B). The specific uptake by the LNCaP tumors, especially in the fluid portion, was observed on ARG, with optimal images of the tumor evaluated 4-7 days after administration. The F-values and T/B of the LNCaP tumors were always higher than those of other organs. Especially T/B of fluid portion were of 6.7 and 6.3, evaluated 4 and 7 days after administration of [sup 125]I-[gamma]MAb respectively. These results suggest that the radiolabeled [gamma] MAb is applicable for immunoscintigraphy of adenocarcinoma of the prostate. (author).

  7. Hospital population screening reveals overrepresentation of CD5(-) monoclonal B-cell lymphocytosis and monoclonal gammopathy of undetermined significance of IgM type. (United States)

    Voigtlaender, Minna; Vogler, Birthe; Trepel, Martin; Panse, Jens; Jung, Roman; Bokemeyer, Carsten; Bacher, Ulrike; Binder, Mascha


    Monoclonal B-cell lymphocytosis (MBL) and monoclonal gammopathy of undetermined significance (MGUS) result from clonal expansions of mature B or plasma cells. Here, we set out to determine the immunophenotypic/monoclonal immunoglobulin (M protein) features and co-prevalence of MBL and MGUS in a hospital-based cohort of 1909 non-hematooncological patients. Of the evaluable cases, 3.8 % showed evidence for MBL by immunophenotyping, while 9.8 % were screened positive for M protein by immunofixation. With six concomitant cases (0.4 %), MBL and MGUS were not statistically associated. At least in two of these coincident cases, MBL and MGUS were of different clonal origin since both clones had divergent light chain restriction. CD5(-) MBL (57.1 %) and IgM+ MGUS (24.7 %) were strikingly overrepresented compared to population-based screenings and did not progress to overt lymphoma or myeloma during the observation period (mean follow-up of 117 weeks or 110 weeks, respectively). Prevalence and phenotypes suggest that a substantial proportion of incidental MBL and MGUS in hospitalized patients may be attributed to transiently expanded B-cell clones in the context of disease-related immune stimulation rather than reflecting veritable precursors of clonal B-cell malignancies.

  8. Characterization of the mechanism of protection mediated by CS-D7, a monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB

    Directory of Open Access Journals (Sweden)

    Gregory ePancari


    Full Text Available We previously reported the development of a human monoclonal antibody (CS-D7, IgG1 with specificity and affinity for the iron regulated surface determinant B (IsdB of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17, not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7●N297A. This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII-/- mice and FcγRIII-/- mice, indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV, was lost in mice depleted of superoxide dismutase (SOD in P47phox deletion mice, and was absent in SCID mice. Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of INF-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme

  9. Two courses of rituximab (anti-CD20 monoclonal antibody) for recalcitrant pemphigus vulgaris

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.


    Background Pemphigus vulgaris (PV) is a severe autoimmune blistering disease involving the skin and mucous membranes. The response to therapy varies greatly amongst patients and treatment may be challenging. Rituximab is a chimeric monoclonal antibody that selectively targets cell surface antigen...

  10. Boronated monoclonal antibody 225. 28S for potential use in neutron capture therapy of malignant melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Tamat, S.R.; Moore, D.E.; Patwardhan, A.; Hersey, P. (Univ. of Sydney (Australia))


    The concept of conjugating boron cluster compounds to monoclonal antibodies has been examined by several groups of research workers in boron neutron capture therapy (BNCT). The procedures reported to date for boronation of monoclonal antibodies resulted in either an inadequate level of boron incorporation, the precipitation of the conjugates, or a loss of immunological activity. The present report describes the conjugation of dicesium-mercapto-undecahydrododecaborate (Cs2B12H11SH) to 225.28S monoclonal antibody directed against high molecular weight melanoma-associated antigens (HMW-MAA), using poly-L-ornithine as a bridge to increase the carrying capacity of the antibody and to minimize change in the conformational structure of antibody. The method produces a boron content of 1,300 to 1,700 B atoms per molecule 225.28S while retaining the immunoreactivity. Characterization in terms of the homogeneity of the conjugation of the boron-monoclonal antibody conjugates has been studied by gel electrophoresis and ion-exchange HPLC.

  11. B lymphocyte depletion with the monoclonal antibody rituximab in Graves' disease: a controlled pilot study

    DEFF Research Database (Denmark)

    El Fassi, Daniel; Nielsen, Claus H; Bonnema, Steen J


    Graves' disease (GD) is a common TSH receptor autoantibody (TRAb)-mediated disorder. Because B lymphocytes are important self-antigen presenting cells and precursors for antibody-secreting plasma cells, temporary B-lymphocyte depletion with the monoclonal antibody rituximab (RTX) might...

  12. Competitive adsorption of monoclonal antibodies and nonionic surfactants at solid hydrophobic surfaces

    DEFF Research Database (Denmark)

    Kapp, Sebastian J; Larsson, Iben; van de Weert, Marco


    Two monoclonal antibodies from the IgG subclasses one and two were compared in their adsorption behavior with hydrophobic surfaces upon dilution to 10 mg/mL with 0.9% NaCl. These conditions simulate handling of the compounds at hospital pharmacies and surfaces encountered after preparation, such ...

  13. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T


    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell kil

  14. Acquired von Willebrand syndrome in a patient with monoclonal gammopathy of undetermined significance. (United States)

    Puronen, Camille E; Josephson, Neil C; Broudy, Virginia C


    Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder that typically presents as mucocutaneous bleeding in individuals with no personal or family history of bleeding disorder. Here we present a case in which a patient presented with profound epistaxis and was found to have AVWS in the setting of monoclonal gammopathy of undetermined significance (MGUS).

  15. Long-term follow-up of a population based cohort with monoclonal proteinaemia

    NARCIS (Netherlands)

    Schaar, Cees G.; le Cessie, Saskia; Snijder, Simone; Franck, Paul F. H.; Wijermans, Pierre W.; Ong, Cisca; Kluin-Nelemans, Hanneke


    Prospective studies on the risk of malignant transformation in patients with monoclonal gammopathy of undetermined significance (MGUS) and factors predictive of survival are lacking. The Dutch Comprehensive Cancer Centre West, comprising 1.6 million inhabitants, initiated a prospective hospital-base

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

    Institute of Scientific and Technical Information of China (English)


    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.

  17. B lymphocyte depletion with the monoclonal antibody rituximab in Graves' disease: a controlled pilot study

    DEFF Research Database (Denmark)

    El Fassi, Daniel; Nielsen, Claus H; Bonnema, Steen Joop


    CONTEXT: Graves' disease (GD) is a common TSH receptor autoantibody (TRAb)-mediated disorder. Because B lymphocytes are important self-antigen presenting cells and precursors for antibody-secreting plasma cells, temporary B-lymphocyte depletion with the monoclonal antibody rituximab (RTX) might...

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

    DEFF Research Database (Denmark)

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


    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 aggregat...

  19. Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma

    DEFF Research Database (Denmark)

    van de Donk, Niels W C J; Moreau, Philippe; Plesner, Torben;


    have demonstrated significantly improved progression-free survival when elotuzumab is added to lenalidomide-dexamethasone or bortezomib-dexamethasone. Importantly, there has been no significant additive toxicity when these monoclonal antibodies are combined with other anti-MM agents, other than...

  20. HIV monoclonal antibodies: a new opportunity to further reduce mother-to-child HIV transmission.

    Directory of Open Access Journals (Sweden)

    Yegor Voronin


    Full Text Available Yegor Voronin and colleagues explore how monoclonal antibodies against HIV could provide a new opportunity to further reduce mother-to-child transmission of HIV and propose that new interventions should consider issues related to implementation, feasibility, and access. Please see later in the article for the Editors' Summary.

  1. Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus

    NARCIS (Netherlands)

    Pal, Pankaj; Dowd, Kimberly A.; Brien, James D.; Edeling, Melissa A.; Gorlatov, Sergey; Johnson, Syd; Lee, Iris; Akahata, Wataru; Nabel, Gary J.; Richter, Mareike K. S.; Smit, Jolanda M.; Fremont, Daved H.; Pierson, Theodore C.; Heise, Mark T.; Diamond, Michael S.


    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit

  2. Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7

    DEFF Research Database (Denmark)

    Clausen, Mads Hartvig; Willats, William George Tycho; Knox, J. Paul


    A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens prov...

  3. Comprehensive analysis of varicella-zoster virus proteins using a new monoclonal antibody collection

    NARCIS (Netherlands)

    T.L. Roviš (Tihana Lenac); S.M. Bailer (Susanne); V.R. Pothineni (Venkata R); W.J.D. Ouwendijk (Werner ); H. Šimić (Hrvoje); M. Babić (Marina); K. Miklić (Karmela); S. Malić (Suzana); M.C. Verweij; M. Baiker (Martin); O. Gonzalez (Orland); A. Brunn (Albrecht von); R. Zimmer; K. Früh (Klaus); G.M.G.M. Verjans (George); S. Jonjic (Stipan); J. Haasb (Jürgeni)


    textabstractVaricella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell typetropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251monoclonal antibodies (MAbs) again

  4. Inhibition of middle east respiratory syndrome coronavirus infection by anti-CD26 monoclonal antibody

    NARCIS (Netherlands)

    K. Ohnuma (Kei); B.L. Haagmans (Bart); R. Hatano (Ryo); V.S. Raj (Stalin); H. Mou (Huihui); S. Iwata (Satoshi); R.L. Dang (Rong); B.J. Bosch (Berend Jan); C. Morimoto (Chikao)


    textabstractWe identified the domains of CD26 involved in the binding of Middle East respiratory syndrome coronavirus (MERS-CoV) using distinct clones of anti-CD26 monoclonal antibodies (MAbs). One clone, named 2F9, almost completely inhibited viral entry. The humanized anti-CD26 MAb YS110 also sign

  5. Harnessing the immune system's arsenal: producing human monoclonal antibodies for therapeutics and investigating immune responses (United States)

    Sullivan, Meghan; Kaur, Kaval; Pauli, Noel


    Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease. PMID:21876728

  6. A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17. (United States)

    McMichael, A J; Parham, P; Rust, N; Brodsky, F


    A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kohler and Milstein [1]. This antibody recognizes a new specificity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.

  7. The generation of monoclonal antibodies and their use in rapid diagnostic tests (United States)

    Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

  8. Anti-interleukin-17 monoclonal antibody ixekizumab in chronic plaque psoriasis

    DEFF Research Database (Denmark)

    Leonardi, Craig; Matheson, Robert; Zachariae, Claus;


    Type 17 helper T cells have been suggested to play a pathological role in psoriasis. They secrete several proinflammatory cytokines, including interleukin-17A (also known as interleukin-17). We evaluated the safety and efficacy of ixekizumab (LY2439821), a humanized anti-interleukin-17 monoclonal...... antibody, for psoriasis treatment....

  9. Survey of citrus tristeza virus populations in Central California that react with MCA13 monoclonal antibody (United States)

    The Citrus Pest Detection Program (CPDP) of the Central California Tristeza Eradication Agency monitors Citrus tristeza virus (CTV) in Central California. MCA13 is a severe strain discriminating monoclonal antibody used to screen for potentially virulent CTV isolates. MCA13-reactive CTV isolates are...

  10. Culture and Identification of Monoclonal Neural Stem Cells Derived from Cerebral Cortex

    Institute of Scientific and Technical Information of China (English)

    TAO Kaixiong; CHEN Jingbo; WANG Guobin; SHU Xiaogang


    To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

  11. Intravenous cidofovir for resistant cutaneous warts in a patient with psoriasis treated with monoclonal antibodies.

    LENUS (Irish Health Repository)

    McAleer, M A


    Human papilloma virus is a common and often distressing cutaneous disease. It can be therapeutically challenging, especially in immunocompromised patients. We report a case of recalcitrant cutaneous warts that resolved with intravenous cidofovir treatment. The patient was immunocompromised secondary to monoclonal antibody therapy for psoriasis.

  12. Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation.

    NARCIS (Netherlands)

    Mulder, A.; Kardol, M.J.; Arn, J.S.; Eijsink, C.; Franke, M.E.; Schreuder, G.M.; Haasnoot, G.W.; Doxiadis, I.I.; Sachs, D.H.; Smith, D.M.; Claas, F.H.


    Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (

  13. A phase II trial of chimeric monoclonal antibody G250 for advanced renal cell carcinoma patients.

    NARCIS (Netherlands)

    Bleumer, I.; Knuth, A.; Oosterwijk, E.; Hofmann, R.; Varga, Z.; Lamers, C.B.H.W.; Kruit, W.; Melchior, S.; Mala, C.; Ullrich, S.; Mulder, P.; Mulders, P.F.A.; Beck, J.L.M.


    Chimeric monoclonal antibody G250 (WX-G250) binds to a cell surface antigen found on >90% of renal cell carcinoma (RCC). A multicentre phase II study was performed to evaluate the safety and efficacy of WX-G250 in metastatic RCC (mRCC) patients. In all, 36 patients with mRCC were included. WX-G250 w

  14. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    NARCIS (Netherlands)

    J. Groen (Jan); N. Juntti; J.S. Teppema; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert); G.F. Rimmelzwaan (Guus)


    textabstractImmuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infe

  15. Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody. (United States)

    Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan


    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues.

  16. Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding

    Directory of Open Access Journals (Sweden)

    Jeremy H. Lakey


    Full Text Available Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated.


    NARCIS (Netherlands)



    In this study, the effect of antibody adsorption on physicochemical properties of Streptococcus sobrinus was studied. Bacteria were preincubated with polyclonal antibodies or with OMVU10, a monoclonal antibody (MAb) reactive with S. sobrinus. The zeta potentials and the hydrophobicity as determined

  18. A monoclonal antibody to feruloylated (1→4)-β-D-galactan

    DEFF Research Database (Denmark)

    Clausen, Mads Hartvig; Ralet, Marie-Christine; Willats, William G. T.


    We report the isolation and characterization of a monoclonal antibody, designated LM9, against feruloylated-(1-->4)-beta-D-galactan. This epitope is a structural feature of cell wall pectic polysaccharides of plants belonging to the family Amaranthaceae (including the Chenopodiaceae). Immuno...

  19. Vitamin D Deficiency in Human and Murine Sepsis* (United States)

    Parekh, Dhruv; Patel, Jaimin M.; Scott, Aaron; Lax, Sian; Dancer, Rachel C. A.; D’Souza, Vijay; Greenwood, Hannah; Fraser, William D.; Gao, Fang; Sapey, Elizabeth; Perkins, Gavin D.


    Objectives: Vitamin D deficiency has been implicated as a pathogenic factor in sepsis and ICU mortality but causality of these associations has not been demonstrated. To determine whether sepsis and severe sepsis are associated with vitamin D deficiency and to determine whether vitamin D deficiency influences the severity of sepsis. Design, Setting, and Patients: Sixty-one patients with sepsis and severe sepsis from two large U.K. hospitals and 20 healthy controls were recruited. Murine models of cecal ligation and puncture and intratracheal lipopolysaccharide were undertaken in normal and vitamin D deficient mice to address the issue of causality. Measurements and Main Results: Patients with severe sepsis had significantly lower concentrations of 25-hydroxyvitamin D3 than patients with either mild sepsis or age-matched healthy controls (15.7 vs 49.5 vs 66.5 nmol/L; p = 0.0001). 25-hydroxyvitamin D3 concentrations were significantly lower in patients who had positive microbiologic culture than those who were culture negative (p = 0.0023) as well as those who died within 30 days of hospital admission (p = 0.025). Vitamin D deficiency in murine sepsis was associated with increased peritoneal (p = 0.037), systemic (p = 0.019), and bronchoalveolar lavage (p = 0.011) quantitative bacterial culture. This was associated with reduced local expression of the cathelicidin-related antimicrobial peptide as well as evidence of defective macrophage phagocytosis (p = 0.029). In the intratracheal lipopolysaccharide model, 1,500 IU of intraperitoneal cholecalciferol treatment 6 hours postinjury reduced alveolar inflammation, cellular damage, and hypoxia. Conclusions: Vitamin D deficiency is common in severe sepsis. This appears to contribute to the development of the condition in clinically relevant murine models and approaches to correct vitamin D deficiency in patients with sepsis should be developed. PMID:27632669

  20. Characterization of a Novel Murine Model to Study Zika Virus (United States)

    Rossi, Shannan L.; Tesh, Robert B.; Azar, Sasha R.; Muruato, Antonio E.; Hanley, Kathryn A.; Auguste, Albert J.; Langsjoen, Rose M.; Paessler, Slobodan; Vasilakis, Nikos; Weaver, Scott C.


    The mosquito-borne Zika virus (ZIKV) is responsible for an explosive ongoing outbreak of febrile illness across the Americas. ZIKV was previously thought to cause only a mild, flu-like illness, but during the current outbreak, an association with Guillain–Barré syndrome and microcephaly in neonates has been detected. A previous study showed that ZIKV requires murine adaptation to generate reproducible murine disease. In our study, a low-passage Cambodian isolate caused disease and mortality in mice lacking the interferon (IFN) alpha receptor (A129 mice) in an age-dependent manner, but not in similarly aged immunocompetent mice. In A129 mice, viremia peaked at ∼107 plaque-forming units/mL by day 2 postinfection (PI) and reached high titers in the spleen by day 1. ZIKV was detected in the brain on day 3 PI and caused signs of neurologic disease, including tremors, by day 6. Robust replication was also noted in the testis. In this model, all mice infected at the youngest age (3 weeks) succumbed to illness by day 7 PI. Older mice (11 weeks) showed signs of illness, viremia, and weight loss but recovered starting on day 8. In addition, AG129 mice, which lack both type I and II IFN responses, supported similar infection kinetics to A129 mice, but with exaggerated disease signs. This characterization of an Asian lineage ZIKV strain in a murine model, and one of the few studies reporting a model of Zika disease and demonstrating age-dependent morbidity and mortality, could provide a platform for testing the efficacy of antivirals and vaccines. PMID:27022155

  1. Forced recombination of psi-modified murine leukaemia virus-based vectors with murine leukaemia-like and VL30 murine endogenous retroviruses

    DEFF Research Database (Denmark)

    Mikkelsen, J G; Lund, Anders Henrik; Duch, M;


    -impaired Akv-MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5' untranslated region...... harbouring the supposed RNA dimer-forming cis -elements and (ii) that copackaged retroviral RNAs can recombine despite pronounced sequence dissimilarity at the cross-over site(s) and within parts of the genome involved in RNA dimerization, encapsidation and strand transferring during reverse transcription....... We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species....

  2. Adrenaline influences the release of interleukin-6 from murine pituicytes

    DEFF Research Database (Denmark)

    Christensen, J D; Hansen, E W; Frederiksen, C


    In this study, we examined the effect of adrenaline and interleukin-1beta on interleukin-6 secretion from cultured murine neurohypophyseal cells. Cells were cultured from neurohypophyses of 3- to 5-week-old mice and experiments were performed after 13 days in culture. Interleukin-6 was measured...... in 24-h samples using a sandwich fluoroimmunoassay. Unstimulated cells released 19+/-3 fmol interleukin-6/neurohypophysis/24 h (mean +/- S.E.M., n = 42). Adrenaline and interleukin-1beta increased the release of interleukin-6 from the cells in a concentration-dependent manner. Incubation with adrenaline...

  3. Drosophila homolog of the murine Int-1 protooncogene.



    We have isolated phage clones from Drosophila melanogaster genomic and cDNA libraries containing a sequence homologous to the murine Int-1 protooncogene. The Drosophila gene is represented by a single locus at position 28A1-2 on chromosome 2. The gene is expressed as a 2.9-kilobase-long polyadenylylated mRNA in embryo, larval, and pupal stages. It is hardly detectable in adult flies. The longest open reading frame of the cDNA clone corresponds to a protein 469 amino acids long. Alignment of t...

  4. Investigation of Murine Models for Sleep, Wakefulness and Target Discovery (United States)


    subjective day, 7pm-7am w R NR 0 50 100 150 200 250 state c o u n t s F2 Females F2 Males Different from humans, mice exhibit polyphasic sleep with many...early trends, e.g., in the 1p.m. (6 hrs of subjective night) group, became non-significant with additional mice tested • The polyphasic sleep ...REPORT Final report - Investigation of Murine Models for Sleep , Wakefulness and Target Discovery 14. ABSTRACT 16. SECURITY CLASSIFICATION OF

  5. Sexual transmission of Trypanosoma cruzi in murine model. (United States)

    Ribeiro, Marcelle; Nitz, Nadjar; Santana, Camilla; Moraes, Aline; Hagström, Luciana; Andrade, Rafael; Rios, Adriano; Sousa, Alessandro; Dallago, Bruno; Gurgel-Gonçalves, Rodrigo; Hecht, Mariana


    Trypanosoma cruzi is mainly transmitted by blood-sucking triatomines, but other routes also have epidemiological importance, such as blood transfusion and congenital transmission. Although the possibility of sexual transmission of T. cruzi has been suggested since its discovery, few studies have been published on this subject. We investigated acquisition of T. cruzi by sexual intercourse in an experimental murine model. Male and female mice in the chronic phase of Chagas disease were mated with naive partners. Parasitological, serological and molecular tests demonstrated the parasites in tissues and blood of partners. These results confirm the sexual transmission of T. cruzi in mice.

  6. Polysaccharides PS-G and protein LZ-8 from Reishi (Ganoderma lucidum) exhibit diverse functions in regulating murine macrophages and T lymphocytes. (United States)

    Yeh, Chen-Hao; Chen, Hsiao-Chin; Yang, Jeng-Je; Chuang, Wen-I; Sheu, Fuu


    Bioactive components in Ganoderma lucidum mainly include polysaccharides (PS-G) and immunomodulatory protein Ling Zhi-8 (LZ-8). These components may have diverse regulatory functions in the immune system. However, the PS-G preparations from different procedures still contained partial LZ-8 residue, indicating that the specific target and regulating function of PS-G and LZ-8 were not fully understood. In the present study, PS-G was subjected to 15% TCA for removing proteins and the LZ-8 detection using anti-LZ-8 monoclonal antibodies showed a remarkable 89.7% protein reduction of the deproteinized PS-G (dpPS-G). The Saccharomyces cerevisiae which expressed recombinant LZ-8 protein (rLZ-8) without glycosylation was generated and then compared with dpPS-G in the induction toward murine primary macrophage and T lymphocytic cells. The peritoneal macrophages from TLR4-deficient and wild type mice revealed that TLR4 was a putative receptor of dpPS-G, mediating the TNF-alpha, IL-1beta and IL-12p70 cytokine production and CD86, MHC II expression on macrophages, while rLZ-8 enhanced the production of IL-1beta, IL-12p70, CD86, and MHC II expression by another obscure route. rLZ-8-treated macrophages enhanced the release of IFN-gamma and IL-2 by murine CD4(+) and CD8(+) T cells, whereas dpPS-G treatment did not enhance the release of IFN-gamma and IL-2. Furthermore, although the direct rLZ-8-treatment conduced dramatic CD154, CD44 expression on CD3(+) T cells and increased IL-2, IFN-gamma secretion on CD4(+) and CD8(+) T cells, the dpPS-G was incapable of priming CD3(+), CD4(+) or CD8(+) T cells unitarily. Taken together, these results demonstrated that LZ-8 could activate murine macrophages and T lymphocytes but PS-G was merely the activator for macrophages, suggesting their diverse roles in activating the innate and adaptive immunity.

  7. Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria.

    Directory of Open Access Journals (Sweden)

    Friederike S Rossmann

    Full Text Available Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA. At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium, a mouse peritonitis model (using S. aureus Newman and LAC and a rat endocarditis model (using E. faecalis 12030 and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.

  8. Immunological evidence of monoclonal gammopathy in North India: a hospital based study

    Directory of Open Access Journals (Sweden)

    Kalpana Singh


    Full Text Available Kalpana Singh1, Bhawna Singh2, Sarika Arora2, Alpana Saxena11Department of Biochemistry, Maulana Azad Medical College and LN Hospital, New Delhi, India; 2Department of Biochemistry, GB Pant Hospital, New Delhi, IndiaBackground: Monoclonal gammopathy of unknown significance (MGUS is a condition in which a paraprotein is found in the blood during standard laboratory tests. It is age-related and characterized by accumulation of bone marrow plasma cells derived from a single abnormal clone. The aim of this study was to investigate the pattern of MGUS in North Indian urban population.Methods: Serum and urine samples were collected from 320 suspected cases of gammopathy, were analyzed by sensitive immunological technique based protein electrophoresis followed by immunofixation for detection and type of monoclonal/polyclonal gammopathies. Twenty-five healthy subjects were included as controls.Results: Gammopathies were observed in 38 (11.88% patients. Out of these 7.5% were ¬monoclonal and 4.3% were polyclonal. Overall age of presentation of these monoclonal ¬gammopathies in both sexes was between 21 and 76 years. Gender-related ratio (men:women for these gammopathies was 1:1.18. Predominant heavy chain isotype was IgG (62.5% followed by IgA (37.5%. Among light chains, kappa (κ and lambda (λ chains appeared in 91.6% and 8.4% gammopathies respectively. Paraprotein fractions obtained were IgGκ (58.3%, IgGλ (4.16%, IgAκ (33.3%, and IgAλ (4.16% with 25% samples being positive for Bence Jones proteinuria.Conclusions: Clinical laboratories play an important role in confirming the immunological diagnosis of gammopathies. Determination of nature of paraproteinemia and its associated diseases calls for more extensive studies in India.Keywords: monoclonal gammopathy, immunoelectrophoresis, multiple myeloma, bence jones protein, immunoglobulins

  9. Model-based prediction of monoclonal antibody retention in ion-exchange chromatography. (United States)

    Guélat, Bertrand; Delegrange, Lydia; Valax, Pascal; Morbidelli, Massimo


    In order to support a model-based process design in ion-exchange chromatography, an adsorption equilibrium model was adapted to predict the protein retention behavior from the amino acid sequence and from structural information on the resin. It is based on the computation of protein-resin interactions with a colloidal model and accounts for the contribution of each ionizable amino acid to the protein charge. As a verification of the protein charge model, the experimental titration curve of a monoclonal antibody was compared to its predicted net charge. Using this protein charge model in the computation of the protein-resin interactions, it is possible to predict the adsorption equilibrium constant (i.e. retention factor or Henry constant) with an explicit pH and salt dependence. The application of the model-based predictions for an in silico screening of the protein retention on various stationary phases or, alternatively, for the comparison of various monoclonal antibodies on a given cation-exchanger was demonstrated. Furthermore, considering the structural differences between charge variants of a monoclonal antibody, it was possible to predict their individual retention times. The selectivity between the side variants and the main isoform of the monoclonal antibody were computed. The comparison with the experimental data showed that the model was reliable with respect to the identification of the operating conditions maximizing the selectivity, i.e. the most promising conditions for a monoclonal antibody variant separation. Such predictions can be useful in reducing the experimental effort to identify the parameter space.

  10. CHO cell line specific prediction and control of recombinant monoclonal antibody N-glycosylation. (United States)

    Grainger, Rhian K; James, David C


    Here we demonstrate that it is possible to predict and control N-glycan processing of a secreted recombinant monoclonal antibody during manufacturing process development using a combination of statistical modelling and comparative measurement of cell surface glycans using fluorescent lectins. Using design of experiments--response surface modelling (DoE-RSM) methodology to adjust the relative media concentrations of known metabolic effectors of galactosylation (manganese, galactose, and uridine) we have shown that β1,4-galactosylation of the same recombinant IgG4 monoclonal antibody produced by different CHO cell lines can be precisely controlled in a cell line specific manner. For two cell lines, monoclonal antibody galactosylation could be increased by over 100% compared to control, non-supplemented cultures without a reduction in product titre and with minimal effect on cell growth. Analysis of galactosylation effector interactions by DoE-RSM indicated that Mn²⁺ alone was necessary but not sufficient to improve galactosylation, and that synergistic combinations of Gal and Urd were necessary to maximize galactosylation, whilst minimizing the deleterious effect of Urd on cell growth. To facilitate rapid cell culture process development we also tested the hypothesis that substrate-level control of cellular galactosylation would similarly affect both cell surface and secreted monoclonal antibody glycans, enabling facile indirect prediction of product glycan processing. To support this hypothesis, comparative quantitation of CHO cell surface β1,4-galactosylation by flow cytometry using fluorescent derivatives of RCA and ConA lectins revealed that substrate-controlled variation in monoclonal antibody galactosylation and cell surface galactosylation were significantly correlated. Taken together, these data show that precision control of a complex, dynamic cellular process essential for the definition of protein product molecular heterogeneity and bioactivity is

  11. Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin 2: stimulation of molecular programs of cytotoxic killer cells and induction of tumor regression. (United States)

    Nakajima, F; Khanna, A; Xu, G; Lagman, M; Haschemeyer, R; Mouradian, J; Wang, J C; Stenzel, K H; Rubin, A L; Suthanthiran, M


    Adoptive cellular immunotherapy, infusions of interleukin 2 (IL-2) in conjunction with in vitro-activated killer cells, has brought new hope to patients with cancer. The broad application of this strategy, however, is constrained by the need for repeated leukapheresis and by the labor-intensive process of in vitro activation of cells. Also, current protocols generally use nonphysiological and toxic concentrations of IL-2. Identification of an in vivo stimulant that renders T cells responsive to physiologic concentrations of IL-2 represents a potential improvement over existing approaches. We have determined whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 induces T-cell responsiveness to IL-2, stimulates cytolytic molecular programs of natural killer cells and cytotoxic T cells, and induces tumor regression. These hypotheses were explored in a murine hepatic MCA-102 fibrosarcoma model. We report that in vivo administration of anti-CD3 mAbs plus IL-2 results in intrahepatic expression of mRNA-encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor alpha. Anti-CD3 mAbs alone or IL-2 alone failed to induce or induced minimal expression of these molecular mediators of cytotoxicity. The anti-CD3 mAbs plus IL-2 regimen also resulted in a significantly smaller number of hepatic metastases and a significantly longer survival time of tumor-bearing mice, compared to treatment with anti-CD3 mAbs alone or IL-2 alone. Our findings suggest that a regimen of anti-CD3 mAbs plus IL-2 is a more effective antitumor regimen compared with anti-CD3 mAbs alone or IL-2 alone and advance an alternative immunotherapy strategy of potential value for the treatment of cancer in humans. Images PMID:8058730

  12. Treatment of steroid-refractory acute graft-versus-host disease with anti-CD147 monoclonal antibody ABX-CBL. (United States)

    Deeg, H J; Blazar, B R; Bolwell, B J; Long, G D; Schuening, F; Cunningham, J; Rifkin, R M; Abhyankar, S; Briggs, A D; Burt, R; Lipani, J; Roskos, L K; White, J M; Havrilla, N; Schwab, G; Heslop, H E


    ABX-CBL, an immunoglobulin M murine monoclonal antibody, recognizes CD147 and initiates cell killing through complement-mediated lysis. In a dose-finding trial, 27 patients with steroid-refractory acute graft-versus-host disease (GVHD) received ABX-CBL at 0.01 (presumed no effect dose), 0.1, 0.2, or 0.3 mg/kg per day, and an additional 32 patients were given ABX-CBL at 0.2 or 0.15 mg/kg per day. All patients had undergone allogeneic transplantation for malignant or nonmalignant disorders and received GVHD prophylaxis, generally with methotrexate- and cyclosporine-containing regimens. None responded to methylprednisolone, given for a minimum of 3 days. ABX-CBL was started 20 to 236 (median, 47) days after transplantation; it was given for 7 consecutive days and was followed by 2 infusions per week for 2 more weeks. Among 51 patients evaluable for efficacy, 26 (51%) responded, including 13 with complete responses (CR) and 13 with partial responses (PR). CR lasting 14 days or longer or PR lasting 7 days or longer occurred in 21 (41%; 8 CR, 13 PR) patients, including 19 of 43 (44%) patients who received 0.1 to 0.3 mg/kg ABX-CBL and 2 of 8 (25%) patients given 0.01 mg/kg per day. Myalgias at doses 0.2 mg/kg or greater were dose limiting and resolved without sequelae. Causes of death included organ failure, progressive GVHD, and infection. No death was attributed to ABX-CBL. At 6 months after the initiation of ABX-CBL therapy, 26 (44%) patients were surviving. These results are encouraging. Further studies on the use of ABX-CBL in the management of GVHD are warranted.

  13. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody. (United States)

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter


    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

  14. Production of a monoclonal antibody by in vitro immunization that recognizes a native chondroitin sulfate epitope in the embryonic chick limb and heart. (United States)

    Capehart, A A; Wienecke, M M; Kitten, G T; Solursh, M; Krug, E L


    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.

  15. Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide. gamma. -chain-specific monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ioannides, C.G.; Itoh, K.; Fox, F.E.; Pahwa, R.; Good, R.A.; Platsoucas, C.D.


    The authors developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the lambda chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, they identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from /sup 125/I-labeled denatured lysates of T3/sup +/ WT31/sup -/ lymphocytes expanded in culture from a SCID patient. Chemical crosslinking of /sup 125/I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These experiments were done with polyclonal cell populations. Cloned T3/sup +/ WT31/sup -/ cell populations are required to determine whether the TCR contains two lambda polypeptide chains. Using the same 9D7 anti-P18K mAb and immunoblotting analysis, they identified a 35 kDa ..gamma..-chain polypeptide under reducing conditions expressed on purified L3T4/sup -/ Lyt2/sup -/ BALB/c mouse thymocytes. This ..gamma..-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.

  16. Analysis of the capacity to produce IL-3 in murine AIDS

    DEFF Research Database (Denmark)

    Neuenschwander, A U; Marker, O; Thomsen, Allan Randrup


    Adult C57BL/6 mice infected with LP-BM5 murine leukaemia virus represent a model of murine AIDS (MAIDS). In this study we have analysed the capacity of CD4+ T cells from infected mice to produce IL-3 following stimulation with ConA for 24-72 h. In contrast to the position with IL-2, the production...

  17. Species difference of CD137 ligand signaling in human and murine monocytes.

    Directory of Open Access Journals (Sweden)

    Qianqiao Tang

    Full Text Available BACKGROUND: Stimulation of CD137 ligand on human monocytes has been shown to induce DC differentiation, and these CD137L-DCs are more potent than classical DCs, in stimulating T cell responses in vitro. To allow an in vivo evaluation of the potency of CD137L-DCs in murine models we aimed at generating murine CD137L-DCs. METHODOLOGY/PRINCIPAL FINDINGS: When stimulated through CD137 ligand murine monocytes responded just as human monocytes with an increased adherence, morphological changes, proliferation and an increase in viable cell numbers. But CD137 ligand signaling did not induce expression of inflammatory cytokines and costimulatory molecules in murine monocytes and these cells had no T cell stimulatory activity. Murine monocytes did not differentiate to inflammatory DCs upon CD137 ligand signaling. Furthermore, while CD137 ligand signaling induces maturation of human immature classical DCs it failed to do so with murine immature classical DCs. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that both human and murine monocytes become activated by CD137 ligand signaling but only human and not murine monocytes differentiate to inflammatory DCs.

  18. Murine anti-vaccinia virus D8 antibodies target different epitopes and differ in their ability to block D8 binding to CS-E.

    Directory of Open Access Journals (Sweden)

    Michael H Matho


    Full Text Available The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV. Here we identified the optimal D8 ligand to be chondroitin sulfate E (CS-E. CS-E is characterized by a disaccharide moiety with two sulfated hydroxyl groups at positions 4' and 6' of GalNAc. To study the role of antibodies in preventing D8 adhesion to CS-E, we have used a panel of murine monoclonal antibodies, and tested their ability to compete with CS-E for D8 binding. Among four antibody specificity groups, MAbs of one group (group IV fully abrogated CS-E binding, while MAbs of a second group (group III displayed widely varying levels of CS-E blocking. Using EM, we identified the binding site for each antibody specificity group on D8. Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E.

  19. 小鼠抗食蟹猴IgG单克隆抗体的制备%Preparation Monoclonal Antibodies Of Mice Against Macaca fascicularis IgG

    Institute of Scientific and Technical Information of China (English)

    张段玲; 张彦龙; 白素英


    目的 制备抗食蟹猴、恒河猴等非人灵长类实验动物免疫球蛋白二级抗体,开展对其传染病血清学快速诊断方法的建立.方法 采用饱和硫酸铵盐析、Agarose-Protein G亲和层析技术,从食蟹猴血清中提纯IgG.经SDS-PAGE电泳鉴定,采用常规法免疫C57BL/6小鼠,三次免疫后取脾细胞与Sp2/0-Agl4骨髓瘤细胞通过PEG4000融合制备杂交瘤细胞,利用间接ELISA、Western blot等方法进行筛选、鉴定.结果 得到5株阳性杂交瘤,分别命名为2B6、2B7、2D9、3B2、5E4,并且5株杂交瘤分泌的抗体均与恒河猴的IgG或血清发生交叉反应,而与其他物种如东北虎、犬等动物的IgG或血清无交叉反应.结论 5株杂交瘤产生的单克隆抗体(McAb)具有较好免疫活性,且能长期、稳定地分泌抗体.此项研究工作为后续研究食蟹猴、恒河猴传染病血清学诊断方法奠定基础.%Objective In order to prepare imraunoglobulin G (IgG) secondary antibody of non-human primates such as Macaca fascicularis and Macaca mulatta and establish a quick serological method to diagnose their diseases. Methods Macaco fascicularis IgG was isolated and purified from Macaco fascicularis serum by precipitated use saturated ammonium sulfate and affinity chromatography technology with Agarose-Protein G. After identified by SDS-PAGE electrophoresis murine monoclonal antibody ( McAb) were produced by conventional immune method and hybridoma technology. Monoclonal antibody was produced when C57BL/6 mice was immunized three times and its spleen cells were fused with SP2/0-Agl4 myeloma cells. Indirect ELISA and Western blot methods were used for identification. Result 5 strains of myeloma cells acquired which were named 2B6, 2B7, 3B2, 5E4, 2D9. The antibodies they secret can all react well with both IgG and their serum of Macaco fascicularis and Macaca mulatto. There are no cross reaction with other species such as east-northern tigers and dogs. Conclusion The 5

  20. A rat monoclonal antibody that recognizes pro- and active MMP-7 indicates polarized expression in vivo

    DEFF Research Database (Denmark)

    Fingleton, Barbara; Powell, William C; Crawford, Howard C


    different species and, by immunohistochemistry, can detect MMP-7 of human, murine, porcine, and gerbil origin. Immunostaining of MMP-7 in normal tissues or benign tumors of intestinal, breast, and prostatic origin indicates that this protein is normally localized luminally in glandular epithelium...

  1. Great efficacy of sulfachloropyrazine-sodium against acute murine toxoplasmosis

    Institute of Scientific and Technical Information of China (English)

    Yan-Bo Zeng; Bing Huang; Shun-Hai Zhu; Hui Dong; Hong-Yu Han; Lian-Lian Jiang; Quan Wang; Jun Cheng; Qi-Ping Zhao; Wei-Jiao Ma


    Objective: To identify more effective and less toxic drugs to treat animal toxoplasmosis.Methods:Efficacy of seven kinds of sulfonamides against Toxoplasma gondii (T. gondii) in an acute murine model was evaluated. The mice used throughout the study were randomly assigned to many groups (10 mice each), which either remained uninfected or were infected intraperitoneally with tachyzoites of T. gondii (strains RH and CN). All groups were then treated with different sulfonamides and the optimal treatment protocol was determined candidates. Sulfadiazine-sodium (SD) was used for comparison. Results: The optimal therapy involved gavaging mice twice per day with 250 mg/kg bw of sulfachloropyrazine-sodium (SPZ) for five days. Using this protocol, the average survival time and the time-point of 50% fatalities were prolonged significantly compared with SD treatment. Treatment with SPZ protected 40% of mice from death, and the heart and kidney tissue of these animals was parasite-free, as determined by nested-PCR. SPZ showed excellent therapeutic effects in the treatment of T. gondii in an acute murine model and is therefore a promising drug candidate for the treatment and prevention of T. gondii in animals. Conclusions: It can be concluded that the effective drug sulfachloropyrazine may be the new therapeutic options against animal toxoplasmosis.

  2. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B;


    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did not a...... treatment caused a shift of the isoforms, but no reduction in isoform number. Two-dimensional gel electrophoresis confirmed the existence of multiple isoforms of human SAP monomers.......Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... not affect their number. When the acute-phase response was analysed in three mouse strains, CBA/J and C3H/HeN initially showed seven SAP isoforms in serum and C57BL/6 J three or four. The responses in all three strains peaked at day 2 and were normalized within 14 days. On days 2 and 4, CBA/J and C3H...

  3. Rapamycin improves lymphoproliferative disease in murine autoimmune lymphoproliferative syndrome (ALPS). (United States)

    Teachey, David T; Obzut, Dana A; Axsom, Kelly; Choi, John K; Goldsmith, Kelly C; Hall, Junior; Hulitt, Jessica; Manno, Catherine S; Maris, John M; Rhodin, Nicholas; Sullivan, Kathleen E; Brown, Valerie I; Grupp, Stephan A


    Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of abnormal lymphocyte survival caused by defective Fas-mediated apoptosis, leading to lymphadenopathy, hepatosplenomegaly, and an increased number of double-negative T cells (DNTs). Treatment options for patients with ALPS are limited. Rapamycin has been shown to induce apoptosis in normal and malignant lymphocytes. Since ALPS is caused by defective lymphocyte apoptosis, we hypothesized that rapamycin would be effective in treating ALPS. We tested this hypothesis using rapamycin in murine models of ALPS. We followed treatment response with serial assessment of DNTs by flow cytometry in blood and lymphoid tissue, by serial monitoring of lymph node and spleen size with ultrasonography, and by enzyme-linked immunosorbent assay (ELISA) for anti-double-stranded DNA (dsDNA) antibodies. Three-dimensional ultrasound measurements in the mice correlated to actual tissue measurements at death (r = .9648). We found a dramatic and statistically significant decrease in DNTs, lymphadenopathy, splenomegaly, and autoantibodies after only 4 weeks when comparing rapamycin-treated mice with controls. Rapamycin induced apoptosis through the intrinsic mitochondrial pathway. We compared rapamycin to mycophenolate mofetil, a second-line agent used to treat ALPS, and found rapamycin's control of lymphoproliferation was superior. We conclude that rapamycin is an effective treatment for murine ALPS and should be explored as treatment for affected humans.

  4. Scanning electron microscopy of the neuropathology of murine cerebral malaria

    Directory of Open Access Journals (Sweden)

    Brenneis Christian


    Full Text Available Abstract Background The mechanisms leading to death and functional impairments due to cerebral malaria (CM are yet not fully understood. Most of the knowledge about the pathomechanisms of CM originates from studies in animal models. Though extensive histopathological studies of the murine brain during CM are existing, alterations have not been visualized by scanning electron microscopy (SEM so far. The present study investigates the neuropathological features of murine CM by applying SEM. Methods C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. When typical symptoms of CM developed perfused brains were processed for SEM or light microscopy, respectively. Results Ultrastructural hallmarks were disruption of vessel walls, parenchymal haemorrhage, leukocyte sequestration to the endothelium, and diapedesis of macrophages and lymphocytes into the Virchow-Robin space. Villous appearance of observed lymphocytes were indicative of activated state. Cerebral oedema was evidenced by enlargement of perivascular spaces. Conclusion The results of the present study corroborate the current understanding of CM pathophysiology, further support the prominent role of the local immune system in the neuropathology of CM and might expose new perspectives for further interventional studies.

  5. Filaria-induced IL-10 suppresses murine cerebral malaria. (United States)

    Specht, Sabine; Ruiz, Daniel Fernández; Dubben, Bettina; Deininger, Susanne; Hoerauf, Achim


    Filarial nematodes achieve long survival in their hosts due to their capacity to modulate immune responses. Therefore, immunomodulation by filarial nematodes may alter responses to concomitant infections such as malaria. Cerebral malaria (CM), a severe complication of Plasmodium falciparum infections, is triggered as a consequence of the immune response developed against malaria parasites. The question arises whether prior infection with helminth parasites is beneficial against CM. In the present work a murine model for subsequent has been used to assess this hypothesis. C57BL/6 mice were infected with the rodent filarial parasite Litomosoides sigmodontis and the murine model parasite for CM, Plasmodium berghei ANKA. Previously filaria-infected C57BL/6 mice showed significantly reduced CM rates. CD8(+) T cell recruitment to the brain, a hallmark for CM development, was reduced in protected mice. Furthermore, in contrast to P. berghei single-infected animals, filaria-infected mice had significantly higher levels of circulating IL-10. The requirement for IL-10 in CM protection was demonstrated by the lack of protection in IL-10 KO mice. This suggests that the anti-inflammatory IL-10 elicited by filarial nematodes is able to suppress the overwhelming inflammatory reaction otherwise triggered against malaria parasites in C57BL/6 mice, preventing full progress to CM.

  6. Neurological Disorders in a Murine Model of Chronic Renal Failure

    Directory of Open Access Journals (Sweden)

    Jean-Marc Chillon


    Full Text Available Cardiovascular disease is highly prevalent in patients with chronic renal failure (CRF. However, data on the impact of CRF on the cerebral circulatory system are scarce—despite the fact that stroke is the third most common cause of cardiovascular death in people with CRF. In the present study, we examined the impact of CRF on behavior (anxiety, recognition and ischemic stroke severity in a well-defined murine model of CRF. We did not observe any significant increases between CRF mice and non-CRF mice in terms of anxiety. In contrast, CRF mice showed lower levels of anxiety in some tests. Recognition was not impaired (vs. controls after 6 weeks of CRF but was impaired after 10 weeks of CRF. Chronic renal failure enhances the severity of ischemic stroke, as evaluated by the infarct volume size in CRF mice after 34 weeks of CRF. Furthermore, neurological test results in non-CRF mice tended to improve in the days following ischemic stroke, whereas the results in CRF mice tended to worsen. In conclusion, we showed that a murine model of CRF is suitable for evaluating uremic toxicity and the associated neurological disorders. Our data confirm the role of uremic toxicity in the genesis of neurological abnormalities (other than anxiety.

  7. Immunological impact of magnetic nanoparticles (Ferucarbotran) on murine peritoneal macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Chen-Hao [National Taiwan University, Department of Horticulture (China); Hsiao, Jong-Kai [National Taiwan University Hospital and College of Medicine, Department of Medical Imaging (China); Wang, Jaw-Lin [National Taiwan University, Institute of Biomedical Engineering (China); Sheu, Fuu, E-mail: [National Taiwan University, Department of Horticulture (China)


    Ferucarbotran, a clinically used superparamagnetic iron oxide, is widely developed as a magnetic resonance imaging (MRI) contrast agent and has the potential to improve the monitoring of macrophage recirculation in vivo. However, the biological effect of Ferucarbotran or magnetic nanoparticles (MNPs) on macrophage is not clearly understood yet. This study is aimed to examine the immunological impact of Ferucarbotran toward murine peritoneal macrophages. Cells treated with Ferucarbotran demonstrated a dose-responsive increase of granularity in the cytoplasm. After 24 h of incubation, viability and cytotoxicity in macrophages treated with 200 {mu}g Fe/mL of Ferucarbotran were not affected. Macrophages loaded with Ferucarbotran above 100 {mu}g Fe/mL showed a significant (p < 0.01) increase in cytokine (TNF-{alpha}, IL-1{beta}, IL-6) secretion and mRNA expression, followed by nitric oxide (NO) secretion and iNOS mRNA expression. Chemotactic responses of Ferucarbotran-preloaded macrophages toward CX3CL1 were significantly (p < 0.05) lower than those of untreated macrophages. Taking together, Ferucarbotran at high dose (100 {mu}g Fe/mL) could induce murine peritoneal macrophages activation in pro-inflammatory cytokine secretion and NO production.

  8. Data on the characterization of follicle-stimulating hormone monoclonal antibodies and localization in Japanese eel pituitary

    Directory of Open Access Journals (Sweden)

    Dae-Jung Kim


    In support of our recent publication, "Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica" [1], it was important to characterize the specificity of eel follicle-stimulating hormone antibodies. Here, the production and ELISA system of these monoclonal antibodies are presented. The affinity-purified monoclonal antibodies specifically detected eel rec-FSH in ELISA and on western blots of rec-FSH produced from CHO cells. Immunohistochemical analysis revealed that FSH staining was specifically localized in the eel pituitary.

  9. Distinct antigenic characteristics of murine parietal yolk sac laminin

    DEFF Research Database (Denmark)

    Wewer, U M; Tichy, D; Damjanov, A;


    Two monoclonal antibodies (LAM-A and LAM-B) specific for laminin from normal and neoplastic parietal yolk sac (PYS) cells were produced in rats immunized with a mouse yolk sac carcinoma cell line. Both antibodies immunoprecipitated the 400,000- and 200,000-Da chains of laminin and reacted...... with purified PYS laminin in ELISA. LAM-A reacted with mouse and rat PYS laminin, whereas LAM-B reacted only with mouse PYS laminin. Formaldehyde- and methanol-fixed adult and fetal somatic tissues were immunohistochemically unreactive with either of the two antibodies. In acetone-fixed tissue sections, both...... antibodies reacted weakly with some medullary tubules of the kidney, the follicular basement membrane of the ovaries, and the seminiferous tubules. The antibodies appear to react with the polypeptide determinants residing on the terminal portion of the long arm of laminin. It is concluded that laminin...

  10. Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis

    Directory of Open Access Journals (Sweden)

    Want MY


    Full Text Available Muzamil Y Want,1 Mohammad Islammudin,1 Garima Chouhan,1 Hani A Ozbak,2 Hassan A Hemeg,2 Asoke P Chattopadhyay,3 Farhat Afrin2 1Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard, Hamdard University, New Delhi, India; 2Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia; 3Department of Chemistry, University of Kalyani, Kalyani, India Abstract: Visceral leishmaniasis (VL is a fatal, vector-borne disease caused by the intracellular protozoa of the genus Leishmania. Most of the therapeutics for VL are toxic, expensive, or ineffective. Sesquiterpenes are a new class of drugs with proven antimicrobial and antiviral activities. Artemisinin is a sesquiterpene lactone with potent antileishmanial activity, but with limited access to infected cells, being a highly lipophilic molecule. Association of artemisinin with liposome is a desirable strategy to circumvent the problem of poor accessibility, thereby improving its efficacy, as demonstrated in a murine model of experimental VL. Nanoliposomal artemisinin (NLA was prepared by thin-film hydration method and optimized using Box–Behnken design with a mean particle diameter of 83±16 nm, polydispersity index of 0.2±0.03, zeta potential of -27.4±5.7 mV, and drug loading of 33.2%±2.1%. Morphological study of these nanoliposomes by microscopy showed a smooth and spherical surface. The mechanism of release of artemisinin from the liposomes followed the Higuchi model in vitro. NLA was free from concomitant signs of toxicity, both ex vivo in murine macrophages and in vivo in healthy BALB/c mice. NLA significantly denigrated the intracellular infection of Leishmania donovani amastigotes and the number of infected macrophages ex vivo with an IC50 of 6.0±1.4 µg/mL and 5.1±0.9 µg/mL, respectively. Following treatment in a murine model of VL, NLA demonstrated superior efficacy compared to artemisinin with a

  11. One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-Gel blue chromatography. (United States)

    Bruck, C; Portetelle, D; Glineur, C; Bollen, A


    Monoclonal antibodies can be purified directly from ascitic fluids by chromatography on a DEAE Affi-gel blue column. Optimal conditions were determined for the recovery of immunoglobulins free of contaminating protease and nuclease activities.

  12. Screening a hybridoma producing a specific monoclonal antibody to HLA-A24+Bw4 antigen by cytotoxicity inhibition assay. (United States)

    Hiroishi, S; Kaneko, T; Arita, J


    A hybridoma secreting a monoclonal antibody (Tsa-1, IgG3) reacting specifically to HLA-A24+Bw4 was screened by cytotoxicity inhibition assay and micrototoxicity test. The R value of the antibody was 0.843.

  13. A case of scleromyxedema without monoclonal gammopathy responded to treatment with IVIG

    Directory of Open Access Journals (Sweden)

    Ayşegül Turan


    Full Text Available Lichen myxedematosus’’ is a primary inflammatory dermatosis characterized by fibroblast proliferation and mucin deposition in the skin. Scleromyxedema is the most frequent variant of lichen myxedematosus and is accompanied by papular lesions, diffuse sclerosis, and erythema. It is usually associated with paraproteinemias. Scleromyxedema without monoclonal gammopathy is a rare and atypical variant of lichen myxedematosus. Systemic involvement and sometimes fatal course can be seen with scleromyxedema. Although several treatment modalities have been tried as case reports for the disease, there isn’t any definitive treatment yet. Intravenous immunoglobulin’s mechanism of action in scleromyxedema is still not clear as well as etiopathogenesis of scleromyxedema. Herein we describe a scleromyxedema case without monoclonal gammopathy and his response to the treatment with intravenous immunoglobulin.

  14. Unique association of Waldenström macroglobulinemia with optic neuritis and monoclonal T cell expansion. (United States)

    Morita, Ken; Yoshimi, Akihide; Masuda, Akiko; Ichikawa, Motoshi; Yatomi, Yutaka; Kurokawa, Mineo


    Waldenström macroglobulinemia is a lymphoplasmacytic lymphoma characterized by production of the immunoglobulin M (IgM) monoclonal protein. Commonly involved sites are the bone marrow, lymph nodes, and spleen. Lymphoplasmacytic infiltration of the central nervous system (CNS), in contrast, is referred to as Bing-Neel syndrome, and is an extremely rare phenomenon. Here, we present a unique case of Waldenström macroglobulinemia with optic neuritis accompanied by monoclonal expansion of T cells, which recovered after administration of CNS-targeting chemotherapy. Although the underlying causal relationships in this case remain obscure, aberrantly expanded T cells may have contributed to the development of optic neuritis, and we should be reminded that some types of cranial neuropathy in Waldenström macroglobulinemia may be reversible.


    Institute of Scientific and Technical Information of China (English)


    Objective: Preparation of anti-human androgen receptor(hAR) monoclonal antibody (McAb). Methods: Four cells lines of hybridoma secreting specific monoclonal antibodies against AR were first established by fusion SP2/0 cell with spleen cell from BALB/c mice immunized with the coupling complex of hAR-KLH. Results: Paraffin-embedded sections of 45 prostate cancers were detected. There was an overall concordance of 91% using Immunohistochemistry between AR polyclonal antibody from Zymed and hAR-N McAb selfmade. Conclusion: The results show that the McAb obtained in this study would be a useful tool to detect the AR status in prostate cancer.

  16. [Monoclonal gammopathy of undetermined significance and asymptomatic multiple myelom in the year 2014 ]. (United States)

    Adam, Zdeněk; Krejčí, Marta; Pour, Luděk; Sevčíková, Eva; Křivanová, Andrea; Rehák, Zdeněk; Koukalová, Renata; Cermáková, Zdeňka; Vaníček, Jíří; Sevčíková, Sabina


    Presence of monoclonal immunoglobulin in serum or urine is a relatively common event affecting about 3.2 % of people over 50. Isolated increase of only one type of free light chain, either κ or λ, is detected in 0.7-0.8 % of people over 50. Most people with monoclonal immunoglobulin meet the criteria of the so-called "mono-clonal gammopathy of undetermined significance (MGUS)". MGUS is defined by concentration of monoclonal immunoglobulin in serum < 30 g/l, number of plasma cells in the bone marrow < 10 % and the absence of symptoms of multiple myeloma and other lymphoproliferative diseases. A proportion of people with MGUS gradually progresses from asymptomatic into symptomatic myeloma or other malignant lymphoproliferative disease requiring treatment. Therefore, MGUS is considered to be one of the most common premalignant conditions with an average risk of transformation into malignant disease of 1 % per year. Monoclonal gammopathy of IgG and IgA subtype can develop into multiple myeloma. Light chain monoclonal gammopathy can develop not only into light chain multiple myeloma but also into AL-amyloidosis and light chain deposition disease (amorphous deposits of light chains damaging organs). IgM monoclonal gammopathy may develop into Waldenstrom macroglobulinemia or other lymphoproliferative disorder, or into rare IgM subtype of multiple myeloma. Unfortunately, people with MGUS are threatened by more than an increased risk of transformation into multiple myeloma or other severe hematologic disease. Pre-malignant clone of plasma cells in the bone marrow causes changes in the bone marrow that directly affect the person. For people with MGUS, there is an increased incidence of osteoporosis and increased fracture risk when compared to the general population. People with MGUS also have an increased risk of bacterial infections and thromboembolic complications compared with the same age population without MGUS. Clonal plasma cells, which are the basis of MGUS, may in

  17. beta-Adrenergic agonist activity of a monoclonal anti-idiotypic antibody. (United States)

    Guillet, J G; Kaveri, S V; Durieu, O; Delavier, C; Hoebeke, J; Strosberg, A D


    Hybridoma cells bearing monoclonal antibody against the beta-adrenergic ligand alprenolol were used as an immunogen to raise monoclonal anti-idiotypic antibodies. Of six anti-idiotypic antibodies, which inhibit ligand binding, three were able to recognize beta-adrenergic receptors. One of them, mAb2B4, an IgM that could be amplified into ascites, binds to the beta-adrenergic catecholamine receptors of intact epidermoid A431 cells and precipitates receptors solubilized from plasma membranes by digitonin. This antibody identifies the beta 2-adrenergic receptor of A431 cells as a single 55-kDa protein and stimulates adenylate cyclase activity. This stimulation is inhibited by the beta-adrenergic antagonist propranolol.

  18. Down-Turner Syndrome: A Case with Double Monoclonal Chromosomal Abnormality (United States)

    Alvarez-Manassero, Denisse; Merino-Luna, Alfredo


    Introduction. The coexistence of Down and Turner syndromes due to double chromosome aneuploidy is very rare; it is even more rare to find the presence of a double monoclonal chromosomal abnormality. Objective. To report a unique case of double monoclonal chromosomal abnormality with trisomy of chromosome 21 and an X ring chromosome in all cells studied; no previous report has been found. Case Report. Female, 28 months old, with pathological short stature from birth, with the following dysmorphic features: tilted upward palpebral fissures, short neck, brachycephaly, and low-set ears. During the neonatal period, the infant presented generalized hypotonia and lymphedema of hands and feet. Karyotype showed 47,X,r(X),+21 [30]. Conclusion. Clinical features of both Down and Turner syndromes were found, highlighting short stature that has remained below 3 z score from birth to the present, associated with delayed psychomotor development. G-banded karyotype analysis in peripheral blood is essential for a definitive diagnosis. PMID:27672470

  19. Characterization of a Novel Neutralizing Monoclonal Antibody Against Ebola Virus GP. (United States)

    Reynard, Olivier; Volchkov, Viktor E


    Ebola virus is the etiological agent of a severe hemorrhagic fever with a high mortality rate. As the only protein exposed on the surface of viral particles, the spike glycoprotein GP is the unique target for neutralizing monoclonal antibodies. In this study, we demonstrate the strong neutralization capacity of the monoclonal antibody #3327 and characterize its activity. GP residues that are required for recognition and neutralization were found to be located both in the internal fusion loop and in the receptor-binding domain. Analysis of Ebola virus entry in the presence of #3327 allows us to hypothesize that this antibody binds to the virus particle before internalization and endosomal processing of GP and likely prevents the final viral fusion step. Importantly, #3327 is able to block entry of virions bearing GP that contain the Q508 escape mutation common to a number of virus-neutralizing antibodies, and therefore provides future perspectives for treatment strategies against Ebola virus infection.

  20. Introduction to the application of QbD principles for the development of monoclonal antibodies. (United States)

    Finkler, Christof; Krummen, Lynne


    Quality by Design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter introduces a publication series on the application of Quality by Design for biopharmaceuticals, with a focus on the development of recombinant monoclonal antibodies. The development of and overview on the QbD concept applied by Roche and Genentech is described and essential QbD elements are presented.

  1. Monoclonal antibodies against proteins of the IBR virus nucleocapside and their assessing by ELISA

    Directory of Open Access Journals (Sweden)

    Jeannette Navarrete 0.


    Full Text Available Monoclonal antibodies were produced against the nucleocapside of a field IBR virus strain. The virus was growth in MOBK cell line. Viral components were purified and concentrated in a continuous 30% sacarose gradient followed by chemical precipitation. Nucleocapsides were run in a 10% SDS-PAGE gel. Positive hybridomes were tested using a direct ELISA developed in our laboratory. As a result eigth ELISA positive clones were obtained, from these five were also positive to seronetralization and immunodot. The clones recognize a 39.8Kda protein. Aditionally, a capture-ELISA was developed using the monoclonal antobodies from this research. This ELISA is useful to detect a reference as a field nm virus strain.

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

    Directory of Open Access Journals (Sweden)

    J. Ivanyi


    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.

  3. Limitations of safranin 'O' staining in proteoglycan-depleted cartilage demonstrated with monoclonal antibodies. (United States)

    Camplejohn, K L; Allard, S A


    The intensity of safranin 'O' staining is directly proportional to the proteoglycan content in normal cartilage. Safranin 'O' has thus been used to demonstrate any changes that occur in articular disease. In this study, staining patterns obtained using monoclonal antibodies against the major components of cartilage proteoglycan chondroitin sulphate (anti CS) and keratan sulphate (anti KS), have been compared with those obtained with safranin 'O' staining, in both normal and arthritic tissues. In cartilage where safranin 'O' staining was not detectable, the monoclonal antibodies revealed the presence of both keratan and chondroitin sulphate. Thus, safranin 'O' is not a sensitive indicator of proteoglycan content in diseases where glycosaminoglaycan loss from cartilage has been severe.

  4. Down-Turner Syndrome: A Case with Double Monoclonal Chromosomal Abnormality. (United States)

    Manassero-Morales, Gioconda; Alvarez-Manassero, Denisse; Merino-Luna, Alfredo


    Introduction. The coexistence of Down and Turner syndromes due to double chromosome aneuploidy is very rare; it is even more rare to find the presence of a double monoclonal chromosomal abnormality. Objective. To report a unique case of double monoclonal chromosomal abnormality with trisomy of chromosome 21 and an X ring chromosome in all cells studied; no previous report has been found. Case Report. Female, 28 months old, with pathological short stature from birth, with the following dysmorphic features: tilted upward palpebral fissures, short neck, brachycephaly, and low-set ears. During the neonatal period, the infant presented generalized hypotonia and lymphedema of hands and feet. Karyotype showed 47,X,r(X),+21 [30]. Conclusion. Clinical features of both Down and Turner syndromes were found, highlighting short stature that has remained below 3 z score from birth to the present, associated with delayed psychomotor development. G-banded karyotype analysis in peripheral blood is essential for a definitive diagnosis.

  5. Monoclonal gammopathy-associated pauci-immune extracapillary-proliferative glomerulonephritis successfully treated with bortezomib. (United States)

    Grundmann, Franziska; Witthus, Marco; Göbel, Heike; Kisner, Tuelay; Siewert, Rainer; Benzing, Thomas; Kurschat, Christine E


    Extracapillary-proliferative glomerulonephritis is a rare complication of multiple myeloma. Partial remission of kidney involvement with cyclophosphamide therapy has previously been described. We report the case of a 60-year-old male patient diagnosed with rapidly progressive glomerulonephritis associated with IgG kappa monoclonal gammopathy. His kidney biopsy revealed pauci-immune extracapillary-proliferative glomerulonephritis without cryoglobulinaemia. Treatment with the proteasome inhibitor bortezomib induced rapid clinical and histological remission of his kidney disease. The patient's renal function remained stable on bortezomib maintenance therapy. Our findings suggest that bortezomib is a promising therapeutic approach to ameliorate severe kidney damage in monoclonal gammopathy- and myeloma-associated pauci-immune extracapillary-proliferative glomerulonephritis.

  6. Down-Turner Syndrome: A Case with Double Monoclonal Chromosomal Abnormality

    Directory of Open Access Journals (Sweden)

    Gioconda Manassero-Morales


    Full Text Available Introduction. The coexistence of Down and Turner syndromes due to double chromosome aneuploidy is very rare; it is even more rare to find the presence of a double monoclonal chromosomal abnormality. Objective. To report a unique case of double monoclonal chromosomal abnormality with trisomy of chromosome 21 and an X ring chromosome in all cells studied; no previous report has been found. Case Report. Female, 28 months old, with pathological short stature from birth, with the following dysmorphic features: tilted upward palpebral fissures, short neck, brachycephaly, and low-set ears. During the neonatal period, the infant presented generalized hypotonia and lymphedema of hands and feet. Karyotype showed 47,X,r(X,+21 [30]. Conclusion. Clinical features of both Down and Turner syndromes were found, highlighting short stature that has remained below 3 z score from birth to the present, associated with delayed psychomotor development. G-banded karyotype analysis in peripheral blood is essential for a definitive diagnosis.

  7. Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody. (United States)

    Zhao, Yinli; Li, Guoxi


    A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1-128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93-97.5%, and the coefficient of variation [CV (%)] were from 5.55-8.38%. For interassay reproducibility, recoveries were from 89.5-95.1%, the coefficient of variation [CV (%)] were from 5.26-9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.

  8. Discrimination between Fibrin and Fibrinogen by a Monoclonal Antibody against a Synthetic Peptide (United States)

    Scheefers-Borchel, Ursula; Muller-Berghaus, Gert; Fuhge, Peter; Eberle, Reinhard; Heimburger, Nobert


    Circulating soluble fibrin, observed in the blood of patients with ongoing intravascular coagulation, is generated from the plasma protein fibrinogen by the limited proteolytic action of thrombin. We report the production of a monoclonal antibody that discriminates between fibrin and fibrinogen in blood. The synthetic hexapeptide Gly-Pro-Arg-Val-Val-Glu, representing the amino terminus of the α chain of human fibrin, was used as immunogen. This hexapeptide is located within the Aα chain of fibrinogen but becomes the amino terminus of the fibrin α chain, after fibrinopeptide A is removed by the action of thrombin, and thus becomes accessible for antibody binding. The monoclonal antibody we have prepared can discriminate between fibrin and fibrinogen and thus can be used in assay systems to quantitate soluble fibrin or, potentially, to image fibrin-rich thrombi.

  9. Immunomodulatory therapies for relapsing-remitting multiple sclerosis: monoclonal antibodies, currently approved and in testing. (United States)

    Craddock, Jessica; Markovic-Plese, Silva


    Relapsing-remitting multiple sclerosis (RRMS), a CNS inflammatory demyelinating disease, is one of the most prevalent causes of chronic disability in young adults. Studies of the disease pathogenesis have identified multiple therapeutic targets. The number of approved disease modifying therapies has almost doubled within the past 5 years, which creates a challenge for medical professionals to stay abreast of their use in everyday practice. This manuscript provides an overview of available injectable, oral, and intravenous therapies for RRMS, and offers guidance in selecting an appropriate therapy. Focus is on the recently approved and emerging monoclonal antibody therapies, because they offer more selective and superior therapeutic efficacy compared with injectable and oral disease modifying therapies. We discuss the outlook for monoclonal antibodies and their role in RRMS treatment in the future.

  10. [Evaluation of the relations between serum proteins electrophoresis and other laboratory tests in monoclonal gammopathies (author's transl)]. (United States)

    Ramacciotti, P G; Lazzari, L; Minardi, P


    We have considered interesting to determine monoclonal gammopathies incidence, in 2191 serum proteins electrophoresis performed in our laboratory from January to December 1974. We have found 15 cases of monoclonal gammopathies, some cases combined with Mieloma (3 cases), some other with other with non specific diseases. We have considered the relations between type of gammopathy and other laboratory tests useful for any other diagnose: they are: immunochemical analysis, E.S.R., red and white count, total proteins, Bence Jones protein.

  11. Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies. (United States)

    Winkelmann, D A; Bourdieu, L; Kinose, F; Libchaber, A


    We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement.

  12. Belimumab: anti-BLyS monoclonal antibody; Benlysta; BmAb; LymphoStat-B. (United States)


    Belimumab is a fully human monoclonal antibody for the treatment of autoimmune disorders that is being developed by Human Genome Sciences and GlaxoSmithKline. Two pivotal phase III trials in systemic lupus erythematosus have been concluded with the primary endpoints being met in both studies. A phase II trial in rheumatoid arthritis has also been completed, with positive results. Marketing authorization submissions are being prepared in the major markets worldwide. This review discusses the development history and scientific profile of belimumab.

  13. Novel EphB4 Monoclonal Antibodies Modulate Angiogenesis and Inhibit Tumor Growth


    Krasnoperov, Valery; Kumar, S. Ram; Ley, Eric; Li, Xiuqing; Scehnet, Jeffrey; Liu, Ren; Zozulya, Sergey; Gill, Parkash S.


    EphB4 receptor tyrosine kinase and its cognate ligand EphrinB2 regulate induction and maturation of newly forming vessels. Inhibition of their interaction arrests angiogenesis, vessel maturation, and pericyte recruitment. In addition, EphB4 is expressed in the vast majority of epithelial cancers and provides a survival advantage to most. Here, we describe two anti-EphB4 monoclonal antibodies that inhibit tumor angiogenesis and tumor growth by two distinct pathways. MAb131 binds to fibronectin...

  14. Production of Human Monoclonal Rheumatoid Factor Secreting Hybridomas Derived from Rheumatoid Synovial Cells (United States)


    antisera to human [gM and human IgG heavy chains , kappa and lambda lightTable 2: Rheumatoid synowal cell tRF subelass speclicity profiles chains , and...antisera to whole mouse Ig including light chains .(ELISA)frompantie MKin Table 1& AD7 RF was a human 1gM k monoclonal antibody without Well IgGI gG2

  15. Protection Against Clostridium difficile Infection With Broadly Neutralizing Antitoxin Monoclonal Antibodies



    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50...

  16. Monoclonal gammopathy of undetermined significance: Using risk stratification to guide follow-up. (United States)

    Uddin, Zia; Maennle, Diane; Russell, Kimberly; Boltri, John M


    Varying combinations of 3 measurable factors determine a patient's risk of progressing toward multiple myeloma and influence monitoring decisions. This review--and accompanying algorithm--can guide your approach. For monoclonal gammopathy of undetermined significance (MGUS) patients at low risk, repeat serum protein electrophoresis (SPE) in 6 months. If no significant elevation of M-protein is found, repeat SPE every 2 to 3 years.

  17. The laboratory of clinical virology in monitoring patients undergoing monoclonal antibody therapy. (United States)

    Cavallo, R


    The relevant efficacy of monoclonal antibodies (mAbs) has resulted in the successful treatment of several diseases, although susceptibility to infections remains a major problem. This review summarizes aspects of the literature regarding viral infections and mAbs, specifically addressing the risk of infection/reactivation, the measures that can reduce this risk, and the role played by the laboratory of clinical virology in monitoring patients undergoing mAb therapy.

  18. Epitope Mapping of Dengue-Virus-Enhancing Monoclonal-Antibody Using Phage Display Peptide Library


    Chung-I Rai; Huan-Yao Lei; Yee-Shin Lin; Hsiao-Sheng Liu; Shun-Hua Chen; Lien-Cheng Chen; Trai-Ming Yeh


    The Antibody-Dependent Enhancement (ADE) hypothesis has been proposed to explain why more severe manifestations of Dengue Hemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS) occur predominantly during secondary infections of Dengue Virus (DV) with different serotypes. However, the epitopes recognized by these enhancing antibodies are unclear. Recently, anti-pre-M monoclonal antibody (mAb 70-21), which recognized all DV serotypes without neutralizing activity, were generated and demonstrated...

  19. The Efficacy of an anti-CD4 Monoclonal Antibody for HIV-1 Treatment


    Fessel, W. Jeffrey; Anderson, Brooke; Follansbee, Stephen E.; Winters, Mark A.; Lewis, Stanley; Weinheimer, Steven; Christos J Petropoulos; Shafer, Robert W.


    The availability of 24 antiretroviral (ARV) drugs within six distinct drug classes has transformed HIV-1 infection (AIDS) into a treatable chronic disease. However, the ability of HIV-1 to develop resistance to multiple classes continues to present challenges to the treatment of many ARV treatment-experienced patients. In this case report, we describe the response to ibalizumab, an investigational CD4-binding monoclonal antibody (mAb), in a patient with advanced immunodeficiency and high-leve...

  20. Characterization of Hemolysin of Moraxella bovis Using a Hemolysis-Neutralizing Monoclonal Antibody



    A concentrated bacterial culture supernatant from the hemolytic Moraxella bovis strain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity of M. bovis and recognized a 94-kDa protein by Western blot analysis in hemolytic M. bovis strains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells to M. bovis concentrated culture supernatants demonstrated a role for an exotoxin in the...