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Sample records for anti-a murine monoclonal

  1. Heterohybridoma for the production of non murine monoclonal antibodies

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    Kh.Victoria Chanu and M. Ayub Ali

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

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    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...

  3. Purification of Murine Monoclonal IgM Antibody

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    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.

  4. 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

    2007-01-01

    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.

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

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

    2013-01-01

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

  6. Monoclonal antibody to murine PECAM-1 (CD31) blocks acute inflammation in vivo

    OpenAIRE

    1994-01-01

    A murine model of peritonitis was used to test the role of platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31) in acute inflammation. A monoclonal antibody (mAb) specific for murine PECAM-1 injected intravenously 4 h before the intraperitoneal injection of thioglycollate broth blocked leukocyte emigration into the peritoneal cavity for up to 48 h. This block was particularly evident for neutrophils. Control mAb, including one that bound to murine CD18 without blocking its function, f...

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

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Kamstrup, Søren; Oleksiewicz, M.B.; Eriksen, L.

    cognate antigen in the pigs were used. Enzyme-linked immunosorbent assays (ELISA) were used to quantitate the circulating m-mAbs, as well as the induced pig anti-mouse antibodies (PAMA), in serum samples from m-mAb-treated pigs. As expected, we generally saw vigorous PAMA responses within 10 days aft er......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...

  8. 99m-technetium labelling of a tumor associated murine monoclonal antibody for immunoscintigraphic studies in man

    International Nuclear Information System (INIS)

    The present study refers to the preparation of a 99m-Technetium labelled murine monoclonal antibody for clinical application. The monoclonal antibody was incubated with a 20fold molar excess of 2-iminothiolane. The free thiol groups created, were capable of binding reduced technetium. Labelling took place through an exchange reaction with 99m-Technetium-Glucoheptonate. The labelling conditions were studied extensively. (Author)

  9. Clinical assay stage I clinical trial with the murine monoclonal antibody IOR-T1: Pharmacokinetic and immune answers

    International Nuclear Information System (INIS)

    As part of the stage I clinical trial with the murine monoclonal antibody IOR-T1 at repeated doses (200-800 mg) in patients carriers of cutaneous T-cell lymphoma, the pharmacokinetics and the response against the mouse protein (HAMA) were studied in the 10 patients under treatment. It was observed a great individual variation in the maximum concentration in serum, which was estimated at 2 hours. The mean life time of the monoclonal antibody was between 13.93 and 19.6 hours. Most of the patients developed antibodies against the monoclonal antibody IOR-T1. The presence of this second antibody did not alter significantly the pharmacokinetics of the administered monoclonal antibody

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

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    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.

  11. In vivo tumor localization using tumor-specific monkey xenoantibody, alloantibody, and murine monoclonal xenoantibody

    International Nuclear Information System (INIS)

    Specific in vivo localization of antibodies reactive with human melanoma cell membrane tumor associated antigens (TAA) has been attempted using congenitally athymic nude mice bearing subcutaneous human melanoma tumor xenografts as the experimental model. IgG fractions were prepared from each of several immune and control sera. Antimelanoma antibody sources included human alloantibody obtained from melanoma patients immunized against allogeneic melanoma cells, a monkey antiserum raised by immunization against a single human melanoma cell line, and a murine monoclonal antimelanoma antibody-secreting hybridoma cell line. Localization of these radiolabeled antibodies and of control IgG preparations to tumor tissue was determined by whole body scintigraphy and by differential tissue counting. Compared with the different control IgG preparations, each of the antimelanoma IgG preparations exhibited significant specific accumulation within the melanoma tissue. However, variation existed in the ability of each antimelanoma IgG to tumor preparation to localize despite attempts to control model parameters such as tumor source, in vivo passage number and mass. This variation appears to reflect basic biologic differences between tumors in different animals and possibly differences in the antigen-binding capacities of each IgG preparation following radioiodination. This technique for tumor localization is very promising and has obvious potential for clinical application

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

    International Nuclear Information System (INIS)

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

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

    International Nuclear Information System (INIS)

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

  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;

    2015-01-01

    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...

  15. In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: Comparison with its murine parent

    International Nuclear Information System (INIS)

    The mutant version of the epidermal growth factor receptor EGFRvIII has been found on gliomas and other tumors, but not on normal tissues. Radioiodinated murine (mu) L8A4 monoclonal antibody (MAb) specifically targets EGFRvIII xenografts in vivo when labeled using N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC). A chimeric (ch) MAb consisting of the variable region of muL8A4 and the constant domains of human IgG2 has been developed that has an affinity and radioiodinated immunoreactive fraction comparable to muL8A4. In vitro, both MAbs were internalized and processed by EGFRvIII expressing cell lines (U87MGΔEGFR or NR6M) at similar rates (maximum intracellular retention, 35-40%). In paired-label tissue distribution studies in athymic mice bearing U87MGΔEGFR tumor xenografts, the ch:mu L8A4 uptake ratio in normal tissues rose to greater than 2:1, whereas in tumor, the ratio remained 1:1 throughout the experiment. These results indicate that chL8A4 exhibits similar binding and internalization properties as its murine parent, but suggest different intracellular processing and/or deposition of catabolites in normal tissues for chL8A4

  16. Probing Structural Variability at the N Terminus of the TSH Receptor with a Murine Monoclonal Antibody That Distinguishes between Two Receptor Conformational Forms

    OpenAIRE

    Hamidi, Sepehr; Chen, Chun-Rong; Murali, Ramachandran; McLachlan, Sandra M.; Rapoport, Basil

    2012-01-01

    Despite elucidation of the crystal structure of M22, a human thyroid-stimulating autoantibody (TSAb) bound to the TSH receptor (TSHR) leucine-rich repeat domain (LRD), the mechanism by which TSAs activate the TSHR and cause Graves' disease remains unknown. A nonstimulatory murine monoclonal antibody, 3BD10, and TSAb interact with the LRD N-terminal cysteine cluster and reciprocally distinguish between two different LRD conformational forms. To study this remarkable phenomenon, we investigated...

  17. Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins.

    OpenAIRE

    Daugherty, B L; DeMartino, J A; Law, M F; Kawka, D W; Singer, I I; Mark, G E

    1991-01-01

    Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs po...

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

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    Fátima Reyes-López

    2015-11-01

    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.

  19. A murine monoclonal antibody (VM-1) against human basal cells inhibits the growth of human keratinocytes in culture.

    Science.gov (United States)

    Oseroff, A R; Pfendt, E A; DiCicco, L; Morhenn, V B

    1985-04-01

    Using epidermal cells from psoriatic plaques as the immunogen, an IgG1 murine monoclonal antibody, VM-1, has been produced which stains basal keratinocytes on frozen sections of skin obtained from normal individuals and from psoriatic plaques. In some areas of both normal and psoriatic epidermis, the cell layer immediately above the basal cells is also stained. Cells in the external root sheath of the hair follicles also bind VM-1. The antibody binding site is trypsin-resistant, and is not blocked by bullous pemphigoid serum. If dispersed epidermal cells are preincubated with VM-1 for 1 h or more before plating, the majority of the cells do not attach and spread out on a collagen-coated Petri dish surface or on a fibroblast feeder layer. When added to attached, preconfluent cultures of keratinocytes, VM-1 inhibits growth and alters cell morphology. The growth inhibition is specific for keratinocytes, and viability studies show that it is not due to an immediate toxic effect of the antibody. The VM-1-induced inhibition of keratinocyte growth is not reversed by soy bean or lima bean trypsin inhibitors added at the time of cell plating or at the time of addition of antibody. PMID:3981036

  20. Immunoradiometric assay for the detection of circulating antibodies to murine monoclonal antibodies in humans (HAMA)

    International Nuclear Information System (INIS)

    The increasing clinical use of monoclonal antibodies (MAb) has focussed attention on the importance of the generation of human anti-mouse antibodies (HAMA). HAMA can not only be life threatening but can also be associated with reduced image quality and accelerated MAb clearance in vivo as well as interfere with in vitro MAb based assays. The development of a two step immunoradiometric assay (IRMA) for detecting circulating HAMA is reported. Preliminary results have been generated with specific MAb coated polystyrene wells. The appropriately diluted serum sample is first incubated with the MAb coated wells followed by a wash step and a second incubation with 125I labeled goat anti-human IgG. After a final wash, the wells are assayed for 125I and the results expressed as percent of the input bound. The prototype assay is compared with an existing commercially available ELISA kit using patient sera obtained at various time periods up to 7 months after IV MAb injection for radioimmunoscintigraphy. 18 refs., 2 tabs., 1 fig

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

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

    1984-01-01

    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 ..mu..Cl of I-131 MrSSl and 15 ..mu..Cl 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.

  2. Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours.

    Science.gov (United States)

    Quinn, K A; Morton, H

    1992-01-01

    Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s.c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 micrograms anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effects of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s.c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [3H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factor circulating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated. PMID:1537058

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

    International Nuclear Information System (INIS)

    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

  4. 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: neri@pharma.ethz.ch [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland)

    2014-09-10

    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

  5. 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.

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

    Science.gov (United States)

    Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom; Machado, Camila Maria L; Horta, Bruno Brasil; Buchpiguel, Carlos; Moro, Ana Maria; Okamoto, Oswaldo Keith; Jacobsson, Lars; Cederkrantz, Elin; Washiyama, Kohshin; Aneheim, Emma; Palm, Stig; Jensen, Holger; Tuma, Maria Carolina B; Chammas, Roger; Hultborn, Ragnar; Albertsson, Per

    2015-01-01

    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. PMID:25970341

  7. Neutralization of lymphokine-mediated antirickettsial activity of fibroblasts and macrophages with monoclonal antibody specific for murine interferon gamma.

    OpenAIRE

    Jerrells, T R; Turco, J; Winkler, H H; Spitalny, G L

    1986-01-01

    Lymphokine-mediated inhibition of Rickettsia prowazekii multiplication in L929 fibroblasts was eliminated by treatment of the lymphokine with a monoclonal antibody specific for interferon-gamma. Soluble monoclonal antibody and antibody conjugated to Sepharose beads were equally effective. Macrophage activation to limit the multiplication of Rickettsia conorii was eliminated with antibody-conjugated beads; however, neutralization of the ability to activate macrophages with soluble antibody was...

  8. COMPARISON OF FOUR METHODS TO GENERATE IMMUNOREACTIVE FRAGMENTS OF A MURINE MONOCLONAL ANTIBODY OC859 AGAINST HUMAN OVARIAN EPITHELIAL CANCER ANTIGEN

    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

    1995-01-01

    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.

  9. A humanised murine monoclonal antibody protects mice from Venezuelan equine encephalitis virus, Everglades virus and Mucambo virus when administered up to 48 h after airborne challenge

    International Nuclear Information System (INIS)

    Currently there are no licensed antiviral treatments for the Alphaviruses Venezuelan equine encephalitis virus (VEEV), Everglades virus and Mucambo virus. We previously developed a humanised version of the mouse monoclonal antibody 1A3B-7 (Hu1A3B-7) which exhibited a wide range of reactivity in vitro and was able to protect mice from infection with VEEV. Continued work with the humanised antibody has now demonstrated that it has the potential to be a new human therapeutic. Hu1A3B-7 successfully protected mice from infection with multiple Alphaviruses. The effectiveness of the humanisation process was determined by assessing proliferation responses in human T-cells to peptides derived from the murine and humanised versions of the VH and VL domains. This analysis showed that the number of human T-cell epitopes within the humanised antibody had been substantially reduced, indicating that Hu1A3B-7 may have reduced immunogenicity in vivo.

  10. Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies

    DEFF Research Database (Denmark)

    Rasch, M.G.; Pass, J.; Illemann, M.;

    2008-01-01

    The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological...... murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this...... mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in...

  11. Biodistribution and pharmacokinetics of monoclonal antibody T1h and variant anti-CD6 murine 10D12 in healthy animals and in experimental arthritis model

    International Nuclear Information System (INIS)

    Biodistribution and pharmacokinetic of two radio labeled monoclonal antibodies was performed with the help of imaging techniques. Isotopic labeling was carried out by means of standardized methods. Pharmacokinetic evaluation was performed using the population approach and sparse data design. Introduction: Targeted therapy with monoclonal antibodies (MAb) is an efficient option for the treatment of rheumatoid arthritis. Th1 is a MAb anti human CD6 developed for the treatment of autoimmune disease and 10D12 is its counterpart anti murine CD6 developed as a pharmacological tool to get deep into the response mechanisms in animals models of rheumatoid arthritis.To investigate the behavior of both antibodies in the assay system, molecules were labeled with 125I to evaluate pharmacokinetic in healthy animals and with 99mTc to evaluate the antibody uptake in inflamed area of induced arthritis. Materials and methods: Antibodies were supplied by the Center of Molecular immunology. Iodination was performed by the iodogen method and technetium labeling was carried out directly by Schwarz method. Female C57BL6 from CENPALAB were used for experiments. Biodistribution and pharmacokinetic was performed by a sparse data design using the population approach. Uptake in region of inflammation was quantified by gammagraphy at the same time points of blood sampling. A compartmental model was build to quantify uptake kinetic. Pharmacokinetic profiles were analyzed using MONOLIX software version 4.2. Results: Minor pharmacokinetic differences were found between monoclonal antibodies labeled with 125I and 99mTc. As a humanized antibody, T1h shows a faster clearance than 10D12 and a biodistribution pattern reflecting preference for excretion mechanisms. The arthritis accumulation was not consistent with a targeted mediated uptake. On the other hand, radio labeled 10D12 shows an accumulation profile in arthritis with two peaks of maximum concentration representing an initial transit to

  12. 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

    2011-08-01

    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.

  13. 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

    2003-11-01

    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.

  14. A Single Dose of Neuron-Binding Human Monoclonal Antibody Improves Spontaneous Activity in a Murine Model of Demyelination

    OpenAIRE

    Denic, Aleksandar; Macura, Slobodan I.; Warrington, Arthur E.; Pirko, Istvan; Grossardt, Brandon R.; Pease, Larry R.; Rodriguez, Moses

    2011-01-01

    Our laboratory demonstrated that a natural human serum antibody, sHIgM12, binds to neurons in vitro and promotes neurite outgrowth. We generated a recombinant form, rHIgM12, with identical properties. Intracerebral infection with Theiler's Murine Encephalomyelitis Virus (TMEV) of susceptible mouse strains results in chronic demyelinating disease with progressive axonal loss and neurologic dysfunction similar to progressive forms of multiple sclerosis. To study the effects of rHIgM12 on the mo...

  15. Characterization of periplasmic protein BP26 epitopes of Brucella melitensis reacting with murine monoclonal and sheep antibodies.

    Directory of Open Access Journals (Sweden)

    Jinlang Qiu

    Full Text Available 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.

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

    Science.gov (United States)

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

    2015-10-21

    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 < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole 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. PMID:26416205

  17. A humanised murine monoclonal antibody protects mice from Venezuelan equine encephalitis virus, Everglades virus and Mucambo virus when administered up to 48 h after airborne challenge

    Energy Technology Data Exchange (ETDEWEB)

    O' Brien, Lyn M., E-mail: lmobrien@dstl.gov.uk; Goodchild, Sarah A.; Phillpotts, Robert J.; Perkins, Stuart D.

    2012-05-10

    Currently there are no licensed antiviral treatments for the Alphaviruses Venezuelan equine encephalitis virus (VEEV), Everglades virus and Mucambo virus. We previously developed a humanised version of the mouse monoclonal antibody 1A3B-7 (Hu1A3B-7) which exhibited a wide range of reactivity in vitro and was able to protect mice from infection with VEEV. Continued work with the humanised antibody has now demonstrated that it has the potential to be a new human therapeutic. Hu1A3B-7 successfully protected mice from infection with multiple Alphaviruses. The effectiveness of the humanisation process was determined by assessing proliferation responses in human T-cells to peptides derived from the murine and humanised versions of the V{sub H} and V{sub L} domains. This analysis showed that the number of human T-cell epitopes within the humanised antibody had been substantially reduced, indicating that Hu1A3B-7 may have reduced immunogenicity in vivo.

  18. A Novel Murine Anti-Lactoferrin Monoclonal Antibody Activates Human Polymorphonuclear Leukocytes through Membrane-Bound Lactoferrin and TLR4

    Directory of Open Access Journals (Sweden)

    Xiao-Min Hu

    2015-01-01

    Full Text Available Soluble lactoferrin (LTF is a versatile molecule that not only regulates the iron homeostasis, but also harbors direct microbicidal and immunomodulating abilities in mammalian body fluids. In contrast, little is known about the function of membrane-bound LTF (mbLTF, although its expression on human polymorphonuclear leukocytes (huPMNs has been reported for decades. Given that LTF/anti-LTF antibodies represent a potential diagnostic/prognostic biomarker and a therapeutic target in patients with immune disorders, we wished, in the present study, to generate a novel human LTF- (huLTF- specific mAb suitable for detailed analyses on the expression and function of mbLTF as well as for deciphering the underlying mechanisms. By using the traditional hybridoma cell fusion technology, we obtained a murine IgG1 (kappa mAb, M-860, against huLTF. M-860 recognizes a conformational epitope of huLTF as it binds to natural, but not denatured, huLTF in ELISA. Moreover, M-860 detects mbLTF by FACS and captures endogenous huLTF in total cell lysates of huPMNs. Functionally, M-860 induces the activation of huPMNs partially through TLR4 but independently of phagocytosis. M-860 is thus a powerful tool to analyze the expression and function of human mbLTF, which will further our understanding of the roles of LTF in health and disease.

  19. A single dose of neuron-binding human monoclonal antibody improves spontaneous activity in a murine model of demyelination.

    Science.gov (United States)

    Denic, Aleksandar; Macura, Slobodan I; Warrington, Arthur E; Pirko, Istvan; Grossardt, Brandon R; Pease, Larry R; Rodriguez, Moses

    2011-01-01

    Our laboratory demonstrated that a natural human serum antibody, sHIgM12, binds to neurons in vitro and promotes neurite outgrowth. We generated a recombinant form, rHIgM12, with identical properties. Intracerebral infection with Theiler's Murine Encephalomyelitis Virus (TMEV) of susceptible mouse strains results in chronic demyelinating disease with progressive axonal loss and neurologic dysfunction similar to progressive forms of multiple sclerosis. To study the effects of rHIgM12 on the motor function of TMEV-infected mice, we monitored spontaneous nocturnal activity over many weeks. Nocturnal behavior is a sensitive measure of rodent neurologic function because maximal activity changes are expected to occur during the normally active night time monitoring period. Mice were placed in activity boxes eight days prior to treatment to collect baseline spontaneous activity. After treatment, activity in each group was continuously recorded over 8 weeks. We chose a long 8-week monitoring period for two reasons: (1) we previously demonstrated that IgM induced remyelination is present by 5 weeks post treatment, and (2) TMEV-induced demyelinating disease in this strain progresses very slowly. Due to the long observation periods and large data sets, differences among treatment groups may be difficult to appreciate studying the original unfiltered recordings. To clearly delineate changes in the highly fluctuating original data we applied three different methods: (1) binning, (2) application of Gaussian low-pass filters (GF) and (3) polynomial fitting. Using each of the three methods we showed that compared to control IgM and saline, early treatment with rHIgM12 induced improvement in both horizontal and vertical motor function, whereas later treatment improved only horizontal activity. rHIgM12 did not alter activity of normal, uninfected mice. This study supports the hypothesis that treatment with a neuron-binding IgM not only protects neurons in vitro, but also influences

  20. A single dose of neuron-binding human monoclonal antibody improves spontaneous activity in a murine model of demyelination.

    Directory of Open Access Journals (Sweden)

    Aleksandar Denic

    Full Text Available Our laboratory demonstrated that a natural human serum antibody, sHIgM12, binds to neurons in vitro and promotes neurite outgrowth. We generated a recombinant form, rHIgM12, with identical properties. Intracerebral infection with Theiler's Murine Encephalomyelitis Virus (TMEV of susceptible mouse strains results in chronic demyelinating disease with progressive axonal loss and neurologic dysfunction similar to progressive forms of multiple sclerosis. To study the effects of rHIgM12 on the motor function of TMEV-infected mice, we monitored spontaneous nocturnal activity over many weeks. Nocturnal behavior is a sensitive measure of rodent neurologic function because maximal activity changes are expected to occur during the normally active night time monitoring period. Mice were placed in activity boxes eight days prior to treatment to collect baseline spontaneous activity. After treatment, activity in each group was continuously recorded over 8 weeks. We chose a long 8-week monitoring period for two reasons: (1 we previously demonstrated that IgM induced remyelination is present by 5 weeks post treatment, and (2 TMEV-induced demyelinating disease in this strain progresses very slowly. Due to the long observation periods and large data sets, differences among treatment groups may be difficult to appreciate studying the original unfiltered recordings. To clearly delineate changes in the highly fluctuating original data we applied three different methods: (1 binning, (2 application of Gaussian low-pass filters (GF and (3 polynomial fitting. Using each of the three methods we showed that compared to control IgM and saline, early treatment with rHIgM12 induced improvement in both horizontal and vertical motor function, whereas later treatment improved only horizontal activity. rHIgM12 did not alter activity of normal, uninfected mice. This study supports the hypothesis that treatment with a neuron-binding IgM not only protects neurons in vitro, but

  1. Systemic CD8+ T cell-mediated tumoricidal effects by intratumoral treatment of oncolytic herpes simplex virus with the agonistic monoclonal antibody for murine glucocorticoid-induced tumor necrosis factor receptor.

    Directory of Open Access Journals (Sweden)

    Mikiya Ishihara

    Full Text Available Oncolytic virotherapy combined with immunomodulators is a novel noninvasive strategy for cancer treatment. In this study, we examined the tumoricidal effects of oncolytic HF10, a naturally occurring mutant of herpes simplex virus type-1, combined with an agonistic DTA-1 monoclonal antibody specific for the glucocorticoid-induced tumor necrosis factor receptor. Two murine tumor models were used to evaluate the therapeutic efficacies of HF10 virotherapy combined with DTA-1. The kinetics and immunological mechanisms of DTA-1 in HF10 infection were examined using flow cytometry and immunohistochemistry. Intratumoral administration of HF10 in combination with DTA-1 at a low dose resulted in a more vigorous attenuation of growth of the untreated contralateral as well as the treated tumors than treatment with either HF10 or DTA-1 alone. An accumulation of CD8(+ T cells, including tumor- and herpes simplex virus type-1-specific populations, and a decrease in the number of CD4(+ Foxp3(+ T regulatory cells were seen in both HF10- and DTA-1-treated tumors. Studies using Fc-digested DTA-1 and Fcγ receptor knockout mice demonstrated the direct participation of DTA-1 in regulatory T cell depletion by antibody-dependent cellular cytotoxicity primarily via macrophages. These results indicated the potential therapeutic efficacy of a glucocorticoid-induced tumor necrosis factor receptor-specific monoclonal antibody in oncolytic virotherapy at local tumor sites.

  2. Non-depleting anti-CD4 monoclonal antibody induces immune tolerance to ERT in a murine model of Pompe disease

    Directory of Open Access Journals (Sweden)

    Baodong Sun

    2014-01-01

    Full Text Available Approximately 35–40% of patients with classic infantile Pompe disease treated with enzyme replacement therapy (ERT develop high, sustained antibody titers against the therapeutic enzyme alglucosidase alfa, which abrogates the treatment efficacy. Induction of antigen-specific immune tolerance would greatly enhance ERT for these patients. Here we show that a short-course treatment with non-depleting anti-CD4 monoclonal antibody successfully induced long-term ERT-specific immune tolerance in Pompe disease mice. Our data suggest an effective adjuvant therapy to ERT.

  3. Monoclonal antibodies specific for oncofetal antigen – immature laminin receptor protein: Effects on tumor growth and spread in two murine models

    OpenAIRE

    McClintock, Shannon D.; Warner, Roscoe L.; Ali, Saqib; Chekuri, Apurupa; Dame, Michael K.; Attili, Durga; Knibbs, Randall K; Aslam, Muhammad Nadeem; Sinkule, Joseph; Morgan, Alton Charles; Barsoum, Adel; Smith, Lauren B.; Beer, David G.; Johnson, Kent J.; Varani, James

    2015-01-01

    The oncofetal antigen – immature laminin receptor protein (OFA/iLRP) has been linked to metastatic tumor spread for several years. The present study, in which 2 highly-specific, high-affinity OFA/iLRP-reactive mouse monoclonal antibodies were examined for ability to suppress tumor cell growth and metastatic spread in the A20 B-cell leukemia model and the B16 melanoma model, provides the first direct evidence that targeting OFA/iLRP with exogenous antibodies can have therapeutic benefit. While...

  4. Interference in a CA 125 assay by human anti-mouse antibodies (HAMA) in patients imaged with the murine monoclonal antibody OC 125

    International Nuclear Information System (INIS)

    The appearance of human anti-mouse antibodies (HAMA) in the sera of patients after injection of MAb OC 125 was evaluated in 11 adenocarcinoma patients (10 ovarian and 1 fallopian tube) who received 1 mg of 131I labeled OC 125 F(ab')2 for immunoscintigraphy. HAMA levels were determined using a two step immunoradiometric assay (Biomira) employing OC 125 (F(ab')2 coated wells and 125I labeled goat anti-human IgG. CA 125 levels were also assessed and were measured using the ELSA CA 125 assay (ID-CIS, Dreieich, West Germany). In four of these 11 patients, a second injection of OC 125 was performed due to suspicion of recurrent disease. Thereafter a significant rise in HAMA levels corresponded with a sudden increase in CA 125 serum levels was recorded. In addition, it is mentioned that other serum assays (FSH, LH) performed at the same time, using kits based on murine MAb, demonstrated a similar effect. It was concluded that these increases were caused by interference of HAMA with the murine MAb. 21 refs., 2 tabs., 1 fig

  5. Monoclonal antibodies specific for oncofetal antigen – immature laminin receptor protein: Effects on tumor growth and spread in two murine models

    Science.gov (United States)

    McClintock, Shannon D; Warner, Roscoe L; Ali, Saqib; Chekuri, Apurupa; Dame, Michael K; Attili, Durga; Knibbs, Randall K; Aslam, Muhammad Nadeem; Sinkule, Joseph; Morgan, Alton Charles; Barsoum, Adel; Smith, Lauren B; Beer, David G; Johnson, Kent J; Varani, James

    2015-01-01

    The oncofetal antigen – immature laminin receptor protein (OFA/iLRP) has been linked to metastatic tumor spread for several years. The present study, in which 2 highly-specific, high-affinity OFA/iLRP-reactive mouse monoclonal antibodies were examined for ability to suppress tumor cell growth and metastatic spread in the A20 B-cell leukemia model and the B16 melanoma model, provides the first direct evidence that targeting OFA/iLRP with exogenous antibodies can have therapeutic benefit. While the antibodies were modestly effective at preventing tumor growth at the primary injection site, both antibodies strongly suppressed end-organ tumor formation following intravenous tumor cell injection. Capacity of anti-OFA/iLRP antibodies to suppress tumor spread through the blood in the leukemia model suggests their use as a therapy for individuals with leukemic disease (either for patients in remission or even as part of an induction therapy). The results also suggest use against metastatic spread with solid tumors. PMID:25799942

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

    International Nuclear Information System (INIS)

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

  7. In vitro characterization of an iodine-125 labeled anti-epidermal growth factor receptor murine monoclonal antibody (MAB-425) in human high grade glioma cells: Binding, uptake, transport and localization

    International Nuclear Information System (INIS)

    The aim of this study was to determine intracellular accumulation and possible nuclear translocation of 125I-labeled murine monoclonal antibody (MAb) 425 in human high grade glioma cells following internalization of this antibody, a prerequisite for the induction of radiotoxic effects. Four human high grade glioma cell lines known to express epidermal growth factor receptor (EGF-R) and a colorectal carcinoma cell line with negligible EGF-R expression were incubated for various time periods with a saturating concentration of 125I-MAb 425. No measurable intranuclear uptake of radioactivity was detected within an incubation period of less than 6 hr. After an incubation of 24-28 hr, only 0.5-2% of the internalized radioactivity was detected in the nuclear fraction. It may be possible to inhibit 125I-MAb 425 degradation with the addition of chloroquine. Overall, the data indicate that the major portion of the 125I-MAb 425 is degraded following internalization. Inhibition of lysosomal activity results in a significant increase in intracellular, but more importantly, intranuclear accumulation of 125I-MAb 425. Since radioactivity is released from the glioma cells following 125I-MAb 425 internalization, discontinuous gel electrophoresis was performed to determine the nature of the released cell products. The cell supernatants were examined at 0, 24 and 48 hr post incubation to determine the nature of any cell-associated radioactivity released from the cell surface or interior. A protein band corresponding to a molecular weight of 167 kDa was detected in all cell supernatants. Parallel electrophoretic gels were processed for autoradiographic studies in order to indicate corresponding radioactive components. In addition, immunostaining by Western blot with peroxidase-labeled goat anti-mouse antibody was performed to confirm the antibody nature of the protein bands

  8. In vitro characterization of an iodine-125 labeled anti-epidermal growth factor receptor murine monoclonal antibody (MAB-425) in human high grade glioma cells: Binding, uptake, transport and localization

    Energy Technology Data Exchange (ETDEWEB)

    Emrich, J.G.

    1993-01-01

    The aim of this study was to determine intracellular accumulation and possible nuclear translocation of [sup 125]I-labeled murine monoclonal antibody (MAb) 425 in human high grade glioma cells following internalization of this antibody, a prerequisite for the induction of radiotoxic effects. Four human high grade glioma cell lines known to express epidermal growth factor receptor (EGF-R) and a colorectal carcinoma cell line with negligible EGF-R expression were incubated for various time periods with a saturating concentration of [sup 125]I-MAb 425. No measurable intranuclear uptake of radioactivity was detected within an incubation period of less than 6 hr. After an incubation of 24-28 hr, only 0.5-2% of the internalized radioactivity was detected in the nuclear fraction. It may be possible to inhibit [sup 125]I-MAb 425 degradation with the addition of chloroquine. Overall, the data indicate that the major portion of the [sup 125]I-MAb 425 is degraded following internalization. Inhibition of lysosomal activity results in a significant increase in intracellular, but more importantly, intranuclear accumulation of [sup 125]I-MAb 425. Since radioactivity is released from the glioma cells following [sup 125]I-MAb 425 internalization, discontinuous gel electrophoresis was performed to determine the nature of the released cell products. The cell supernatants were examined at 0, 24 and 48 hr post incubation to determine the nature of any cell-associated radioactivity released from the cell surface or interior. A protein band corresponding to a molecular weight of 167 kDa was detected in all cell supernatants. Parallel electrophoretic gels were processed for autoradiographic studies in order to indicate corresponding radioactive components. In addition, immunostaining by Western blot with peroxidase-labeled goat anti-mouse antibody was performed to confirm the antibody nature of the protein bands.

  9. 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

    2006-06-01

    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

  10. Monoclonal antibodies against chicken interleukin-6

    Science.gov (United States)

    Monoclonal antibodies (mAb) were produced against a recombinant (r) chicken interleukin-6 (IL-6). Eight mAbs that were produced were tested for isotype; ability to inhibit recombinant forms of chicken (ch), human (h) and murine (m) IL-6; and recognition of rchIL-6 by Western immunoblotting. The mA...

  11. Application of Monoclonal Antibodies in Veterinary Parasitology

    Directory of Open Access Journals (Sweden)

    Gupta A.

    2011-08-01

    Full Text Available The discovery of hybridoma technology by Kohler and Milstein in 1975, heralded a new era in antibody research. Mouse hybridomas were the first reliable source of monoclonal antibodies. The generation of monoclonal antibodies from species other than rats and mice, has developed slowly over the last 30 years. The advent of antibody engineering and realization of the advantages of non murine antibodies has increased their relevance recently. However, in the area of veterinary parasitology, monoclonal antibodies are just beginning to fulfill the promises inherent in their great specificity for recognizing and selectively binding to antigens. This review describes the recent advances in the application of monoclonal antibodies for immunodiagnosis / prophylaxis and immunotherapy of parasitic diseases. [Vet. World 2011; 4(4.000: 183-188

  12. Monoclonal antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MAbs) are antibodies having single specificity for a given antigen site (epitope). The development of hybridoma technology and the relative ease by which MAbs can be prepared has revolutionized many aspects of serological applications in diagnosis and differentiation of disease producing agents. The property of monospecificity offers advantages in diagnostic applications over polyclonal sera in that tests can be defined exactly with regard to the antigen detected and the affinity of reaction between the given antigenic site and the monoclonal reagent. In addition, MAbs offer better possibilities for test standardization, because the same reagent can be used in different laboratories. Such an MAb can be supplied by a central laboratory or 'grown' from hybridoma cells, ensuring that the resultant product is identical from laboratory to laboratory and that the part of the test involving the MAb reaction is the same. The methodologies for inoculation regimes, mice, cloning methods, selection of fusion partners, etc., have been validated extensively in developed country laboratories. The decision to establish a MAb production facility must be examined on a strict cost-benefit basis, since it is still expensive to produce a product. There are many MAbs available that should be sought to allow exploitation in developing tests. If a production facility is envisaged, it should produce reagents for national needs, i.e. there should be a clear problem oriented approach whereby exact needs are defined. In the field of veterinary applications, MAbs are the central reagent in many immunoassays based on the enzyme linked immunosorbent assay (ELISA). The development of specific tests for diagnosing diseases is dominated by MAbs and has been fuelled by a strong research base, mainly in developed countries allied to developing countries through the study of related diseases. Thus, there are very many assays dependent on MAbs, some of which form the basis of

  13. Monoclonal antibodies.

    Science.gov (United States)

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  14. Monoclonal antibodies for treating cancer

    International Nuclear Information System (INIS)

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

  15. A monoclonal antibody against leptin.

    Science.gov (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin. PMID:23098305

  16. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents

    OpenAIRE

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-01-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine m...

  17. Inhibitory mechanism of peptides and antibodies targeting murine urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Liu, Zhuo

    2012-01-01

    high affinity and specificity of mupain-1-16 makes it a suitable inhibitor for targeting of murine uPA in order to investigate the importance of uPA in murine disease models. Secondly, two high affinity monoclonal antibodies targeting murine uPA (mU1 and mU3) were studied. These antibodies showed...... be important for future tumour model studies and the development of more efficient inhibitors against uPA....

  18. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    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…

  19. Monoclonal antibody analysis of specific antigenic similarities among pathogenic Treponema pallidum subspecies.

    OpenAIRE

    Marchitto, K S; Jones, S A; Schell, R F; Holmans, P L; Norgard, M V

    1984-01-01

    Murine monoclonal antibodies directed against a 47,000-dalton immunodominant surface-exposed antigen of Treponema pallidum subsp. pallidum (Nichols) were isolated. These monoclonal antibodies cross-reacted with analogous 47,000-dalton antigens of two other virulent treponemes, T. pallidum subsp. pertenue and T. pallidum subsp. endemicum (Bosnia A), as determined by radioimmunoassay and immunoblot analyses. Immunoelectron microscopy confirmed that the 47,000-dalton antigen of T. pallidum subsp...

  20. PRODUCTION OF MONOCLONAL ANTIBODIES

    Directory of Open Access Journals (Sweden)

    TOLKOVA E.S.

    2015-01-01

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

  1. PRODUCTION OF MONOCLONAL ANTIBODIES

    OpenAIRE

    TOLKOVA E.S.

    2015-01-01

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

  2. Monoclonal antibodies and cancer

    International Nuclear Information System (INIS)

    The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111In, 67Ga and 131I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

  3. Administration of an anti-interleukin 2 receptor monoclonal antibody prolongs cardiac allograft survival in mice

    OpenAIRE

    1985-01-01

    Administration of the monoclonal antibody M7/20, which binds to the murine interleukin-2 (IL) receptor, significantly prolongs cardiac allograft survival in two H-2-incompatible strain combinations of inbred mice. The results support the important role of the IL-2 receptor in the mechanism of graft rejection, and suggest its suitability as a target for immunosuppressive therapy.

  4. 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; Gottschalk, M.

    1996-01-01

    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...

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    97%]) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal...... antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application...... for the in situ diagnosis of systemic bovine zygomycosis....

  6. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

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

    2015-01-01

    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. Monoclonal antibody as radiopharmaceutical

    International Nuclear Information System (INIS)

    The purification of anti-CEA monoclonal antibody 4C11 belonging to IgG sub(2a) subclass from mouse ascitis, donated by Ludwig Institute, Brazil was developed. The fragmentation of purified IgG sub(2a) by pepsin digestion and analytical studies by polyacrilamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) were done as preliminary assessment for their specific application in immunoscintigraphy. (author)

  8. Monoclonal antibody against a genus-specific antigen of Chlamydia species: location of the epitope on chlamydial lipopolysaccharide.

    OpenAIRE

    Caldwell, H D; Hitchcock, P J

    1984-01-01

    Monoclonal antibodies were prepared by the fusion of murine myeloma NS1 cells with spleen cells of BALB/c mice immunized with Formalin-killed elementary bodies of the Chlamydia trachomatis L2 serovar. The specificity of these monoclonal antibodies was determined with a solid-phase immunoassay in which HeLa 229 cells infected with C. trachomatis serovars D, G, H, I, L2 and the Chlamydia psittaci meningopneumonitis strain Cal-10 were used. An immunoglobulin G3 monoclonal antibody (L2I-6) was id...

  9. Monoclonal antibody against a Burkitt lymphoma-associated antigen.

    OpenAIRE

    Wiels, J; Fellous, M.; Tursz, T

    1981-01-01

    A monoclonal antibody, referred to as 38.13, was obtained by fusing murine myeloma cells with Lewis rat splenocytes sensitized with Daudi cells (human Burkitt lymphoma containing Epstein--Barr virus genome but lacking HLA-A, -B, and -C and beta 2-microglobulin molecules at the cell surface). 38.13 antibody was demonstrated to be a rat IgM. By complement-dependent microcytotoxicity and indirect immunofluorescence assays, 38.13 antibody was shown to react specifically with cells derived from Bu...

  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

    1987-01-01

    (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...... human beta 2-m; and the modifying activity of murine MLC responder cells was blocked in an intermediary step by an alloantibody, which reacts specifically with murine major histocompatibility complex, class I-associated beta 2-m. These findings suggest that the modification process is preceded by an...

  11. Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices

    OpenAIRE

    Moreno Tamarit, Mª José; PLANA ANDANI, EMMA; Manclus Ciscar, Juan José; Montoya Baides, Ángel

    2014-01-01

    [EN] A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against (RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl-1,1,2,2-tetrafluoroethyl ether (tetraconazole), 2-(4-triazolyl)benzimidazole (thiabendazole), and (RS)-1-(be...

  12. Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy

    OpenAIRE

    1983-01-01

    Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

  13. Monoclonal antibodies to Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Halpern, J L; Lundgren, B; Swan, J C; Parrillo, J E; Masur, H

    1989-01-01

    To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii...... reacted with human P. carinii, and none of four monoclonal antibodies to human P. carinii reacted with rat P. carinii. Two antibodies to human P. carinii reacted by immunofluorescence with only one human P. carinii isolate. Immunoblot studies identified major antigens of rat P. carinii with molecular...

  14. Monoclonal gammopathy associated with visceral leishmaniasis

    Directory of Open Access Journals (Sweden)

    Vishal Sharma

    2010-06-01

    Full Text Available Monoclonal gammopathy can accompany diverse conditions and is usually benign. It should be distinguished from monoclonal gammopathy of undetermined significance (MGUS which can rarely turn malignant. Visceral leishmaniasis has only rarely been associated with monoclonal gammopathy. We describe the case of a 55-year-old male who had monoclonal gammopathy associated with visceral leishmanisais, which reversed with stibogluconate therapy.

  15. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

    OpenAIRE

    Broze, G J; S. Hickman; Miletich, J P

    1985-01-01

    Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

  16. Coeliac disease: characterisation of monoclonal antibodies raised against a synthetic peptide corresponding to amino acid residues 206-217 of A-gliadin.

    OpenAIRE

    Ellis, H. J.; Doyle, A. P.; Sturgess, R P; Ciclitira, P J

    1992-01-01

    A dodecapeptide of A-gliadin, which shares amino acid homologies with the E1b protein of adenovirus 12, was used to produce murine monoclonal antibodies. Five monoclonal antibodies were produced and were screened by enzyme linked immunosorbant assay, immunodot assay, and immunoblotting. The antibodies were tested against whole wheat gliadin and its alpha, beta, gamma, and omega subfractions, and the prolamins of rye, barley, oats, maize, millet, rice, and sorghum. Four of the five antibodies ...

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

    OpenAIRE

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

    1990-01-01

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

  18. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    OpenAIRE

    Hehle, Verena K; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2015-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    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, 5.1...... in the IFAT in a similar way to the Danish reference strain of T. gondii, SSI-119....

  20. Monoclonal antibody against Klebsiella capsular polysaccharide reduces severity and hematogenic spread of experimental Klebsiella pneumoniae pneumonia.

    OpenAIRE

    Held, T K; Trautmann, M.; Mielke, M E; Neudeck, H; Cryz, S J; Cross, A S

    1992-01-01

    Klebsiella pneumoniae is an important nosocomial pathogen causing severe pulmonary infections. The majority of clinical Klebsiella isolates produce a high-molecular-weight capsular polysaccharide (CPS) which is one of the dominant virulence factors. In the present study, we examined the potency of a murine immunoglobulin M monoclonal antibody (MAb) with specificity to Klebsiella type 2 CPS to protect rats against experimental Klebsiella pneumonia. The MAb did not prevent the invasion of virul...

  1. Detection of complement activation using monoclonal antibodies against C3d

    OpenAIRE

    Thurman, Joshua M.; Kulik, Liudmila; Orth, Heather; Wong, Maria; Renner, Brandon; Sargsyan, Siranush A.; Mitchell, Lynne M.; Hourcade, Dennis E.; Hannan, Jonathan P.; Kovacs, James M.; Coughlin, Beth; Woodell, Alex S.; Pickering, Matthew C.; Rohrer, Bärbel; Holers, V. Michael

    2013-01-01

    During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monocl...

  2. Monoclonal antibody therapy for Junin virus infection.

    Science.gov (United States)

    Zeitlin, Larry; Geisbert, Joan B; Deer, Daniel J; Fenton, Karla A; Bohorov, Ognian; Bohorova, Natasha; Goodman, Charles; Kim, Do; Hiatt, Andrew; Pauly, Michael H; Velasco, Jesus; Whaley, Kevin J; Altmann, Friedrich; Gruber, Clemens; Steinkellner, Herta; Honko, Anna N; Kuehne, Ana I; Aman, M Javad; Sahandi, Sara; Enterlein, Sven; Zhan, Xiaoguo; Enria, Delia; Geisbert, Thomas W

    2016-04-19

    Countermeasures against potential biothreat agents remain important to US Homeland Security, and many of these pharmaceuticals could have dual use in the improvement of global public health. Junin virus, the causative agent of Argentine hemorrhagic fever (AHF), is an arenavirus identified as a category A high-priority agent. There are no Food and Drug Administration (FDA) approved drugs available for preventing or treating AHF, and the current treatment option is limited to administration of immune plasma. Whereas immune plasma demonstrates the feasibility of passive immunotherapy, it is limited in quantity, variable in quality, and poses safety risks such as transmission of transfusion-borne diseases. In an effort to develop a monoclonal antibody (mAb)-based alternative to plasma, three previously described neutralizing murine mAbs were expressed as mouse-human chimeric antibodies and evaluated in the guinea pig model of AHF. These mAbs provided 100% protection against lethal challenge when administered 2 d after infection (dpi), and one of them (J199) was capable of providing 100% protection when treatment was initiated 6 dpi and 92% protection when initiated 7 dpi. The efficacy of J199 is superior to that previously described for all other evaluated drugs, and its high potency suggests that mAbs like J199 offer an economical alternative to immune plasma and an effective dual use (bioterrorism/public health) therapeutic. PMID:27044104

  3. Occult choriocarcinoma: Detection using radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Occult choriocarcinoma, manifested only by an elevated B-hCG level, can be a difficult management problem. The authors evaluated the ability of I-131-labeled 5F9.3, a murine monoclonal antibody reactive with choriocarcinomas but not hCG, to detect foci of choriocarcinoma in five patients referred with elevated B-hCG levels but in whom the location of residual disease was uncertain. I-131 5F9.3, 0.5-1.0 mCi, was injected intravenously in each patient and images with dynamic background subtraction of TcHSA were obtained at later time points. In four patients chest studies were true positive (confirmed surgically in all), the chest CT scans in these patients had been interpreted as not definitely showing active disease. In the fifth patient no abnormal focus of uptake was seen and subsequent B-hCG levels normalized. In two of the patients with chest lesions, foci of abdominal uptake were seen that were not due to tumor. One of these patients had a partial small bowel obstruction; the other appeared to have a false-positive study. I-131 5F9.3 is a promising agent for the detection of occult choriocarcinomas

  4. Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

    Science.gov (United States)

    Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

    2016-08-25

    Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes. PMID:27330002

  5. Uses of monoclonal antibody 8H9

    Science.gov (United States)

    Cheung, Nai-Kong V.

    2013-04-09

    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.

  6. Detection of Campylobacter species using monoclonal antibodies

    Science.gov (United States)

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

    1999-01-01

    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.

  7. Phase 1 study of monoclonal antibody I-131 3F8 targeted radiation therapy of human neuroblastoma

    International Nuclear Information System (INIS)

    This study is phase I of monoclonal antibody 131-I 3F8 targeted radiotherapy of human neuroblastoma. A murine IgG3 monoclonal antibody specific for ganglioside GD2(3F8), has unusually high (0.08% ID/g) tumor localization in patients and restricted distribution in normal tissues. Nine patients with refractory neuroblastoma (seven with soft tissue masses, four with bone disease, and three with bone marrow disease) have been treated with intravenous I-131 3F8 (10 μg 3F8) and oral saturated solution of potassium iodide and potassium perchlorate. Results are presented

  8. Preparation and Evaluation of Norcantharidin-encapsulated Liposomes Modified with a Novel CD19 Monoclonal Antibody 2E8

    Institute of Scientific and Technical Information of China (English)

    张晶樱; 汤永民; 钱柏芹; 沈红强

    2010-01-01

    In this study,norcanthridin(NCTD)-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8(2E8-NCTD-liposomes) and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19+ leukemia cells were evaluated.BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody(mAb).NCTD-liposomes were prepared by using film dispersion method.2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology.Flow cytometry show...

  9. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

    Science.gov (United States)

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

    1981-05-01

    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.

  10. Radiolabeled monoclonal antibodies: a review

    International Nuclear Information System (INIS)

    Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author)

  11. Tumor imaging with monoclonal antibodies

    International Nuclear Information System (INIS)

    Many monoclonal antibodies directed against tumor-associated antigens have been identified, but so far none of these are tumor specific. Polyclonal and monoclonal antibodies have been used for imaging of a wide variety of tumors with success. Radiolabeling of antibody is usually done with iodine isotopes of which 123I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111In, which is the best radioisotope for imaging with monoclonal antibodies due to its favorable half-life of 2.5 days. Usually imaging cannot be performed within 24 h after injection, but clearance of antibody can be increased by using F(ab)2 of Fab. Another approach is to clear non-bound antibody by a second antibody, directed against the first. The detection limit of immunoimaging is about 2 cm, but will be improved by tomography or SPECT. There is still a high false positive and false negative rate, which makes it impossible to use radioimmunodetection as the only technique for diagnosis of tumors. In combination with other detection techniques, tumor imaging with monoclonal antibodies can improve diagnosis. 44 refs.; 3 tabs

  12. Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

    Science.gov (United States)

    De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G

    1993-04-01

    The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

  13. Anti-CD45 Radioimmunotherapy with 90Y but Not 177Lu Is Effective Treatment in a Syngeneic Murine Leukemia Model

    OpenAIRE

    Orozco, Johnnie J.; Ethan R. Balkin; Gooley, Ted A.; Kenoyer, Aimee; Hamlin, Donald K.; Wilbur, D. Scott; Fisher, Darrell R.; Hylarides, Mark D.; Shadman, Mazyar; Green, Damian J.; Gopal, Ajay K.; Press, Oliver W.; Pagel, John M.

    2014-01-01

    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 CD4...

  14. Monoclonal antibodies technology. Protocols

    International Nuclear Information System (INIS)

    Full text: Immunization. The first step in preparing useful monoclonal antibodies (MAbs) is to immunize an animal (Balb/c for example) with an appropriate antigen. Methods (only for soluble antigen): Solubilize selected antigen in Phosphate buffer solution (PBS) at pH 7.2-7.4, ideally at a final concentration per animal between 10 to 50 μg/ml. It is recommended that the antigen under consideration be incorporated into the emulsion adjuvants in 1:1 volumetric relation. We commonly use Frend's adjuvant (FA) to prepared immunized solution. The first immunization should be prepared with complete FA, and the another could be prepared with incomplete FA. It is recommended to inject mice with 0.2 ml intraperitoneal (ip) or subcutaneous (sc). Our experience suggests the sc route is the preferred route. A minimum protocol for immunizing mice to generate cells for preparing hybridomas is s follows: immunize sc on day 0, boost sc on day 21, take a trial bleeding on day 26; if antibody titters are satisfactory, boost ip on day 35 with antigen only, and remove the spleen to obtain cells for fusion on day 38. Fusion protocol. The myeloma cell line we are using is X63 Ag8.653. At the moment of fusion myeloma cells need a good viability (at least a 95%). 1. Remove the spleen cells from immunized mice using sterile conditions. An immune spleen should yield between 7 a 10x107 nucleated cells. 2. Place the spleen in 20 ml of serum-free RPMI 1640 in a Petri dish. Using a needle and syringe, inject the spleen with medium to distend and disrupt the spleen stroma and free the nucleated cells. 3. Flush the cell suspension with a Pasteur pipet to disperse clumps of cells. 4. Centrifuge the spleen cell suspension at 250g for 10 min. Resuspend the pellet in serum-free RPMI 1640. Determine cell concentration using Neuhabuer chamber. 5. Mix the myeloma cells and spleen cells in a conical 50-ml tube in serum-free RPMI 1640, 1 x107 spleen cells to 1x106 myeloma cells (ratio 10:1). Centrifuge

  15. Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

    Science.gov (United States)

    Hongo, J A; Tsai, S P; Moffat, B; Schroeder, K A; Jung, C; Chuntharapai, A; Lampe, P A; Johnson, E M; de Sauvage, F J; Armanini, M; Phillips, H; Devaux, B

    2000-08-01

    Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors. PMID:11001403

  16. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody.

    OpenAIRE

    Barbour, A. G; Tessier, S L; Todd, W J

    1983-01-01

    Ixodid tick-associated spirochetes have been implicated as the etiological agents of Lyme disease. We raised a murine monoclonal antibody (H5332) against a spirochete, strain B31, isolated from Ixodes dammini ticks. In indirect immunofluorescence assays and western blot analyses, H5332 reacted with whole cells or isolated components of not only strain B31 but also spirochetes isolated from Ixodes ricinus ticks, a field mouse, a raccoon, and patients with Lyme disease. In contrast, H5332 did n...

  17. Use of monoclonal antibodies for the characterization of novel DNA- binding proteins recognized by human autoimmune sera

    OpenAIRE

    1985-01-01

    Autoantibodies to a DNA-binding heterodimer consisting of 70,000 and 80,000 dalton subunits were identified in 30-50% of human autoimmune sera from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), and scleroderma. Three murine monoclonal antibodies (mAb) against the heterodimer were produced in BALB/c mice by immunizing with isolated human B cell nuclei. By immunofluorescence, the mAb and autoimmune sera demonstrated both speckled nucleoplasmic stainin...

  18. Monoclonal Antibody Therapies against Anthrax

    OpenAIRE

    Zhaochun Chen; Mahtab Moayeri; Robert Purcell

    2011-01-01

    Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. It not only causes natural infection in humans but also poses a great threat as an emerging bioterror agent. The lethality of anthrax is primarily attributed to the two major virulence factors: toxins and capsule. An extensive effort has been made to generate therapeutically useful monoclonal antibodies to each of the virulence components: protective antigen (PA), lethal factor (LF) and ede...

  19. 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

    2009-11-01

    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

  20. Monoclonal antibodies in targeted therapy

    Directory of Open Access Journals (Sweden)

    Beata Powroźnik

    2012-09-01

    Full Text Available Targeted therapy is a new therapeutic method consisting in the inhibition of specific molecular pathways. In modern therapy, the key role is played by monoclonal antibodies, included in the group of biological agents. The success of molecularly targeted therapy is to define the proper “molecular target”, selecting the right drug active against a specific “target” and selecting a group of patients who benefit from treatment. Introduction of targeted therapy resulted in improved results of the treatment of many serious and chronic diseases. In general, targeted molecular therapies have good toxicity profiles, but some patients are exquisitely sensitive to these drugs and can develop particular and severe toxicities. Patient selection and proper monitoring significantly decrease the risk of life-threatening adverse events. Data concerning late side effects are still unavailable because of the short follow-up of molecularly targeted therapy. Currently in the U.S. and Europe there are approximately 31 registered therapeutic monoclonal antibodies, while 160 are subjected to clinical trials. This paper presents an overview of therapeutic monoclonal antibodies currently used in therapy and the present state of knowledge about them. 

  1. 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

    2013-01-01

    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

  2. Monoclonal antibodies against antigens displayed on a progressively growing mammary tumor.

    OpenAIRE

    Tax, A; Manson, L A

    1981-01-01

    We have produced lymphocyte hybridomas between mouse myeloma cells and either spleen cells of C3H/f/C57BL/6 mice bearing the Mm5mt/c1 tumor-producing murine mammary tumor virus (MMTV) or spleen cells from Fisher rats inoculated with the same tumor. Two classes of hybridoma-secreted monoclonal antibodies were obtained. In the first class are IVC11, IIIA1, and VE7, each of which precipitated a 52,000-dalton protein from 125I-labeled purified preparations of MMTV and [3H]glucosamine-labeled Mm5m...

  3. Monoclonal Antibodies for Lipid Management.

    Science.gov (United States)

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

    In recent years, biochemical and genetic studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) as a major mediator of low-density lipoprotein cholesterol (LDL-c) levels and thereby a potential novel target for reducing risk of coronary heart disease (CHD). These observations led to the development of PCSK9 inhibitors, which lower LDL-c levels more than any other non-invasive lipid-lowering therapy presently available. The PCSK9 inhibitors furthest along in clinical trials are subcutaneously injected monoclonal antibodies. These PCSK9 inhibitors have demonstrated LDL-c-lowering efficacy with acceptable safety in phase III clinical trials and may offer a useful therapy in addition to maximally tolerated HMG-CoA reductase inhibitors (statins) in certain patient groups. Longer-term data are required to ensure sustained efficacy and safety of this new class of medications. This review provides an overview of the biology, genetics, development, and clinical trials of monoclonal antibodies designed to inhibit PCSK9. PMID:27221501

  4. Radioimmunoguided surgery using monoclonal antibody

    International Nuclear Information System (INIS)

    The potential proficiency of radioimmunoguided surgery in the intraoperative detection of tumors was assessed using labeled monoclonal antibody B72.3 in 66 patients with tissue-proved tumor. Monoclonal antibody B72.3 was injected 5 to 42 days preoperatively, and the hand-held gamma-detecting probe was used intraoperatively to detect the presence of tumor. Intraoperative probe counts of less than 20 every 2 seconds, or tumor-to-adjacent normal tissue ratios less than 2:1 were considered negative (system failure). Positive probe counts were detected in 5 of 6 patients with primary colon cancer (83 percent), in 31 of 39 patients with recurrent colon cancer (79 percent), in 4 of 5 patients with gastric cancer (80 percent), in 3 of 8 patients with breast cancer (37.5 percent), and in 4 of 8 patients with ovarian cancer (50 percent) undergoing second-look procedures. Additional patients in each group were scored as borderline positive. Overall, radioimmunoguided surgery using B72.3 identified tumors in 47 patients (71.2 percent), bordered on positive in 6 patients (9.1 percent), and failed to identify tumor in 13 patients (19.7 percent). Improved selection of patients for antigen-positive tumors, the use of higher affinity second-generation antibodies, alternate routes of antibody administration, alternate radionuclides, and more sophisticatedly bioengineered antibodies and antibody combinations should all lead to improvements in radioimmunoguided surgery

  5. Monoclonal antibodies to Leptospira interrogans serovar pomona.

    OpenAIRE

    Ainsworth, A J; Lester, T L; Capley, G

    1985-01-01

    Three monoclonal antibodies produced against Leptospira interrogans serovar pomona have been studied for their diagnostic usefulness. All three monoclonals reacted strongly in the enzyme-linked immunosorbent assay and indirect fluorescent antibody test with serovar pomona and did not react with serovars grippotyphosa, canicola, icterohaemorrhagiae and hardjo.

  6. Cuban Monoclonal Antibodies for Radioimmunodiagnosis and Radioimmunotherapy of Cancer Diseases

    International Nuclear Information System (INIS)

    The Centre of Molecular Immunology produces monoclonal antibodies for treating cancer diseases. We are mainly focus on two target systems; one is the epidermal growth factor receptor (EGF-R) because there is a tremendous relationship between the EGF/EGF-R system and several human tumours such as lung, head and neck, ovarian breast and brain cancers; the second one is the ganglioside system, the relevance of certain gangliosides in tumour growth and metastatic dissemination has been well documented, GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues. Nimotuzumab (h-R3) is a humanized monoclonal antibody (mAb) that was obtained by complementarity-determining regions grafting of a murine mAb (ior egf/r3) to a human framework having remarkable antiproliferative, pro-apoptotic, and antiangiogenic effects. A Phase I clinical trial was performed to evaluate the toxicity and clinical effect of an intracavitary (intracerebral) administration of a single dose of nimotuzumab (h-R3) labelled with increasing doses of 188Re. All patients bearing astrocytomas grade III/IV should be treated previously with conventional therapies and have an EGF-R overexpression in the tumour, demonstrated by immunohistochemical study. Maximal tolerated dose was 3 mg of the h-R3 labelled with 10 mCi of 188Re. The radioimmunoconjugate showed a high retention in the surgical created resection cavity and the brain adjacent tissues with a mean value of 85.5% of the injected dose one hour post-administration. This radioimmunoconjugate may be relatively safe and a promising therapeutic approach for treating high grade gliomas. GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues according to immunohistochemical studies, using either polyclonal or monoclonal antibodies. But both immunohistochemical and biochemical methods have strongly suggested its over-expression in human breast and colon

  7. Selection of monoclonal antibody E48 IgG or U36 IgG for adjuvant radioimmunotherapy in head and neck cancer patients.

    OpenAIRE

    van Bree, R.; Roos, J C; Plaizier, M. A.; Quak, J.J.; Van Kamp, G J; den Hollander, W.; Snow, G. B.; van Dongen, G. A.

    1997-01-01

    Preliminary data from recent clinical radioimmunoscintigraphy studies indicate that 99mTc-labelled murine monoclonal antibodies (MAbs) E48 and U36 have a similar ability to target squamous cell carcinoma of the head and neck (HNSCC) selectively. In the present study we describe additional aspects of murine and chimeric MAb (mMAb and cMAb) E48 and U36, which might influence the selection of one MAb for adjuvant radioimmunotherapy. To make direct comparison possible, ten patients received 11.2 ...

  8. Capto MMC mixed-mode chromatography of murine and rabbit antibodies.

    Science.gov (United States)

    Arakawa, Tsutomu; Kurosawa, Yasunori; Storms, Michael; Maruyama, Toshiaki; Okumura, C J; Kita, Yoshiko

    2016-11-01

    Murine antibodies have weak affinity for Protein-A. Here, we have tested binding of murine monoclonal antibody (mAb) to Protein-A or Protein-A/Protein-G mixture under salting-out conditions. The addition of ammonium sulfate to HEK conditioned medium (CM) expressing murine mAb resulted in complete binding, leading to its elution by low pH or neutral arginine solution. Alternatively, a mixed-mode chromatography using Capto MMC resin was developed as a capture step. Binding of murine mAb occurred at neutral pH. The bound mAb was eluted with a gradient from 0.3 M NaCl to 0.3 M arginine/0.3 M NaCl at pH 7.0. The Capto MMC-purified murine mAb was further purified by hydroxyl apatite chromatography. Similarly, rabbit mAb was processed with some modifications. Binding of rabbit mAb to Capto MMC required a lower pH. Elution of the bound rabbit mAb was achieved by a gradient to 0.3 M NaCl, pH 7.0. PMID:27444249

  9. Experimental comparison of three background subtraction agents for monoclonal antibody imaging of ovarian carcinoma xenografts

    International Nuclear Information System (INIS)

    Dual-isotope background subtraction imaging can be used to enhance lesion detection in imaging studies using monoclonal antibodies. The best agent for background subtraction has not been determined, however. The author compared Tc-99m human serum albumin, Tc-99m red blood cells, and I-123-labeled nonspecific murine myeloma immunoglobulin (UPC-10) for their ability to enhance imaging of ovarian carcinoma xenografts in groups of nude mice 24 hours after they were injected with I-131 5G6.4 tumor-specific intact immunoglobulin. Using dual-isotope dynamic background subtraction, tumor imaging was most clear with Tc-99m red blood cells, second best with Tc-99m human serum algumin, and least clear with I-123 UPC-10. while verification in humans will be essential, these animal findings suggest that dual-isotope monoclonal antibody imaging of tumors is enhanced most with Tc-99m red blood cells and least with an isotype-matched monoclonal antibody

  10. Relationship between hyperthyroidism and monoclonal gammapathy

    International Nuclear Information System (INIS)

    A 66-year-old man with primary hyperparathyroidism (PHPT) and monoclonal gammapathy associated to it of uncertain significance (MGUS). A possible pathogenic relationship between HPTP and MGUS is analyzed. Interleukin 6 could play a pivotal role.

  11. Antibodies and Selection of Monoclonal Antibodies.

    Science.gov (United States)

    Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

    2016-01-01

    Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology. PMID:27236550

  12. Cloning murine antibody V-genes with non-degenerate primers and conversion to a recombinant antibody format.

    Science.gov (United States)

    Bialon, Magdalena; Schellenberg, Ludmila; Herzog, Nicolas; Kraus, Stefan; Jörißen, Hannah; Fischer, Rainer; Stein, Christoph; Nähring, Jörg; Barth, Stefan; Püttmann, Christiane

    2014-12-01

    Monoclonal antibodies are produced in cultured hybridoma cell lines, but these cells tend to be unstable; it is therefore necessary to rescue the corresponding genetic information. Here we describe an improved method for the amplification of antibody variable gene (V-gene) information from murine hybridoma cells using a panel of specific, non-degenerate primers. This primer set allows sequences to be rescued from all murine V-genes, except the lambda light chain genes, which rarely contribute to murine immune diversity. We tested the primers against a range of antibodies and recovered specific amplification products in all cases. The heavy and light chain variable regions were subsequently joined by a two-step cloning strategy or by splice overlap extension PCR. PMID:25545205

  13. Prolongation of murine skin allograft survival by immunologic manipulation with anti-interleukin 2 receptor antibody

    International Nuclear Information System (INIS)

    Administration of a rat monoclonal antibody (M7/20) directed against the murine interleukin 2 (IL 2) receptor in combination with sublethal x-irradiation of the recipient significantly enhanced the survival of skin allografts, both when the grafts were MHC disparate from the hosts and when only minor histocompatibility differences were present compared with untreated controls. No prolongation in graft survival was seen with either treatment alone at the dose employed. M7/20 and x-ray-treated allograft recipients also displayed significantly decreased alloantigen-specific reactivity against donor-strain spleen cells in both delayed-type hypersensitivity and cytotoxicity assays. Thus, such combination treatment reduces expression of host immune reactivity against graft determinants by several criteria. This work provides additional evidence that monoclonal antibodies directed against the IL 2 receptor may be useful in clinical transplantation

  14. Engineering, expression in transgenic plants and characterisation of E559, a rabies virus-neutralising monoclonal antibody.

    Science.gov (United States)

    van Dolleweerd, Craig J; Teh, Audrey Y-H; Banyard, Ashley C; Both, Leonard; Lotter-Stark, Hester C T; Tsekoa, Tsepo; Phahladira, Baby; Shumba, Wonderful; Chakauya, Ereck; Sabeta, Claude T; Gruber, Clemens; Fooks, Anthony R; Chikwamba, Rachel K; Ma, Julian K-C

    2014-07-15

    Rabies post-exposure prophylaxis (PEP) currently comprises administration of rabies vaccine together with rabies immunoglobulin (RIG) of either equine or human origin. In the developing world, RIG preparations are expensive, often in short supply, and of variable efficacy. Therefore, we are seeking to develop a monoclonal antibody cocktail to replace RIG. Here, we describe the cloning, engineering and production in plants of a candidate monoclonal antibody (E559) for inclusion in such a cocktail. The murine constant domains of E559 were replaced with human IgG1κ constant domains and the resulting chimeric mouse-human genes were cloned into plant expression vectors for stable nuclear transformation of Nicotiana tabacum. The plant-expressed, chimeric antibody was purified and biochemically characterized, was demonstrated to neutralize rabies virus in a fluorescent antibody virus neutralization assay, and conferred protection in a hamster challenge model. PMID:24511101

  15. Autoradiography-based, three-dimensional calculation of dose rate for murine, human-tumor xenografts

    International Nuclear Information System (INIS)

    A Fast Fourier Transform method for calculating the three-dimensional dose rate distribution for murine, human-tumour xenografts is outlined. The required input includes evenly-spaced activity slices which span the tumour. Numerical values in these slices are determined by quantitative 125I autoradiography. For the absorbed dose-rate calculation, we assume the activity from both 131I- and 90Y-labeled radiopharmaceuticals would be distributed as is measured with the 125I label. Two example cases are presented: an ovarian-carcinoma xenograft with an IgG 2ak monoclonal antibody and a neuroblastoma xenograft with meta-iobenzylguanidine (MIBG). (Author)

  16. 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)

    2010-02-15

    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

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

    OpenAIRE

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

    2013-01-01

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

  18. A Recombinant Humanized Anti-Cocaine Monoclonal Antibody Inhibits the Distribution of Cocaine to the Brain in Rats

    OpenAIRE

    Norman, Andrew B.; Gooden, Felicia C. T.; Tabet, Michael R.; Ball, William J.

    2014-01-01

    The monoclonal antibody (mAb), h2E2, is a humanized version of the chimeric human/murine anti-cocaine mAb 2E2. The recombinant h2E2 protein was produced in vitro from a transfected mammalian cell line and retained high affinity (4 nM Kd) and specificity for cocaine over its inactive metabolites benzoylecgonine (BE) and ecgonine methyl ester. In rats, pharmacokinetic studies of h2E2 (120 mg/kg i.v.) showed a long terminal elimination half-life of 9.0 days and a low volume of distribution at st...

  19. A Molecular Model for Cocaine Binding by the Immunotherapeutic Human/Mouse Chimeric Monoclonal Antibody 2E2

    OpenAIRE

    Lape, Michael; Paula, Stefan; Ball, William J.

    2010-01-01

    Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (γ1 heavy chain)/murine (λ light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2’s ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a...

  20. A monoclonal antibody (8H3) that binds to rat T lineage cells and augments in vitro proliferative responses

    OpenAIRE

    1990-01-01

    A murine monoclonal antibody, designated 8H3, recognizes a cell surface antigen expressed exclusively on rat T lineage cells. 8H3 antibody immunoprecipitated 180-, 120-, and 90-kD components from rat thymocytes as well as splenic T cells under nonreducing conditions. 8H3 antibody specifically inhibited the binding of thymocytes to fibronectin. Furthermore, binding of rat thymocytes to immobilized synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro-Cys-BSA was inhibited by 8H3 antibody as was Gly-Arg-Gl...

  1. Pravastatin stimulates angiogenesis in a murine hindlimb ischemia model: a positron emission tomography imaging study with 64Cu-NOTA-TRC105

    OpenAIRE

    Orbay, Hakan; Hong, Hao; Koch, Jill M.; Valdovinos, Hector F.; Hacker, Timothy A.; Theuer, Charles P.; Barnhart, Todd E.; Cai, Weibo

    2013-01-01

    In this study, 64Cu-NOTA-TRC105 (TRC105 is an anti-CD105 monoclonal antibody that binds to both human and murine CD105) positron emission tomography (PET) was used to assess the response to pravastatin treatment in a murine model of peripheral artery disease (PAD). Hindlimb ischemia was induced by ligation of the right femoral arteries in BALB/c mice under anesthesia, and the left hindlimb served as an internal control. Mice in the treatment group were given intraperitoneal pravastatin daily ...

  2. 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

    2000-01-01

    @@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].

  3. Trends in Malignant Glioma Monoclonal Antibody Therapy

    Science.gov (United States)

    Chekhonin, Ivan; Gurina, Olga

    2015-01-01

    Although new passive and active immunotherapy methods are emerging, unconjugated monoclonal antibodies remain the only kind of biological preparations approved for high-grade glioma therapy in clinical practice. In this review, we combine clinical and experimental data discussion. As antiangiogenic therapy is the standard of care for recurrent glioblastoma multiforme (GBM), we analyze major clinical trials and possible therapeutic combinations of bevacizumab, the most common monoclonal antibody to vascular endothelial growth factor (VEGF). Another humanized antibody to gain recognition in GBM is epidermal growth factor (EGFR) antagonist nimotuzumab. Other antigens (VEGF receptor, platelet-derived growth factor receptor, hepatocyte growth factor and c-Met system) showed significance in gliomas and were used to create monoclonal antibodies applied in different malignant tumors. We assess the role of genetic markers (isocitrate dehydrogenase, O6-methylguanine-DNA methyltransnsferase) in GBM treatment outcome prediction. Besides antibodies studied in clinical trials, we focus on perspective targets and briefly list other means of passive immunotherapy.

  4. Mouse monoclonal antibodies against estrogen receptor.

    Science.gov (United States)

    De Rosa, Caterina; Rossi, Valentina; Abbondanza, Ciro

    2014-01-01

    The production of monoclonal antibodies, by cloning hybridoma derived from the fusion of myeloma cells and spleen lymphocytes, has allowed to obtain great advances in many fields of biological knowledge. The use of specific antibodies to the estrogen receptor, in fact, has been an invaluable method to bring out its mechanisms of action and its effects, both genomic and extra-genomic. Here we describe, step by step, the production of monoclonal antibodies, starting from protocol for antigen preparation to the selection of antibody-secreting hybridoma. PMID:25182770

  5. Radiolabelling of monoclonal antibodies for radiotherapy. Thailand

    International Nuclear Information System (INIS)

    Nuclear medicine is now playing a great role not only in diagnostic application but also in therapy of cancer patients. Under the concept of targeted radiotherapy, a number of radiopharmaceuticals based on radiolabelled biomolecules had been evaluated for treatment of cancer by many investigators. Of these, monoclonal antibodies and some small specific peptides labelled with beta emitting radiometals such as Sm-153, Re-186, Re-188 or Y-90, are being introduced into clinical trials. The objective of this project is to develop laboratory procedures to label monoclonal antibodies, peptide or other proteins with beta emitting radionuclides to prepare radiopharmaceuticals for therapeutic purpose

  6. Radiolabelling of monoclonal antibodies for radiotherapy

    International Nuclear Information System (INIS)

    Nuclear medicine is now playing a great role not only in diagnostic application but also in therapy of cancer patients. Under the concept of targeted radiotherapy, a number of radiopharmaceuticals based on radiolabelled biomolecules had been evaluated for treatment of cancer by many investigators. Of these, monoclonal antibodies and some small specific peptides labelled with beta emitting radiometals such as Sm-153, Re-186, Re-188 or Y-90, are being introduced into clinical trials. The objective of this project is to develop laboratory procedures to label monoclonal antibodies, peptide or other proteins with beta emitting radionuclides to prepare radiopharmaceuticals for therapeutic purpose

  7. Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors

    DEFF Research Database (Denmark)

    Nastainczyk, W; Schmidt-Spaniol, I; Boldyreff, B;

    1995-01-01

    A murine monoclonal anti-protein kinase CK2 beta antibody was isolated and characterized. The antibody detects 1 pmol of purified recombinant CK2 beta-subunit after analysis on SDS-PAGE. Alternatively undenatured CK2 beta-subunit was detected by an ELISA assay either as recombinant CK2 beta-subun...

  8. Criteria for the use of monoclonal antibodies, legislation and ethical considerations

    International Nuclear Information System (INIS)

    The criteria governing the in vivo use of monoclonal antibodies in humans are based upon a number of legal requirements with respect to radiation hygiene, pharmaceutical legislation, radiopharmaceutical legislation and regulations with respect to products arising from biotechnology. This in itself has led to a complicated situation which has undoubtedly restricted the development of valuable diagnostic and potential therapeutic agents. From the ethical point of view there are also important considerations, firstly with respect to the methods of producing antibodies, which has resulted in the discontinuation of the raising of antibodies in murine ascites, and secondly in consideration of the ethics of administering labelled antibodies to healthy volunteers and to patients who may not necessarily benefit personally from the procedure. These factors must be evaluated in the light of the EEC document 'Good clinical practice for trials in medicinal products in the European Community from the CPMP working party on Efficacy of Medicinal Products'. (author)

  9. Chemoradiotherapy of cancer using boronated monoclonal antibodies. Comprehensive progress report, April 1, 1982-October 31, 1984

    International Nuclear Information System (INIS)

    Research to develop boron neutron capture for radiotherapy applications is summarized. Work is reported in the following areas: (1) chemical and biochemical research with B12H11SH2-; (2) chemical and protein-binding studies with B12H11NCO2- and (CH3)3NB10H8NCO1-; (3) effects of neutron irradiation and capture on phytomitogen stimulated lymphocyte blastogenesis; (4) production and characterization of monoclonal antibodies directed against the murine B16 melanoma; (5) reactor description and determination of flux profile; (6) determination of neutron profile; (7) improvement of the neutron beam by the bismuth scatterer method; and (8) in vitro sensitivity of B16 melanoma cells to thermal neutrons and 10B(n,α) capture. 11 references

  10. Radioimmunodetection with 111In-labeled monoclonal antibody Nd2 in patients with pancreatic cancer

    International Nuclear Information System (INIS)

    This report summarizes results from an initial clinical evaluation of radioimmunodetection (RAID) in patients with pancreatic cancer using murine monoclonal antibody Nd2, directed against mucins from pancreatic cancer. Nd2 (2 mg) was labeled with 111In (2 mCi) and injected into 19 patients suspected of having pancreatic cancer. Planar scintigrams were taken 3 days post-infusion. As for final diagnoses after surgery, 14 cases were pancreatic cancer, and one case each was chronic pancreatitis, neurilemmoma, islet cell carcinoma, cholangioma, and apparent absence of suspected recurrent lesion of pancreatic cancer. Of 14 patients with pancreatic cancer, RAID was positive in 10 cases (71.4%). Cases other than pancreatic cancer were all negative, so the specificity was 100%. These results demonstrate that RAID using 111In-Nd2 can be useful in differentiating exocrine pancreatic cancer from benign conditions and other types of carcinomas in the pancreatoduodenal regions. (author)

  11. Immunohistochemical diagnosis of fusariosis with monoclonal antibodies

    DEFF Research Database (Denmark)

    Jensen, H.E.; Aalbæk, B.; Jungersen, Gregers; Hartvig, T.; Moser, C.; Rozell, B.L.; Blennow, O.

    establishing an accurate diagnosis. Although molecular techniques (e.g. in situ hybridization and PCR) have been explored for diagnostic use, the development of specific monoclonal antibodies (Mabs) for immunohistochemical identification of Fusarium spp. will extend the availability of diagnostic options for...

  12. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Science.gov (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.

  13. The development of a radioallergosorbent test (RAST) for murine IgE antibodies

    International Nuclear Information System (INIS)

    A purified monoclonal IgE preparation, isolated from the ascitic fluid of mice bearing a hybridoma secreting IgE with specificity to ovalbumin, was used for the production of goat anti-murine IgE (GAME) antiserum, which was then rendered monospecific for the epsilon chain. Another monoclonal hybridoma IgE preparation with specificity for the 2,4-dinitrophenyl group was isolated from ascitic fluid in a relatively pure state by affinity chromatography and used in the form of an immunosorbent to isolate antibodies from the monospecific goat serum. The GAME of antibodies were 125I-labeled and used to develop a radioallergosorbent test (RAST) for the quantitation of murine IgE antibodies specifically adsorbed onto antigen-coupled paper discs. The RAST was specific for antibodies of the IgE class only and was as sensitive as and more accurate than PCA assay. RAST results on sera of mice treated with tolerogenic conjugates indicated a reduction in the affinity and concentration of the IgE antibody populations on suppression of the IgE response. The effect of interference by non-IgE antibody populations on the RAST curves has been discussed. (Auth.)

  14. Monoclonal antibody against human ovarian tumor-associated antigens

    International Nuclear Information System (INIS)

    Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies

  15. PRODUCTION OF MONOCLONAL ANTIBODY AGAINST HUMAN IMMUNOGLOBULIN

    Directory of Open Access Journals (Sweden)

    J. Majidi

    2000-04-01

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

  16. Selection of therapeutic H5N1 monoclonal antibodies following IgVH repertoire analysis in mice.

    Science.gov (United States)

    Gray, Sean A; Moore, Margaret; VandenEkart, Emily J; Roque, Richard P; Bowen, Richard A; Van Hoeven, Neal; Wiley, Steven R; Clegg, Christopher H

    2016-07-01

    The rapid rate of influenza virus mutation drives the emergence of new strains that inflict serious seasonal epidemics and less frequent, but more deadly, pandemics. While vaccination provides the best protection against influenza, its utility is often diminished by the unpredictability of new pathogenic strains. Consequently, efforts are underway to identify new antiviral drugs and monoclonal antibodies that can be used to treat recently infected individuals and prevent disease in vulnerable populations. Next Generation Sequencing (NGS) and the analysis of antibody gene repertoires is a valuable tool for Ab discovery. Here, we describe a technology platform for isolating therapeutic monoclonal antibodies (MAbs) by analyzing the IgVH repertoires of mice immunized with recombinant H5N1 hemagglutinin (rH5). As an initial proof of concept, 35 IgVH genes were selected using a CDRH3 search algorithm and co-expressed in a murine IgG2a expression vector with a panel of germline murine kappa genes. Culture supernatants were then screened for antigen binding. Seventeen of the 35 IgVH MAbs (49%) bound rH5VN1203 in preliminary screens and 8 of 9 purified MAbs inhibited 3 heterosubtypic strains of H5N1 virus when assayed by HI. Two of these MAbs demonstrated prophylactic and therapeutic activity in virus-challenged mice. This is the first example in which an NGS discovery platform has been used to isolate anti-influenza MAbs with relevant therapeutic activity. PMID:27109194

  17. Localization of pulmonary human sarcoma xenografts in athymic nude mice with indium-111-labeled monoclonal antibodies

    International Nuclear Information System (INIS)

    In order to study localization of metastatic tumors with a radiolabeled monoclonal antibody, a pulmonary metastases model was devised in athymic mice. Metastatic pulmonary sarcoma colonies were verified by histological examination. A murine monoclonal antibody (MAb 19-24) directed against a human sarcoma antigen was labeled with indium-111 (111In) by use of the linker 1-(p-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (SCN-Bz-DTPA). MAb P3 was similarly labeled as a negative control. In the group given MAb 19-24, the percent injected dose per gram lung tissue bearing tumor colonies (30.1%, 29.6%, and 27.7% on Days 1, 2, and 3, respectively) was significantly (p less than 0.05) higher than in those receiving MAb P3. Hepatic activities of both 111In-MAb 19-24 and 111In-MAb P3 were low. The lungs with tumor colonies demonstrated clearest images on Day 3. The specific binding of 111In-SCN-Bz-DTPA-labeled MAb 19-24 to pulmonary xenografts without appreciable liver uptake indicates that it may be useful in the clinical localization of pulmonic metastatic lesions

  18. Identification of murine complement receptor type 2.

    OpenAIRE

    Fingeroth, J D; Benedict, M A; Levy, D.N.; Strominger, J L

    1989-01-01

    A rabbit antiserum reactive with the human complement component C3d/Epstein-Barr virus receptor (complement receptor type 2, CR2) immunoprecipitates a Mr 155,000 murine B-cell surface antigen. The apparent molecular weight and cellular distribution of this murine antigen are similar to those of human CR2. Cells expressing the murine protein bind sheep erythrocytes coated with antibody and murine C1-C3d but do not bind Epstein-Barr virus at all. The monospecific antiserum to human CR2 together...

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

    Science.gov (United States)

    Swanstrom, J. A.; Plante, J. A.; Plante, K. S.; Young, E. F.; McGowan, E.; Gallichotte, E. N.; Widman, D. G.; Heise, M. T.; de Silva, A. M.

    2016-01-01

    ABSTRACT 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. PMID:27435464

  20. Serrumab: a human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom.

    Science.gov (United States)

    Pucca, Manuela Berto; Zoccal, Karina Furlan; Roncolato, Eduardo Crosara; Bertolini, Thaís Barboza; Campos, Lucas Benício; Cologna, Camila Takeno; Faccioli, Lúcia Helena; Arantes, Eliane Candiani; Barbosa, José Elpidio

    2012-01-01

    In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNFα, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom. PMID:22424317

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

    Science.gov (United States)

    Chou, Shu-Fen; Chen, Chien-Yuan

    2004-02-01

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

  2. Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

    Science.gov (United States)

    Rickert, Keith W; Grinberg, Luba; Woods, Robert M; Wilson, Susan; Bowen, Michael A; Baca, Manuel

    2016-04-01

    The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material. PMID:26852694

  3. Studies of monoclonal antibodies IOR-CEA-1 and IOR-EGF/R3 labelled with 99mTc

    International Nuclear Information System (INIS)

    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. 99mTc 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 99mTc. 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 μL MDP, 3.0 μg Sn2+, 1 mL of 99mTc and 30 min. reaction time. With these conditions the labelling yield was always higher than 95% and the maximum activity of 99mTc was about 2220 MBq (60 mCi). The evidences of the efficiency and quality of the methods here

  4. Monoclonal antibodies for radioimmunoimaging: Current perspectives

    International Nuclear Information System (INIS)

    The ability to image tumor using radiolabeled monoclonal antibody products has been widely demonstrated. The questions of safety and efficacy remain open and require further experience, but in some clinical situations, radioimmunoimaging has provided clinically useful information. This paper deals with a set of current problems in imaging with radiolabeled monoclonal antibodies and current perspectives on the possible solutions to these problems. The major areas discussed here are the following: (a) The selection process. How might we choose the ''best'' antibody for imaging from among the multitude now available and what form (i.e., which fragments) may be useful? (b) The imaging procedure: What are the basic optimal imaging parameters and how does the data produced by this modality interface with information obtained by more standard methods of imaging? (c) Quantitative techniques: How can noninvasive quantitative techniques provide information useful to the antibody selection process and to the diagnostic and therapeutic applications

  5. Recent developments in monoclonal antibody radiolabeling techniques

    International Nuclear Information System (INIS)

    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

  6. Notch pathway inhibition controls myeloma bone disease in the murine MOPC315.BM model

    International Nuclear Information System (INIS)

    Despite evidence that deregulated Notch signalling is a master regulator of multiple myeloma (MM) pathogenesis, its contribution to myeloma bone disease remains to be resolved. Notch promotes survival of human MM cells and triggers human osteoclast activity in vitro. Here, we show that inhibition of Notch through the γ-secretase inhibitor XII (GSI XII) induces apoptosis of murine MOPC315.BM myeloma cells with high Notch activity. GSI XII impairs murine osteoclast differentiation of receptor activator of NF-κB ligand (RANKL)-stimulated RAW264.7 cells in vitro. In the murine MOPC315.BM myeloma model GSI XII has potent anti-MM activity and reduces osteolytic lesions as evidenced by diminished myeloma-specific monoclonal immunoglobulin (Ig)-A serum levels and quantitative assessment of bone structure changes via high-resolution microcomputed tomography scans. Thus, we suggest that Notch inhibition through GSI XII controls myeloma bone disease mainly by targeting Notch in MM cells and possibly in osteoclasts in their microenvironment. We conclude that Notch inhibition is a valid therapeutic strategy in MM

  7. Technological progresses in monoclonal antibody production systems

    OpenAIRE

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

    2009-01-01

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

  8. Detection of enterovirus 70 with monoclonal antibodies.

    OpenAIRE

    Anderson, L J; Hatch, M. H.; Flemister, M R; Marchetti, G E

    1984-01-01

    To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave ...

  9. A monoclonal thyroid-stimulating antibody

    OpenAIRE

    Ando, Takao; Latif, Rauf; Pritsker, Alla; Moran, Thomas; Nagayama, Yuji; Davies, Terry F.

    2002-01-01

    The thyrotropin receptor, also known as the thyroid-stimulating hormone receptor (TSHR), is the primary antigen of Graves disease. Stimulating TSHR antibodies are the cause of thyroid overstimulation and were originally called long-acting thyroid stimulators due to their prolonged action. Here we report the successful cloning and characterization of a monoclonal antibody (MS-1) with TSHR-stimulating activity. The thyroid-stimulating activity of MS-1 was evident at IgG concentrations as low as...

  10. Imaging tumors with radiolabelled monoclonal antibodies

    International Nuclear Information System (INIS)

    Using a metallic radionuclide, either directly bound to a monoclonal antibody, or to a chelating agent (such as di-ethylenetriamine-pentaacetic acid (DTPA)) conjugated to the antibody, a tumor can be traced rapidly and with high specificity. The labelled antibody is injected into the host. In some cases, a localization of distant metastases is possible, giving an indication of tumor spreading. Detection occurs by photoscanning. (Auth.)

  11. The role of adipokines in monoclonal gammopathies

    OpenAIRE

    Oliveira, Luís Alberto Resendes de

    2015-01-01

    In the pathogenesis of monoclonal gammopathies (MG), namely in multiple myeloma (MM), the bone marrow microenvironment displays a critical role. As there is accumulating evidence supporting a link between obesity and MM, it is possible that this association is made through altered adipokines secretion levels. Whereas these hormones are important in several physiologic functions, there are studies showing that they also participate in the carcinogenesis process of some solid tumors. However, a...

  12. Monoclonal antibodies to Bacteroides fragilis lipopolysaccharide.

    OpenAIRE

    Linko-Kettunen, L; Arstila, P; Jalkanen, M; Jousimies-Somer, H; Lassila, O; Lehtonen, O P; Weintraub, A; Viljanen, M K

    1984-01-01

    Monoclonal antibodies (MoAbs) to the lipopolysaccharide (LPS) of Bacteroides fragilis were produced by immunizing mice before hybridization with bacterial outer membranes solubilized with Triton X-100. Nineteen stabile clones were established. They all produced antibodies that reacted more strongly with purified B. fragilis LPS than with crude sonicated antigen in an enzyme immunoassay. Four MoAbs were studied by immunoblotting and enzyme immunoassay inhibition. Immunoblotting confirmed that ...

  13. Radioimmunoscintigraphy with anti-thyroglobulin monoclonal antibodies

    International Nuclear Information System (INIS)

    Monoclonal mouse antibodies to human thyroglobulin were conjugated to the cyclic dianhydride of DTPA. After radiolabelling with 111In this compound was injected into nude mice bearing various human thyroid carcinomas. Repeated imaging studies were carried out 15 min to 50 h after tracer administration. In both papillary and undifferentiated thyroid carcinoma no significant uptake of radiolabelled anti-hTG-MAb was observed. (orig.)

  14. Conjugates of monoclonal antibodies and chelating polymers

    International Nuclear Information System (INIS)

    The primary purpose of protein modification with chelating polymers is to prepare monoclonal antibodies labeled with heavy metal isotopes (alpha-, beta-, and gamma-emitting metal and paramagnetic ions for NMR tomography). Conventional binding of metals to proteins via chelating agents directly coupled to proteins does not permit binding of a large number of metal atoms per protein molecule without causing alterations in the specific properties of the protein molecules. On the other hand, metal ion binding to proteins via intermediate chelating polymers should permit binding of several dozens of the metal atoms per protein molecule without affect the specific properties adversely. Moreover, the biodistribution and clearance rates can be regulated by varying the polymer properties. Modified antibodies may be used successfully in nuclear and NMR diagnostic applications and in radiotherapy. Possible applications of this approach shall be demonstrated with monoclonal antibody R11D10 for visualization of acute myocardial infarction. Use of this modification with other monoclonal antibodies is also discussed. The chemistry of protein modification with these polymers is presented

  15. Monoclonal antibodies as diagnostics; an appraisal

    Directory of Open Access Journals (Sweden)

    Siddiqui M

    2010-01-01

    Full Text Available Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use.

  16. SPECT assay of radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data

  17. [Glomerulopathies with organized monoclonal immunoglobulin deposits].

    Science.gov (United States)

    Touchard, Guy; Bridoux, Frank; Goujon, Jean-Michel

    2016-02-01

    The spectrum of glomerular disorders with organized immunoglobulin (Ig) deposits is heterogeneous. It encompasses 2 mains categories: glomerulopathies with fibrillary deposits are mostly represented by immunoglobulinic amyloidosis (most commonly AL amyloidosis, characterized by monoclonal light chain deposits often of the lambda isotype), and pseudo-amyloid fibrillary glomerulonephritis in which deposits predominantly contain polyclonal IgG4. Glomerulopathies with microtubular deposits include cryoglobulinemic glomerulonephritis (type I and type II, with or without detectable serum cryoglobulin) and glomerulonephritis with organized microtubular monoclonal Ig deposits (GOMMID) also referred to as immunotactoid glomerulopathy. Pathological diagnosis requires meticulous studies by light microscopy (with systematic Congo red staining), immunofluorescence with specific conjugates, and electron microscopy. Ultrastructural studies are required to differentiate amyloid fibrils (8 to 10 nm in external diameter), pseudo-amyloid fibrils (15-20 nm) and microtubules (10 to 50 nm in external diameter, with a central hollow core). Glomerular deposits in type I cryoglobulinemic glomerulonephritis are arranged into parallel straight microtubules similar to those observed in GOMMID, but with different topography that allows distinction between the two entities. Glomerular substructures composed of circulating Igs should be distinguished from collagen fibrils that are commonly observed in glomerular disorders with or without deposition of monoclonal or polyclonal Igs. PMID:26810049

  18. THE CONSTRUCTION AND EXPRESSION OF THE MURINE SCFV GENE IN E.COLI AGAINST HUMAN CERVICAL CANCER

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    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...

  19. Murine Typhus and Febrile Illness, Nepal

    OpenAIRE

    Zimmerman, Mark D.; Murdoch, David R.; Rozmajzl, Patrick J.; Basnyat, Buddha; Woods, Christopher W.; Richards, Allen L.; Belbase, Ram Hari; Hammer, David A.; Anderson, Trevor P.; Reller, L. Barth

    2008-01-01

    Murine typhus was diagnosed by PCR in 50 (7%) of 756 adults with febrile illness seeking treatment at Patan Hospital in Kathmandu, Nepal. Of patients with murine typhus, 64% were women, 86% were residents of Kathmandu, and 90% were unwell during the winter. No characteristics clearly distinguished typhus patients from those with blood culture–positive enteric fever.

  20. Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

    International Nuclear Information System (INIS)

    In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [3H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

  1. The acquisition of cytokine responsiveness by murine B cells

    DEFF Research Database (Denmark)

    Poudrier, J; Owens, T

    1994-01-01

    The mechanism whereby small resting (high buoyant density) murine B cells are induced to express interleukin-2 receptors (IL-2R) and to respond to IL-2 was addressed by staining with anti-IL-2R alpha and -IL-2R beta monoclonal antibodies (mAb), and using receptor-specific cDNA probes. Resting B...... staining and mRNA were induced by the combination of LPS plus IL-5. LPS+IL-5-treated B cells responded to IL-2 by Ig secretion. This indicates that B cells regulate their responsiveness to IL-2 similarly to T cells, via the combined level of expression of IL-2R beta and IL-2R alpha. The synergy between IL...... cells expressed undetectable levels of both IL-2R alpha and beta chains on their surface and did not respond to IL-2, even at supra-physiological concentrations. Sepharose-coupled, but not streptavidin-cross-linked, plastic-adsorbed or soluble, anti-mu up-regulated the expression of IL-2R alpha and beta...

  2. Prevalence of Monoclonal Gammopathy of Undetermined Significance: A Systematic Review

    OpenAIRE

    Wadhera, Rishi K.; Rajkumar, S. Vincent

    2010-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell disorder that is associated with a lifelong risk of multiple myeloma. We conducted a systematic review of all studies investigating the prevalence and incidence of MGUS in the online database PubMed. The review was conducted from January 6, 2009, through January 15, 2010. The following MeSH search headings were used: monoclonal gammopathy, benign and prevalence; monoclonal gammopathy, benign and incidence;...

  3. Laboratory Persistence and Clinical Progression of Small Monoclonal Abnormalities

    OpenAIRE

    Murray, David L.; Seningen, Justin L.; Dispenzieri, Angela; Snyder, Melissa R.; Kyle, Robert A.; Rajkumar, S. Vincent; Katzmann, Jerry A.

    2012-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) that presents with no quantifiable M spike on immunofixation electrophoresis (IFE) can be termed IFE MGUS. We retrospectively identified patients with IFE MGUS who were monitored with at least 1 subsequent assessment that included an IFE, and evaluated the persistence of the monoclonal protein and the progression of disease. Although the monoclonal proteins persisted in the majority of patients, 16% did not experience this persistence,...

  4. Critical evaluation of monoclonal antibody staining in breast carcinoma.

    OpenAIRE

    Parham, D M; Coghill, G; Robertson, A.J.

    1989-01-01

    The immunoperoxidase staining of 84 primary invasive breast carcinomas with four monoclonal antibodies (BRST-1, HMFG1, EMA, B72.3) was evaluated by semiquantitative light microscopical examination and quantitative image analysis. Major differences in the staining of the tumours for each of the monoclonal antibodies was observed. Correlation between monoclonal antibody staining and patient age, survival, histological grade, tumour diameter and cellularity was also carried out. This showed a si...

  5. Murine malignant cells synthesize a 19,000-dalton protein that is physicochemically and antigenically related to the immunosuppressive retroviral protein, P15E.

    Science.gov (United States)

    Cianciolo, G J; Lostrom, M E; Tam, M; Snyderman, R

    1983-09-01

    Murine tumors contain low molecular weight factors that inhibit macrophage accumulation at inflammatory foci. Certain oncogenic murine leukemia viruses contain similar inhibitory activity and the active component of the retroviruses was shown to be the envelope protein P15E. A number of murine malignant and nonmalignant cell lines, as well as primary tumors, have now been examined to determine whether production of retroviral P15E or a related protein is characteristic of neoplastic cells. Tumor lines examined included the Hep 129 hepatocarcinoma, BP8 fibrosarcoma, RL1 lymphoma, and three variants of the B16 melanoma. Tumor lines were virus negative by electron microscopy. Nonmalignant cells examined included ST0, 3T3/BALB, and 3T3/L1 fibroblasts and unstimulated, as well as mitogen-stimulated murine splenocytes. Cells were pulse-labeled with [35S]methionine, proteins immunoprecipitated with two monoclonal antibodies to P15E and analyzed by SDS-PAGE and gel fluorography. All tumor lines synthesized a approximately 19,000-dalton protein that co-migrated with retroviral P15E on SDS-PAGE. None of the nonmalignant cells synthesized this protein. Two-dimensional gel electrophoresis of the proteins precipitated from two B16 melanoma lines by monoclonal anti-P15E showed them to be physicochemically similar to P15E from Rauscher leukemia virus. A competition ELISA assay for P15E was developed and confirmed the results obtained by metabolic labeling and demonstrated P15E-related antigens in the tumor cell lines and also in the ascites fluid of mice injected with Hep 129 cells. More importantly, P15E antigens were expressed in both a spontaneous mammary adenocarcinoma and in a primary methylcholanthrene-induced fibrosarcoma. Nonmalignant tissues from animals bearing these tumors contained no detectable P15E antigen. Extracts from the primary fibrosarcomas, when injected into the thighs of mice, inhibited the intraperitoneal accumulation of inflammatory macrophages. The

  6. Identification of Key Amino Acid Residues in a Thyrotropin Receptor Monoclonal Antibody Epitope Provides Insight into Its Inverse Agonist and Antagonist Properties

    OpenAIRE

    Chen, Chun-Rong; McLachlan, Sandra M.; Rapoport, Basil

    2008-01-01

    CS-17 is a murine monoclonal antibody to the human TSH receptor (TSHR) with both inverse agonist and antagonist properties. Thus, in the absence of ligand, CS-17 reduces constitutive TSHR cAMP generation and also competes for TSH binding to the receptor. The present data indicate that for both of these functions, the monovalent CS-17 Fab (50 kDa) behaves identically to the intact, divalent IgG molecule (150 kDa). The surprising observation that CS-17 competes for TSH binding to the human but ...

  7. Characterization of the mechanism of protection mediated by CS-D7, a monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB)

    OpenAIRE

    Gregory ePancari; Hongxia eFan; Sharon eSmith; Amita eJoshi; Robin eHaimbach; Desmond eClark; Yingzhe eLi; Jin eHua; Troy eMcKelvey; Yangsi eOu; James eDrummond; Leslie eCope; Donna eMontgomery; Tessie eMcNeely

    2012-01-01

    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. Th...

  8. Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

    Science.gov (United States)

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

    2011-09-01

    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. PMID:21752470

  9. Production of Monoclonal Antibody against Human Nestin.

    Science.gov (United States)

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

    2010-04-01

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

  10. Production of Monoclonal Antibody against Human Nestin

    OpenAIRE

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

    2010-01-01

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

  11. Bone marrow dosimetry for monoclonal antibody therapy

    International Nuclear Information System (INIS)

    Immunoglobulins must permeate through the basement membrane of capillaries in order to enter the extracellular space (ECS) of tissue. Since the process is quite slow, the blood plasma activity in various organs contributes considerably to the radiation dose of the dose-limiting tissues. In bone marrow the basement membrane is absent and the blood circulation is functionally open. Therefore, blood plasma and marrow ECS maintain equal concentrations of labeled immunoglobulins. A combination of factors including intravenous administration, slow absorption into most tissues, slow breakdown and elimination of labeled immunoglobulin, and rapid entry into bone marrow ECS as well as known radiosensitivity of marrow led the authors to expect this tissue would prove to be the primary tissue at risk for systemic monoclonal antibody therapy. They have developed and applied in a Phase I clinical study of 131I labeled CEA antibody a procedure for estimation of radiation dose to red bone marrow. Serieal measurements of blood plasma and total body retention are carried out. Binding of labeled antibody to the cellular components of blood is verified to be very low. They have observed bone marrow depression at doses greater than 400 rad. If no special procedures are used to reconstitute marrow after radiation treatment, this level represents a much greater than generally recognized limitation to radiolabeled monoclonal antibody therapy. 25 references, 4 tables

  12. Monoclonal Antibodies Specific for Hippurate Hydrolase of Campylobacter jejuni

    OpenAIRE

    Steele, Marina; Gyles, Carlton; Chan, Voon Loong; Odumeru, Joseph

    2002-01-01

    Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.

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

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    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 ...

  14. Production of monoclonal antibody with Celline-350 bioreactor

    International Nuclear Information System (INIS)

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

  15. Kinetics of expression of interleukin 2 receptors on class I and class II restricted murine T cell clones

    International Nuclear Information System (INIS)

    The kinetics of interleukin 2 receptor (IL-2R) expression has been examined on various class I and class II restricted, influenza specific murine T cell clones. Expression and relative levels of IL-2R were examined by Fluorescence Activated Cell Sorter analysis utilizing 3 anti-murine IL-2R monoclonal antibodies. Receptor expression was analyzed by scatchard analysis using radiolabeled recombinant human interleukin 2 to access the number of high and low affinity IL-2R per cell as well as the affinity of binding. The clones tested bound all 3 monoclonal antibodies and were inhibited in an IL-2 dependent proliferation assay by the addition of the antibodies to the culture. There was, however, differing degrees of inhibition ranging up to 99%, depending on the clone and the antibody used. IL-2R expression was detectable as early as 4-6 hours after antigenic stimulation of quiescent cells. After maximal levels of receptors were expressed, which was about 24 hours after stimulation, expression of IL-2R decreased with time on all clones examined (both class I and class II restricted). Differing rates of receptor loss is seen however, with some class II restricted clones retaining relatively high levels of receptors

  16. Experimental radioimmunotherapy of a xenografted human glioma using 131I-labeled monoclonal antibody to epidermal growth factor receptor

    International Nuclear Information System (INIS)

    131I-labeled F (ab')2 fragments of murine monoclonal antibodies (MAb) 425 specific to the epidermal growth factor receptor expressed on human gliomas were used in experimental human malignant glioma immunotherapy. Two injections of 150 μCi 131I-labeled 425 F(ab')2 achieved growth inhibition of U-87MG human malignant glioma xenografts in nude mice. This radiolabeled specific MAb F(ab')2 was significantly superior to radiolabeled fragments of an anti-hepatitis virus control MAb A5C3 in influencing tumor growth. However, similar treatment of established human malignant glioma xenografts did not inhibit progressive tumor growth significantly. No clear tumor inhibition was produced by unlabeled MAb 425F(ab')2. These studies suggest that 131I-labeled MAbs have a significant antitumor effect where unmodified antibody is ineffective. Multiple doses of antibody may achieve an increase in labeled MAb concentration in tumors. (author)

  17. Development of a Monoclonal Antibody-Based Sandwich ELISA for Peanut Allergen Ara h 1 in Food

    Directory of Open Access Journals (Sweden)

    Chuanlai Xu

    2013-07-01

    Full Text Available We have established a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA based on two monoclonal antibodies (mAb to measure the content of the major peanut allergen Ara h 1 in foods. Two mAbs were selected out of 12 murine hybridoma cells secreting Ara h 1-specific antibody. Using mAb 6 as the capture antibody and HRP-labelled mAb 4 as the detection antibody, the limit of detection (LOD the assay was 0.34 ng/mL. Cross-reaction analysis showed that this method was strongly specific and had no cross-reactions with Ara h 2, pea protein or soy protein. Sample analysis showed that this ELISA was a useful tool to monitor peanut allergens in food products by measuring Ara h 1 content.

  18. 99mTc direct labeling of anti-CEA monoclonal antibodies: Quality control and preclinical studies

    International Nuclear Information System (INIS)

    The anti-carcinoembryonic B2C114 monoclonal antibody was radiolabeled with 99mTc by a direct method and quality control tested in vitro by instant thin layer chromatography, gel column scanning and cellulose acetate electrophoresis and assessed in vivo for radioimmunodetection on a murine spontaneous mammary carcinoma. The optimal results of percent 99mTc bound to protein were obtained at a dithiothreitol: antibody molar ratio ranging from 800:1 to 1000:1 and at a methylene diphosphonate: stannous fluoride weight ratio of 4.3:1. Although cysteine removed up to 18% of the label during the first 4 h, the stability of the tracer appeared to be excellent in human serum at 37 deg. C and when challenged with DTPA. 99mTc-labeled B2C114 demonstrated good and specific in vivo tumor targeting

  19. Development and characterization of a monoclonal antibody to human embryonal carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Khazaeli, M.B.; Beierwaltes, W.H.; Pitt, G.S.; Kabza, G.A.; Rogers, K.J.; LoBuglio, A.F.

    1987-06-01

    A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian, 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with /sup 131/I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21.

  20. 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.

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

    Science.gov (United States)

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

    2015-02-01

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

  2. Monoclonal antibodies in treatment of multiple sclerosis

    Science.gov (United States)

    Rommer, P S; Dudesek, A; Stüve, O; Zettl, UK

    2014-01-01

    Monoclonal antibodies (mAbs) are used as therapeutics in a number of disciplines in medicine, such as oncology, rheumatology, gastroenterology, dermatology and transplant rejection prevention. Since the introduction and reintroduction of the anti-alpha4-integrin mAb natalizumab in 2004 and 2006, mAbs have gained relevance in the treatment of multiple sclerosis (MS). At present, numerous mAbs have been tested in clinical trials in relapsing–remitting MS, and in progressive forms of MS. One of the agents that might soon be approved for very active forms of relapsing–remitting MS is alemtuzumab, a humanized mAb against CD52. This review provides insights into clinical studies with the mAbs natalizumab, alemtuzumab, daclizumab, rituximab, ocrelizumab and ofatumumab. PMID:24001305

  3. SPECT assay of radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    The long-term goal of this research project is to develop methods to improve the utility of single photon emission computed tomography (SPECI) to quantify the biodistribution of monoclonal antibodies (MoAbs) labeled with clinically relevant radionuclides (123I, 131I, and 111In) and with another radionuclide,211At, recently used in therapy. We describe here our progress in developing quantitative SPECT methodology for 111In and 123I. We have focused our recent research thrusts on the following aspects of SPECT: (1) The development of improved SPECT hardware, such as improved acquisition geometries. (2) The development of better reconstruction methods that provide accurate compensation for the physical factors that affect SPECT quantification. (3) The application of carefully designed simulations and experiments to validate our hardware and software approaches

  4. 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.

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

    Science.gov (United States)

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

    2016-05-01

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

  6. Chemoimmunotherapy of murine bladder cancer.

    Science.gov (United States)

    Stogdill, B J; Lamm, D L; Livingston, R B

    1981-11-01

    The lethality of invasive transitional cell carcinoma (TCC) has prompted a search for effective, minimally toxic, adjuvant therapy. Such agents were evaluated in a murine bladder cancer (MBT2) model which parallels the clinical disease. One hundred C3H/He mice were inoculated i.d. with 2.5 x 10(4) viable MBT2 tumor cells and randomized to receive either normal saline (control), cis-Platinum (CPT), cyclophosphamide (CY), methotrexate (MTX), BCG, (CY + MTX), or (CY + MTX + BCG). Chemotherapy was given intraperitoneally weekly starting on day 7 after inoculation. Immunotherapy was given intralesionally on days 1 and 10 only. All mice were treated for 5 weeks followed by 5 weeks of observation. At 5 weeks, tumors of mice receiving cyclophosphamide alone or either of the combinations of therapy were smaller (P less than 0.01) than tumors of controls or other single agents alone. Each regimen increased survival, but only the combination regimen increase survival significantly (P less than 0.01). In the doses and schedule used in this model. Combination chemotherapy and chemoimmunotherapy significantly delay tumor growth and increase duration of survival (P less than 0.01) when compared with controls or single agent groups. PMID:7298287

  7. Apoptosis in irradiated murine tumors.

    Science.gov (United States)

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

    1991-09-01

    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. PMID:1886987

  8. Development of a method to measure kinetics of radiolabelled monoclonal antibody in human tumour with applications to microdosimetry: positron emission tomography studies of iodine-124 labelled 3F8 monoclonal antibody in glioma

    International Nuclear Information System (INIS)

    We present a method to assess quantitatively the immuhological characteristics of tumours using radiolabelled monoclonal antibody and positron emission tomography (PET) to improve dosimetry for radioimmunotherapy. This method is illustrated with a glioma patient who injected with 96.2 MPq of iodine-124 labelled 3FB, a murine antibody (IgG3) specific against the ganglioside GD2. Serial PET scans and plasma samples were taken over 11 days. A three-compartment model was used to estimate the plasma to tumour transfer constant (K1), the tumour to plasma transfer constant k2, the association and dissociation constants (k3, k4) of antibody binding, and the binding potential. Tumour radioactivity peaked at 18 h at 0.0045% ID/g. The kinetic parameters were estimated to be: K1=0.048 ml h-1 g-1, k2=0.16 h-1, k3=0.03 h-1, k4=0.015 h-1 and BP=2.25. Based on these kinetic parameters, the amount of tumour-bound radiolabelled monoclonal antibody was calculated. This method permits estimates of both macrodosimetry and microdosimetry at the cellular level based on in vivo non-invasive measurement. (orig.)

  9. Monoclonal gammopathy: a diagnosis for to keep in mind

    International Nuclear Information System (INIS)

    How to identify monoclonal gammopathies at risk for progression has been studied for the last year. 40 patients were studied in which a monoclonal band had been detected, in some of the cases de novo. The electrophoresis was performed in the Hydrasys system. Of the total of electrophoresis carried out, the 14% was monoclonal gammopathy. In 36% a diagnostic assumption was not stated. Most frequent diagnosis in the group of patients with a diagnosis was multiple myeloma. Average age of patients was 61.5 years and there were differences among percentages for sex

  10. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

    Science.gov (United States)

    Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

    2015-08-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

  11. Monoclonal antibodies and Fc fragments for treating solid tumors

    Directory of Open Access Journals (Sweden)

    Eisenbeis AM

    2012-01-01

    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

  12. Preparation and Evaluation of Human-Murine Chimeric Antibody against Protective Antigen of Bacillus anthracis

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    Lina Hao

    2014-10-01

    Full Text Available The aim of this research is to develop a human/murine chimeric Fab antibody which neutralizes the anthrax toxin, protective antigen (PA. The chimeric Fab was constructed using variable regions of murine anti-PA monoclonal antibody in combination with constant regions of human IgG. The chimeric PA6-Fab was expressed in E. coli. BL21 and evaluated by ELISA and co-immunoprecipitation- mass spectra. The potency of PA6-Fab to neutralize LeTx was examined in J774A.1 cell viability in vitro and in Fisher 344 rats in vivo. The PA6-Fab did not have domain similarity corresponding to the current anti PA mAbs, but specifically bound to anthrax PA at an affinity of 1.76 nM, and was able to neutralize LeTx in vitro and protected 56.9% cells at 20 μg/mL against anthrax LeTx. One hundred μg PA6-Fab could neutralize 300 μg LeTx in vivo. The PA6-Fab has potential as a therapeutic mAb for treatment of anthrax.

  13. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents.

    Science.gov (United States)

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-07-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  14. Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans.

    Science.gov (United States)

    Marchitto, K S; Kindt, T J; Norgard, M V

    1986-09-01

    Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses. PMID:2428519

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

    Directory of Open Access Journals (Sweden)

    Malanchuk O. M.

    2015-06-01

    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.

  16. Friend and Moloney murine leukemia viruses specifically recombine with different endogenous retroviral sequences to generate mink cell focus-forming viruses.

    OpenAIRE

    Evans, L H; Cloyd, M W

    1985-01-01

    A group of mink cell focus-forming (MCF) viruses was derived by inoculation of NFS/N mice with Moloney murine leukemia virus (Mo-MuLV 1387) and was compared to a similarly derived group of MCF viruses from mice inoculated with Friend MuLV (Fr-MuLV 57). Antigenic analyses using monoclonal antibodies specific for MCF virus and xenotropic MuLV envelope proteins and genomic structural analyses by RNase T1-resistant oligonucleotide finger-printing indicated that the Moloney and Friend MCF viruses ...

  17. A monoclonal antibody toolkit for C. elegans.

    Directory of Open Access Journals (Sweden)

    Gayla Hadwiger

    Full Text Available BACKGROUND: Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. METHODOLOGY/PRINCIPAL FINDINGS: We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1, a component of synaptic vesicles; to Rim (UNC-10, a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1, a component of centrosomes; to CENP-C (HCP-4, which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2, a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5; to the nuclear envelope protein lamin (LMN-1; to EHD1 (RME-1 a marker for recycling endosomes; to caveolin (CAV-1, a marker for caveolae; to the cytochrome P450 (CYP-33E1, a resident of the endoplasmic reticulum; to beta-1,3-glucuronyltransferase (SQV-8 that labels the Golgi; to a chaperonin (HSP-60 targeted to mitochondria; to LAMP (LMP-1, a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7 of the 26S proteasome; to dynamin (DYN-1 and to the alpha-subunit of the adaptor complex 2 (APA-2 as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1 and cadherin (HMR-1, both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1, which localized to apical membranes; to an ERBIN family protein (LET-413 which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7 which localizes to the plasma membrane at cell-cell contacts. In addition to

  18. Identification and typing of herpes simplex viruses with monoclonal antibodies.

    OpenAIRE

    Balachandran, N; Frame, B; Chernesky, M; Kraiselburd, E; Kouri, Y; Garcia, D.; Lavery, C; Rawls, W. E.

    1982-01-01

    Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited num...

  19. Production and characterization of yeast killer toxin monoclonal antibodies

    OpenAIRE

    Polonelli, L; Morace, G

    1987-01-01

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

  20. Characterization of human serum spreading factor with monoclonal antibody.

    OpenAIRE

    Barnes, D W; Silnutzer, J; See, C; Shaffer, M

    1983-01-01

    Serum spreading factor is a glycoprotein isolated from human serum that promotes spreading of a variety of cell types on culture dishes. We developed mouse hybridoma lines secreting monoclonal antibody to serum spreading factor that markedly inhibited the rate of serum spreading factor-promoted spreading of both fibroblastic and epithelial cells in culture. Fibronectin-promoted cell spreading was unaffected by monoclonal antibody to serum spreading factor, and the factor appeared to be distin...

  1. Immunophenotypic studies of monoclonal gammopathy of undetermined significance

    OpenAIRE

    McKenna Robert W; Chen Weina; Wang Huan-You; Olteanu Horatiu; Karandikar Nitin J

    2008-01-01

    Abstract Background Monoclonal gammopathy of undetermined significance (MGUS) is a common plasma cell dyscrasia, comprising the most indolent form of monoclonal gammopathy. However, approximately 25% of MGUS cases ultimately progress to plasma cell myeloma (PCM) or related diseases. It is difficult to predict which subset of patients will transform. In this study, we examined the immunophenotypic differences of plasma cells in MGUS and PCM. Methods Bone marrow specimens from 32 MGUS patients ...

  2. Current research status of radioimmunotherapy monoclonal antibody drug

    International Nuclear Information System (INIS)

    Radioimmunotherapy (RIT) was one of the most important progresses in the field of cancer therapy over the past 20 years. It has been successfully applied in the treatment of blood system tumors such as NHL. For the utilization of RIT in therapy of solid tumors, however, development of more effective monoclonal antibodies, labeling methods and so on are needed. The current status of radionuclides, monoclonal antibodies and drugs commonly used in the RIT were briefly reviewed. (authors)

  3. Bone marrow stromal elements in murine leukemia

    International Nuclear Information System (INIS)

    A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNFα in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicity, reduced CSF production. (author)

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

    International Nuclear Information System (INIS)

    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

  5. Technetium-99m labeled monoclonal antibodies in the detection of metastatic melanoma

    International Nuclear Information System (INIS)

    Twenty-six stage II/III malignant melanoma patients with 321 measurable metastatic lesions were imaged using Fab fragments of an IgG murine monoclonal antibody labeled specifically with 10-30 mCi Tc-99m with a bi-functional chelating method (NeoRx, Seattle, WA). There were no side effects or adverse reactions. Immunoscintigraphy demonstrated 66.6% of lesions larger than 1 cm and 92.5% of lesions larger than 3 cm. Most frequently detected metastases were in lymph nodes, subcutaneous areas, and bone. Of lesions less than 1 cm, 23.6% were detected if superficial cutaneous lesions were excluded. The smallest detectable lesion was 4 mm. Twenty-one additional clinically unsuspected sites were visualized in 12 of the 26 patients studied. Of these, 56% were confirmed as metastasis by other tests. There were apparent nonspecific localizations owing to other causes, including fracture, varicosities, skin abscess and pneumonitis. Increased experience in image analysis facilitates correct interpretation of these localizations. This study demonstrates that imaging with Tc-99m labeled antibody fragments detects melanoma lesions in organs routinely surveyed and in other areas not routinely assessed by other imaging techniques. The procedure is readily performed and safe. The principal advantage of the test is its ability to survey the entire body and all organs with a single test. Its principal limitation, in common with other diagnostic imaging procedures, is its poor sensitivity for detecting lesions less than 1 cm

  6. 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: ikuta@biken.osaka-u.ac.jp [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)

    2009-09-11

    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.

  7. Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.

    Science.gov (United States)

    Wang, Zhanhui; Liang, Qi; Wen, Kai; Zhang, Suxia; Shen, Jianzhong

    2014-11-15

    The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). PMID:25261834

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

    International Nuclear Information System (INIS)

    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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Manabe,Yuichi

    1984-04-01

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

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

    Science.gov (United States)

    Zheng, Xiaojing; Li, Yanqing; Zhao, Junli; Wang, Dongyang; Xia, Haibin; Mao, Qinwen

    2016-01-01

    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. PMID:27018871

  12. 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

    2009-06-01

    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

  13. Production of monoclonal antibodies against canine leukocytes.

    Science.gov (United States)

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

    2004-04-01

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

  14. Technological progresses in monoclonal antibody production systems.

    Science.gov (United States)

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

    2010-01-01

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

  15. Monoclonal antibodies to human urinary thrombopoietin

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MA) to a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) were obtained from hybridomas derived from the fusion of P3 x 63/Ag 8 cells and spleen cells from TSF-immunized BALB/c mice. Media from several hybrid cultures were tested in a microantibody detection technique that measured the binding of MA to a 125I-purified TSF preparation from human embryonic kidney (HEK) cells. Hybridized cells were injected into pristane-primed mice and the antibodies produced in the ascites fluid were also shown to bind the 125I-TSF. Compared to the results of normal mouse serum, ascites fluid containing MA was shown to bind the unlabeled TSF from HEK cells. The TSF activity was significantly reduced in the supernatant fluid after precipitating the TSF-anti-TSF immune complex by a second antibody when tested in an immunothrombocythemic mouse assay. After SDS-PAGE, the precipitate from this TSF-Ma conjugate showed that the antiserum bound a single 32,000 mol wt component, indicating the monospecificity of the MA. MA directed toward human TSF will allow studies that were not previously possible

  16. Drug Development of Therapeutic Monoclonal Antibodies.

    Science.gov (United States)

    Mould, Diane R; Meibohm, Bernd

    2016-08-01

    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. PMID:27342605

  17. Characterization of monoclonal antibodies against fowl poxvirus.

    Science.gov (United States)

    Singh, P; Tripathy, D N

    2000-01-01

    Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells. PMID:10879917

  18. Licensed monoclonal antibodies and associated challenges.

    Science.gov (United States)

    Khan, Amjad Hayat; Sadroddiny, Esmaeil

    2015-12-23

    Monoclonal antibodies (mAbs) are the leading class of targeted therapeutics and remarkably effective in addressing autoimmune diseases, inflammations, infections, and various types of cancer. Several mAbs approved by US food and drug administration (FDA), are available on the market and a number are pending for approval. Luckily, FDA approved mAbs have played a pivotal role in the treatment and prevention of lethal diseases. However, claiming that licensed mAbs are 100% safe is still debatable, because infections, malignancies, anaphylactoid, and anaphylactic reactions are the more frequently associated adverse events. To evaluate benefit to risk ratio of mAbs, it is important for the clinical research staff or physicians to monitor and follow-up the patients who are receiving mAbs dozes. It is recommended that patients, physicians, biopharmaceutical companies, and researchers should keep in touch to highlight and resolve antibody-based adverse events. In this review we underscore the associated challenges of mAbs, approved by FDA from 2007-2014. PMID:27472864

  19. Monoclonal antibodies based on hybridoma technology.

    Science.gov (United States)

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

    2013-03-01

    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. PMID:24237029

  20. Monoclonal Antibodies for Systemic Lupus Erythematosus (SLE

    Directory of Open Access Journals (Sweden)

    Gabriella Moroni

    2010-01-01

    Full Text Available A number of monoclonal antibodies (mAb are now under investigation in clinical trials to assess their potential role in Systemic Lupus Erythematosus (SLE. The most frequently used mAb is rituximab, which is directed against CD20, a membrane protein expressed on B lymphocytes. Uncontrolled trials reported an improvement of SLE activity in non-renal patients and other studies even reported an improvement of severe lupus nephritis unresponsive to conventional treatments. However two randomized trials failed to show the superiority of rituximab over conventional treatment in non renal SLE and in lupus nephritis. Preliminary trials reported promising results with epratuzumab, a humanized mAb directed against CD22, and with belimumab, a human mAb that specifically recognizes and inhibits the biological activity of BLyS a cytokine of the tumornecrosis-factor (TNF ligand superfamily. Other clinical trials with mAb directed against TNF-alpha, interleukin-10 (Il-10, Il-6, CD154, CD40 ligand, IL-18 or complement component C5 are under way. At present, however, in spite of good results reported by some studies, no firm conclusion on the risk-benefit profile of these mAbs in patients with SLE can be drawn from the available studies.

  1. Murine Typhus in Child, Yucatan, Mexico

    OpenAIRE

    Zavala-Castro, Jorge E.; Zavala-Velázquez, Jorge E.; Uicab, Justo Eduardo Sulú

    2009-01-01

    A case of murine typhus in Yucatan was diagnosed in a child with nonspecific signs and symptoms. The finding of Rickettsia typhi increases the number of Rickettsia species identified in Yucatan and shows that studies are needed to determine the prevalence and incidence of rickettsioses in Mexico.

  2. Reemergence of Murine Typhus in the US

    Centers for Disease Control (CDC) Podcasts

    2015-04-21

    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.

  3. Radioimmunoscintigraphy of colorectal carcinoma using technetium-99m-labeled, totally human monoclonal antibody 88BV59H21-2.

    Science.gov (United States)

    Gulec, S A; Serafini, A N; Moffat, F L; Vargas-Cuba, R D; Sfakianakis, G N; Franceschi, D; Crichton, V Z; Subramanian, R; Klein, J L; De Jager, R L

    1995-12-01

    Radioimmunoscintigraphy (RIS) using human monoclonal antibodies offers the important clinical advantage of repeated imaging over murine monoclonal antibodies by eliminating the cross-species antibody response. This article reports a Phase I-II clinical trial with Tc-99m-labeled, totally human monoclonal antibody 88BV59H21-2 in patients with colorectal carcinoma. The study population consisted of 34 patients with colorectal cancer (20 men and 14 women; age range, 44-81 years). Patients were administered 5-10 mg antibody labeled with 21-41 mCi Tc-99m by the i.v. route and imaged at 3-10 and 16-24 h after infusion using planar and single-photon emission computed tomographic (CT) techniques. Pathological confirmation was obtained in 25 patients who underwent surgery. Human antihuman antibody (HAHA) titers were checked prior to and 1 and 3 months after the infusion. RIS with Tc-99m-labeled 88BV59H21-2 revealed a better detection rate in the abdomen-pelvis region compared with axial CT. The combined use of both modalities increased the sensitivity in both the liver and abdomen-pelvis regions. Ten patients developed mild adverse reactions (chills and fever). No HAHA response was detected in this series. Tc-99m-labeled human monoclonal antibody 88BV59H21-2 RIS shows promise as a useful diagnostic modality in patients with colorectal cancer. RIS alone or in combination with CT is more sensitive than CT in detecting tumor within the abdomen and pelvis. Repeated RIS studies may be possible, due to the lack of a HAHA response. PMID:7493345

  4. Monoclonal Antibodies to Conformational Epitopes of the Surface Glycoprotein of Caprine Arthritis-Encephalitis Virus: Potential Application to Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detecting Antibodies in Goat Sera

    OpenAIRE

    Özyörük, Fuat; Cheevers, William P.; Hullinger, Gordon A.; McGuire, Travis C.; Hutton, Melinda; Knowles, Donald P

    2001-01-01

    Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79–63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromato...

  5. Monoclonal antibodies to drosophila cytochrome P-450's

    International Nuclear Information System (INIS)

    Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins

  6. Monoclonal antibodies to drosophila cytochrome P-450's

    Energy Technology Data Exchange (ETDEWEB)

    Sundseth, S.S.; Kennel, S.J.; Waters, L.C.

    1987-05-01

    Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins.

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

    Directory of Open Access Journals (Sweden)

    Sophia R. Balderman

    2015-07-01

    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.

  8. 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

    2012-01-01

    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.

  9. Improved iodine radiolabels for monoclonal antibody therapy.

    Science.gov (United States)

    Stein, Rhona; Govindan, Serengulam V; Mattes, M Jules; Chen, Susan; Reed, Linda; Newsome, Guy; McBride, Bill J; Griffiths, Gary L; Hansen, Hans J; Goldenberg, David M

    2003-01-01

    A major disadvantage of (131)iodine (I)-labeled monoclonal antibodies (MAbs) for radioimmunotherapy has been the rapid diffusion of iodotyrosine from target cells after internalization and catabolism of the radioiodinated MAbs. We recently reported that a radioiodinated, diethylenetriaminepentaacetic acid-appended peptide, designated immunomedics' residualizing peptide 1 (IMP-R1), was a residualizing iodine label that overcame many of the limitations that had impeded the development of residualizing iodine for clinical use. To determine the factors governing the therapeutic index of the labeled MAb, as well as the factors required for production of radioiodinated MAb in high yield and with high specific activity, variations in the peptide structure of IMP-R1 were evaluated. A series of radioiodinated, diethylenetriaminepentaacetic acid-appended peptide moieties (IMP-R1 through IMP-R8) that differed in overall hydrophilicity and charge were compared. Radioiodinations of the peptides followed by conjugations to disulfide-reduced RS7 (an anti-epithelial glycoprotein-1 MAb) furnished radioimmunoconjugates in good overall incorporations, with immunoreactivities comparable to that of directly radioiodinated RS7. Specific activities of up to 8 mCi/mg and yields > 80% have been achieved. In vitro processing experiments showed marked increases in radioiodine retention with all of the adducts; radioiodine retention at 45 h was up to 86% greater in cells than with directly iodinated RS7. Each of the (125)I-peptide-RS7 conjugates was compared with (131)I-RS7 (labeled by the chloramine-T method) in paired-label biodistribution studies in nude mice bearing human lung tumor xenografts. All of the residualizing substrates exhibited significantly enhanced retention in tumor in comparison to directly radioiodinated RS7, but the nontarget uptakes differed significantly among the residualizing labels. The best labels were IMP-R4 and IMP-R8, showing superior tumor-to-non-tumor ratios

  10. Radioimmunoimaging of experimental gliomas using radiolabelled monoclonal antibodies

    International Nuclear Information System (INIS)

    The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab')2 fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy at 48 and 96 hours pointed to a superior tumour localisation by the fragments that was clearly related to the dose. Wholebody determinations of the biokinetic behaviour led to the following results: Faster clearance anc more ready elimination from the blood pool for the fragments, preferential uptake in the tumour; intact antibodies; binding in the liver, spleen and lungs. The study confirmed the value of fragments of monoclonal antibodies in the diagnosis of tumours and pointed to the possibility of using intact monoclonal antibodies as carriers of radioisotopes and cytotoxic drugs within the scope of therapeutic programmes. (TRV)

  11. Monoclonal antibodies against human placental glutathione transferase (class pi).

    Science.gov (United States)

    Massoud, R; Lo Bello, M; De Stefano, E; Molino, A; Zelaschi, D; Federici, G

    1991-02-01

    Five monoclonal antibodies (MAbs) were produced in a mouse hybridoma system against human placental glutathione transferase (GST pi). Four of these monoclonal antibodies, named 461 to 464, were of immunoglobulin G class, whereas the monoclonal antibody 465 was of IgA class. All these MAbs specifically recognized the glutathione transferase from human placenta (class pi) showing no cross reactivity against the basic and the neutral forms of GST from human liver. When each MAb was incubated with the GST pi, no inhibition of enzymatic activity towards 1-chloro-2,4-dinitrobenzene was observed except for MAb 465 which showed a slight inhibition to a serial dilution of 1:128. PMID:1709614

  12. Monoclonal gammopathy: The good, the bad and the ugly.

    Science.gov (United States)

    Glavey, Siobhan V; Leung, Nelson

    2016-05-01

    Monoclonal gammopathy of undetermined significance (MGUS) is a condition characterized by the presence of a monoclonal gammopathy (MG) in which the clonal mass has not reached a predefined state in which the condition is considered malignant. It is a precursor to conditions such as multiple myeloma or lymphoma at a rate of ~1%/year. Thus, from a hematologic standpoint, MGUS is a fairly benign condition. However, it is now recognized that organ damage resulting from just the MG without the need MM or lymphoma can occur. One of the most recognized is nephropathy secondary to monoclonal gammopathy of renal significance (MGRS). Other well-recognized conditions include neuropathies, oculopathies and dermopathies. Some conditions such as autoimmune diseases and coagulopathies are less common and recognized. Finally, systemic involvement of multiple organs is well described in several entities. In all of these conditions, the role of the MG is no longer insignificant. Thus, the term MGUS should be avoided when describing these entities. PMID:26732417

  13. Monoclonal regulatory T cells provide insights into T cell suppression.

    Science.gov (United States)

    Gubser, Céline; Schmaler, Mathias; Rossi, Simona W; Palmer, Ed

    2016-01-01

    Regulatory T cells (Tregs) have a crucial role in maintaining lymphocyte homeostasis. However an understanding of how Tregs function at a cellular and molecular level has not yet been fully elucidated. Here, we make use of a T cell receptor (TCR) transgenic, Rag(-/-) mouse expressing a Forkhead-Box-Protein P3 (Foxp3) transgene. This mouse provides a source of monoclonal CD4(+) Foxp3(+) T cells with a defined specificity. Here we show that monoclonal B3K506 Tregs are functional in vitro and in vivo and clearly require cognate antigen to be suppressive. We further show that the strength of Treg stimulation determines the strength of Treg mediated suppression. Finally we analysed various suppressive mechanisms used by monoclonal Tregs and found that Treg-Tconv proximity is a parameter, which correlates with enhanced suppression. PMID:27210828

  14. Efficacy of Wnt-1 monoclonal antibody in sarcoma cells

    International Nuclear Information System (INIS)

    Sarcomas are one of the most refractory diseases among malignant tumors. More effective therapies based on an increased understanding of the molecular biology of sarcomas are needed as current forms of therapy remain inadequate. Recently, it has been reported that Wnt-1/β-catenin signaling inhibits apoptosis in several cancers. In this study, we investigated the efficacy of a monoclonal anti-Wnt-1 antibody in sarcoma cells. We treated cell lines A-204, SJSA-1, and fresh primary cultures of lung metastasis of sarcoma with a monoclonal anti-Wnt-1 antibody. Wnt-1 siRNA treatment was carried out in A-204. We assessed cell death using Crystal Violet staining. Apoptosis induction was estimated by flow cytometry analysis (Annexin V and PI staining). Cell signaling changes were determined by western blotting analysis. We detected Wnt-1 expression in all tissue samples and cell lines. Significant apoptosis induction was found in monoclonal anti-Wnt-1 antibody treated cells compared to control monoclonal antibody treated cells (p < 0.02). Similarly, we observed increased apoptosis in Wnt-1 siRNA treated cells. Blockade of Wnt-1 signaling in both experiments was confirmed by analyzing intracellular levels of Dishevelled-3 and of cytosolic β-catenin. Furthermore, the monoclonal anti-Wnt-1 antibody also induced cell death in fresh primary cultures of metastatic sarcoma in which Wnt-1 signaling was active. Our results indicate that Wnt-1 blockade by either monoclonal antibody or siRNA induces cell death in sarcoma cells. These data suggest that Wnt-1 may be a novel therapeutic target for the treatment of a subset of sarcoma cells in which Wnt-1/β-catenin signaling is active

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

    OpenAIRE

    Marković Ljiljana; Ašanin Ružica; Radojičić Sonja; Isenović Esma

    2007-01-01

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

  16. Studies on radiolabelling of monoclonal antibodies with 99Tcm and other radionuclides for scintigraphy

    International Nuclear Information System (INIS)

    This work performed on the development of radiolabelling of monoclonal antibodies for scintigraphy using direct 99Tcm labelling and other radiolabelling methods of monoclonal antibodies with In-111, Ga-67 or Ru-103

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

    International Nuclear Information System (INIS)

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

  18. Retroviral Transduction of Murine Primary T Lymphocytes

    Science.gov (United States)

    Lee, James; Sadelain, Michel; Brentjens, Renier

    2016-01-01

    Summary In comparison to human T cells, efficient retroviral gene transfer and subsequent expansion of murine primary T cells is more difficult to achieve. Herein, we describe an optimized gene transfer protocol utilizing an ecotropic viral vector to transduce primary murine T cells activated with magnetic beads coated with agonistic anti-CD3 and CD28 antibodies. Activated T cells are subsequently centrifuged (spinoculated) on RetroNectin-coated tissue culture plates in the context of retroviral supernatant. Variables found to be critical to high gene transfer and subsequent efficient T cell expansion included CD3/CD28 magnetic bead to cell ratio, time from T cell activation to initial spinoculation, frequency of T cell spinoculation, interleukin-2 concentration in the medium, and the initial purity of the T cell preparation. PMID:19110621

  19. Murine Typhus: Clinical and epidemiological aspects

    Directory of Open Access Journals (Sweden)

    Gaspar Peniche Lara

    2012-06-01

    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.

  20. Eliminating Murine Norovirus by Cross-Fostering

    OpenAIRE

    Buxbaum, Laurence U.; DeRitis, Pierina C.; Chu, Niansheng; Conti, Pierre A.

    2011-01-01

    Murine norovirus (MNV) is a newly discovered and extremely prevalent pathogen of laboratory mouse colonies. MNV causes severe disease in some immunocompromised mouse strains and can cause persistent infections even in immunocompetent mice. Despite the fact that immunocompetent mice are generally asymptomatic, the possibility that MNV infection might alter immune responses makes its eradication a potentially useful goal for many facilities. Initial attempts by others to use a strategy of testi...

  1. Signaling pathways regulating murine pancreatic development

    DEFF Research Database (Denmark)

    Serup, Palle

    2012-01-01

    The recent decades have seen a huge expansion in our knowledge about pancreatic development. Numerous lineage-restricted transcription factor genes have been identified and much has been learned about their function. Similarly, numerous signaling pathways important for pancreas development have...... been identified and the specific roles have been investigated by genetic and cell biological methods. The present review presents an overview of the principal signaling pathways involved in regulating murine pancreatic growth, morphogenesis, and cell differentiation....

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

    OpenAIRE

    Booman, P.

    1989-01-01

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

  3. Cell-free translation of murine coronavirus RNA.

    OpenAIRE

    Leibowitz, J L; Weiss, S.R.; Paavola, E; Bond, C W

    1982-01-01

    The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These stud...

  4. 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; Ingvarsen, S; Porse, A; Behrendt, N; Cieplak, P; Strongin, A Y

    2013-01-01

    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......, prevents 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...... its well-controlled conversion into the mature MMP-2 enzyme. In sum, understanding of the structural requirements for the 9E8 antibody specificity may pave the way for the focused design of the inhibitory antibodies against other individual MMPs....

  5. Variations of the pharmakocinetic in rabbits of the monoclonal antibody ior t1 produced by the radioiodonation with the chloramina T and iodogen methods

    International Nuclear Information System (INIS)

    The monoclonal antibody ior t1, an IgG2a was labeled with 125I, using the chloramine T and iodogen methods. Immunoreactivity against human lymphocites in vitro was affected in a significant way, mostly with chloramine T methods. In F1 male rabbits, the plasma radioactivity declined in apparently bioexponential manner in the administration of unlabeled ior t1, measured by an specific ELISA to murine IgG, and with the use of chloramine T. A monoexponential declined with the iodogen reagent was observed. We consider the possible of an unspecific binding in blood in the experiment with iodogen reagent. The t-tes student analysis show significant differences between the unlabeled protein and both methods of radioiodination, that differences must be have their origin in the high specific activity when labeled with chloramine T and in the probably of non-specific binding when we employs the iodogen reagents

  6. Indium-111 labeled anti-melanoma monoclonal antibodies

    Science.gov (United States)

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

  7. Palladium-109 labeled anti-melanoma monoclonal antibodies

    Science.gov (United States)

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  9. Radioimmunodetection of human tumors with radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    The present study reports the use of radiolabeled monoclonal antibodies prospectively as a diagnostic method in order to localize tumor sites in patients with a suspicion of recurrence or metastasis based on isolated elevation of serum tumor markers. Results of immunoscintigraphy are compared to data obtained with more conventional investigations including essentially ultra sonography and CT scan. (Auth.)

  10. Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Bo Wang

    2009-01-01

    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.

  11. Monoclonal antibodies to Herpes Simplex Virus Type 2

    International Nuclear Information System (INIS)

    In this thesis the production and characterisation of monoclonal antibodies to Herpes Simplex Virus Type 2 is described. The development of a suitable radioimmunoassay for the detection of anti-HSV-2 antibodies, and the selection of an optimal immunisation schedule, is given. Three assay systems are described and their reliability and sensitivity compared. (Auth.)

  12. Side-effects of monoclonal antibodies during immunoscintigraphy

    International Nuclear Information System (INIS)

    When monoclonal antibodies, most of which are developed from mouse hybridomas, are injected into the patient they are recognized as foreign globulins. The resulting immune response leads to the development of human anti-mouse antibodies or so called side-effects. (author). 1 ref

  13. Novel electrokinetic approaches to improve purification processes with monoclonal antibodies

    OpenAIRE

    Faude, Alexander

    2009-01-01

    This work was focussed on mAb separations using cation exchange and hydrophobic interaction chromatography. Methods to accelerate long winded development strategies of purification processes with monoclonal antibodies were developed facilitated by further improvement of understanding the basic adsorption mechanisms of proteins on chromatographic resins. The new experimental electrokinetic methods introduced are zeta potential determination with proteins via laser light scattering and electro-...

  14. The significance of monoclonal gammopathy of undetermined significance

    OpenAIRE

    Kyle, Robert A.; Kumar, Shaji

    2009-01-01

    Since it is now well established that all patients with multiple myeloma have a preceding monoclonal gammopathy of undetermined significance (MGUS), identification of potential risk factors for the progression becomes most important. In this perspective article, Drs. Kyle and Kumar highlight the need for a better understanding of the etiology and biology of MGUS. See related paper on page 1714.

  15. Prevention of Progression in Monoclonal Gammopathy of Undetermined Significance

    OpenAIRE

    Rajkumar, S. Vincent

    2009-01-01

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

  16. Production and characterization of monoclonal antibodies against mink leukocytes

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  17. MONOCLONAL ANTIBODIES TO IDENTIFY TOMATO MOSAIC TOBAMOVIRUS (TOMV

    Directory of Open Access Journals (Sweden)

    Duarte Keila M.R.

    2001-01-01

    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.

  18. Development of monoclonal antibodies that recognize Treponema pallidum.

    OpenAIRE

    Saunders, J. M.; Folds, J D

    1983-01-01

    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.

  19. Characterization of Binding Epitopes of CA125 Monoclonal Antibodies

    DEFF Research Database (Denmark)

    Marcos-Silva, Lara; Narimatsu, Yoshiki; Halim, Adnan;

    2014-01-01

    The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics ...

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

    Czech Academy of Sciences Publication Activity Database

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

    2003-01-01

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

  1. Specific and nonspecific imaging of localized Fisher immunotype 1 Pseudomonas aeruginosa infection with radiolabeled monoclonal antibody

    International Nuclear Information System (INIS)

    To determine if radiolabeled specific antibodies directed against bacterial antigens could be used to detect sites of infection, gamma camera imaging studies were performed in animals infected with Pseudomonas aeruginosa. Murine monoclonal antibodies (Mabs) directed against Fisher Immunotype 1 Pseudomonas aeruginosa and a nonmicrobial, nonmammalian haptene, p-arsanilic acid, were labeled with 125I by the lodogen-Bead method. Unilateral, deep thigh infections were created by innoculation with 2 X 10(8) Fisher Immunotype 1 P. aeruginosa. Twenty-four hours later, one of the radiolabeled antibodies was injected intravenously at a dose of 0.25 mg/kg (100-150 microCi). Serial gamma imaging was then carried out beginning at 4 hr and at approximately 24-hr intervals thereafter. Beginning as early as 4 hr postinjection, the area of inflammation could be visualized with either the specific or nonspecific Mab, with the images continuing to intensify until 24-48 hr postinjection. At 48 hr, the contrast between lesion and background with the nonspecific Mab began to fade, while the contrast in the specific Mab-generated images continued to intensify until approximately 192 hr postinjection. Clear-cut differentiation between specific and nonspecific Mab-generated images was possible by 72 hr postinjection. We conclude that specific immune imaging of localized infection with Mab's directed against specific microbial antigens is possible and should be clinically useful. In addition, images created by the localization of immunoglobulin non-specifically at the site of inflammation in the first 24-48 hr postinjection may also provide useful information as to the anatomic location of hidden abscesses

  2. Positron emission tomography imaging of tumor angiogenesis with a 66Ga-labeled monoclonal antibody.

    Science.gov (United States)

    Engle, Jonathan W; Hong, Hao; Zhang, Yin; Valdovinos, Hector F; Myklejord, Duane V; Barnhart, Todd E; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

    2012-05-01

    The goal of this study was to develop a (66)Ga-based positron emission tomography (PET) tracer for noninvasive imaging of CD105 expression during tumor angiogenesis, a hallmark of cancer. (66)Ga was produced using a cyclotron with (nat)Zn or isotopically enriched (66)Zn targets. TRC105, a chimeric anti-CD105 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (66)Ga. No difference in CD105 binding affinity or specificity was observed between TRC105 and NOTA-TRC105 based on flow cytometry analysis. Reactivity of (66)Ga for NOTA, corrected to the end of bombardment, was between 74 and 222 GBq/μmol for both target enrichments with 80% radiochemical yield. Serial PET imaging revealed that the murine breast cancer 4T1 tumor uptake of (66)Ga-NOTA-TRC105 was 5.9 ± 1.6, 8.5 ± 0.6, and 9.0 ± 0.6% ID/g at 4, 20, and 36 h postinjection, respectively (n = 4). At the last time point, tumor uptake was higher than that of all organs, which gave excellent tumor contrast with a tumor/muscle ratio of 10.1 ± 1.1. Biodistribution data as measured by gamma counting were consistent with the PET findings. Blocking experiment, control studies with (66)Ga-NOTA-cetuximab, as well as ex vivo histology all confirmed the in vivo target specificity of (66)Ga-NOTA-TRC105. Successful PET imaging with high specific activity (66)Ga (>700 GBq/μmol has been achieved) as the radiolabel opens many new possibilities for future PET research with antibodies or other targeting ligands. PMID:22519890

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

    Directory of Open Access Journals (Sweden)

    Qian Yajuan

    2011-05-01

    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.

  4. Monoclonal antibodies and coupling reagents to cell membrane proteins for leukocyte labeling

    International Nuclear Information System (INIS)

    Current gamma-emitting agents for tagging leukocytes, In-111 oxine or tropolone, label all cell types indiscriminantly, and nuclear localization in lymphocytes results in radiation damage. Coupling reagents and murine monoclonal antibodies (Mab) specific for cell surface antigens of human leukocytes were tried as cell labeling agents to avoid nuclear localization. 10/sup 8/ mixed human leukocytes in Hepes buffer were added to tubes coated with 5 mg of dry cyclic dianhydride of DTPA for 15 minutes at room temperature. After washing, 0.1 ml of In-111 Cl in ACD (pH 6.8) was added. After 30 minutes, a cell labeling yield of 23% was obtained. Washing the cells in an elutriation centrifuge showed that this label was irreversible. Mab for cell surface antigens of human granulocytes were labeled with 300 μCi of I-125 using the Iodobead technic and unbound activity was removed by gel column chromatography. 1-10 μg were added to 10/sup 8/ mixed leukocytes in 0.5 ml plasma or saline for 1 hr. With Mab anti-leu M4 (clone G7 E11), an IgM, the cell labeling yield was 21%, irreversible, and specific for granulocytes. With anti-human leukocyte Mab NEI-042 (clone 9.4), and IgG2a, and anti-granulocyte Mab MAS-065 (clone FMCl1) an IgG1, the cell labeling was relatively unstable. Labeling of leukocyte subpopulations with Mab is feasible, and the binding of multivalent IgM is stronger than that of other immunoglobulins. DTPA cyclic anhydride is firmly bound to cell membranes, but the labeling is non-specific

  5. Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

    International Nuclear Information System (INIS)

    Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

  6. A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells.

    Science.gov (United States)

    Weiher, Hans; Pircher, Haymo; Jansen-Dürr, Pidder; Hegenbarth, Silke; Knolle, Percy; Grunau, Silke; Vapola, Miia; Hiltunen, J Kalervo; Zwacka, Ralf M; Schmelzer, Elmon; Reumann, Kerstin; Will, Hans

    2016-01-01

    Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion. PMID:26921094

  7. Existence of multiple isoforms of HS1-associated protein X-1 in murine and human tissues.

    Science.gov (United States)

    Lees, Delphine M; Hart, Ian R; Marshall, John F

    2008-06-13

    To date, the literature concerning the HS1 (haematopoietic cell-specific protein 1)-associated protein X-1 (HAX1) protein has reported considerable variation regarding its function in mammalian cells, subcellular localisation and binding partners. We show here that HAX1 comprises a family of proteins. Murine tissues express three mRNA variants, encoded by two genes on chromosomes 2 and 3. The chromosome 2 gene is intronless and would encode a protein 100% identical with that encoded by chromosome 3. In humans, alternative splice variants, encoded by the chromosome 1 gene, produce a family of transcripts composed of up to eight members. Based on the sequences published in GenBank and Ensembl, we designed specific primers and detected by PCR three mRNA species in murine tissues and eight variants in human cells. We screened a panel of 19 human cell lines as well as primary fibroblasts, oral keratinocytes and freshly isolated peripheral blood mononuclear cells. All human cells studied expressed at least six of the possible HAX1 mRNA variants. In silico analysis of the variants revealed an open reading frame in all of them, suggesting that murine and human tissues can express two and eight HAX1 proteins, respectively. Analysis of human protein lysates by Western blotting with the use of a monoclonal anti-HAX1 antibody revealed multiple bands. These bands were decreased after treatment of cells with a single small interfering RNA duplex targeting a region common to six of the variants, confirming their identity as HAX1 proteins. Comparison of the human variants with the six HAX1 homologues described to date in the chimpanzee (Pan troglodytes) and the four homologues described in macaque (Macaca mulatta) revealed very high conservation with only one amino acid substitution between human and chimpanzee homologues. Moreover, a number of additional products were amplified and sequenced, which indicated that further human isoforms are likely to exist. These findings are

  8. Murine erythrocytes contain high levels of lysophospholipase activity

    NARCIS (Netherlands)

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

    1984-01-01

    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

  9. Requirements for the triggering of lysis by cloned murine cytolytic T lymphocytes (CTL)

    Energy Technology Data Exchange (ETDEWEB)

    Lancki, D.W.; Weiss, A.; Fitch, F.W.

    1986-03-05

    Cloned murine cytolytic T lymphocytes (CTL) having defined specificity were triggered by the phorbol ester PMA + a calcium ionophore (either A23187 or Ionomycin) to lyse syngeneic or third party target cells efficiently. Neither PMA nor calcium ionophore alone induced efficient lysis. The characteristics of the lytic process induced by these signals are similar to those of antigen specific or lectin facilitated lysis by CTL: lysis is calcium and temperature dependent, and shows directionality and kinetics similar to lysis mediated via the antigen receptor. Helper T lymphocytes were not induced by these agents to lyse target cells efficiently. Activation of L3 CTL clone lysis by PMA + ionophore differed from antigen-mediated activation in specificity and in susceptibility to inhibition by cytochalasin B, an inhibitor of antigen mediated conjugate formation. Anti-LFA-1 monoclonal antibodies inhibited antigen-mediated, lectin-facilitated, or PMA + ionophore induced lysis. Anti-Lyt-2 and anti-receptor antibodies had little if any effect on lysis induced by PMA + ionophore. The triggering of lysis with PMA + ionophore appears to involve intracellular signals similar to those involved in lysis mediated by the antigen receptor.

  10. Radiolabelling of monoclonal antibodies with technetium-99 m via metallothionein

    International Nuclear Information System (INIS)

    Metallothionein (MT), a small cysteine-rich protein, was used as a bifunctional chelating agent in the radiolabelling of monoclonal antibodies with Tc-99m. The efficiency of the conjugation reaction of MT with antibodies (Ab) was found as 58%. The yield of radiolabelling of Tc-99m to MT-Ab by reduction method was higher than 90%, while the unspecific radiolabelling occurred less than 10%. The Tc-99m-MT-Ab has proven to be satisfactory stable in Vitro in the presence of a couple of strong chelating agents. The preliminary biological experimental results in tumor-bearing nude mice indicated that the Tc-99m-labelled anti-colorectal carcinoma monoclonal antibody 2C10 had strong affinity toward tumor and was stable in vivo

  11. Monoclonal antibodies to cell surface antigens of human melanoma

    International Nuclear Information System (INIS)

    The authors have worked with three human melanoma antigens which have been defined by monoclonal mouse antibodies: p97, a glycoprotein that is structurally related to transferrin, a proteoglycan, and a GD3 ganglioside that is slightly different from the GD3 of normal brain. All three antigens can be detected in frozen sections of melanoma, using immunohistological techniques. Antibodies and Fab fragments, specific for either p97 or the proteoglycan antigen, have been radiolabelled with 131I and successfully used for tumor imaging, and Phase I therapeutic trails are underway, using 131I-labelled Fab fragments, specific for p97 or the proteoglycan antigen, to localize a potentially therapeutic dose of radiation into tumors. It may be feasible to use the same monoclonal antibodies, or antibody fragments, as carriers of neutron capturers, such as boron, for possible use in tumor therapy. The initial experiments on this are best carried out by using nude mice (or rats) carrying human melanoma xenografts

  12. Choriocarcinoma: blocking factor and monoclonal antibody iodine 131 imaging

    Energy Technology Data Exchange (ETDEWEB)

    Pattillo, R.A.; Khazaeli, M.B.; Ruckert, A.C.; Hussa, R.O.; Collier, B.D.; Beierwaltes, W.; Mattingly, R.F.

    1984-04-01

    Postoperative iodine 131 monoclonal antibody localization in metastatic choriocarcinoma was accomplished in this study. The monoclonal antibody was prepared to male choriocarcinoma which cross reacted with gestational choriocarcinoma. The antibody was raised against whole choriocarcinoma cells and human chorionic gonadotropin (hCG) cross reactivity was excluded. The purified antibody was iodinated with /sup 131/I and successfully imaged BeWo choriocarcinoma transplanted in nude mice; however, imaging of choriocarcinoma in a patient was verified only after resection. It is our belief that failure to sufficiently concentrate the antibody in the tumor before operation was due to blocking factor in the serum of the patient. Blocking factor and hCG dropped postoperatively. Blocking factor activity in 15 patients with metastatic trophoblastic disease was monitored and, like hCG, was found to be a sensitive indicator of the presence of disease. Its efficacy may be in the small number of patients without hCG but with persistent disease.

  13. Development of radiolabelling techniques of anti-CEA monoclonal antibody

    International Nuclear Information System (INIS)

    The purpose of this work was to label monoclonal and polyclonal antibodies with 99Tcm such as the ior-CEA-1 antibody and polyclonal IgG using a direct method, to check the radiochemical and biological behavior of labelled products, to prepare it under sterile and apyrogenic conditions as a lyophilized kit and to employ it in clinical trials. In addition, a photoactivation method was used to label polyclonal IgG with 99Tcm and to compare with the established method using mercaptoethanol (2-ME) as the reducing agent. Finally polyclonal IgG was labelled using an indirect method in which a chelator was covalently attached to the protein and the 99Tcm added as glucoheptonate complex. The properties of 99Tcm when labelled with monoclonal and polyclonal antibodies by different methods were assessed by in vitro and in vivo studies

  14. Preparation and identification of monoclonal antibodies against Ractopamine

    International Nuclear Information System (INIS)

    In order to prepare the monoclonal antibody against Ractopamine (RCT), RCT-conjugated antigen was produced by methods of mixed acid anhydride. The spleen cells of BALB/c mice immunized by RCT-BSA were fused with SP2/0 plasmacytoma cells using PEG4000. A hybridoma cell line of 3E1-C9-E10 was screened for specificity to RCT and cloned by limited dilution method, which secreted stable monoclonal antibodies against RCT with indirect ELISA titers of 1 x 105 in supernatant, 1 x 107 in ascites. The McAb of 3E1-C9-E10 generally had 24% cross-reactivity to Dobutamine, and showed little or no cross-reactivity to Salbutamol and Clenbuterol. (authors)

  15. 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.

  16. Murine Typhus: Clinical and epidemiological aspects

    Directory of Open Access Journals (Sweden)

    Gaspar Peniche Lara

    2012-06-01

    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.

  17. A monoclonal antibody against a canine CD45 homologue: analysis of tissue distribution, biochemical properties and in vitro immunological activity.

    Science.gov (United States)

    Aguiar, Paulo Henrique Palis; Barrouin-Melo, Stella Maria; Franke, Carlos Roberto; dos Santos, Roberto Robson Borges; Silva, Tânia Maria Correia; Mengel, José O; dos-Santos, Washington Luis Conrado; Pontes-de-Carvalho, Lain

    2007-01-01

    This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs. PMID:16249107

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

    Directory of Open Access Journals (Sweden)

    Jamie Heimburg

    2006-11-01

    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.

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

    Science.gov (United States)

    Yoshinari, K; Arai, K

    1998-02-01

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

  1. Production of mouse monoclonal antibodies for the analysis of idiotypes in serum of patients with chronic lymphatic leukaemia.

    Science.gov (United States)

    de Rie, M A; van Heemstra, D J; Huijgens, P C; Zeijlemaker, W P; Out, T A; Melief, C J; von dem Borne, A E

    1988-01-01

    In this report a simple procedure for the production of murine monoclonal antibodies (MoAb) against the idiotype of malignant B cells is described. Mice were immunized with lymphoid cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL). After fusion of the spleen cells, hybridoma supernatants were screened for anti-idiotypic MoAb in ELISA with immunoglobulins obtained from tumour-cell lysates, xenohybridomas and patients' sera. The anti-idiotypic MoAb were used to study tumour cells and serum immunoglobulins (Ig) from four different patients with B-CLL. It was found that the serum IgM and IgD in one patient shared the same idiotype. Evidence is presented that IgG-secreting cell populations are not restricted to lambda-Ig-light chain-expressing B-CLL cells. With the help of anti-idiotype MoAb accurate measurements of total and idiotype-positive serum immunoglobulin levels during chemotherapy were possible. PMID:3257881

  2. Long-term treatment of pigs with low doses of monoclonal antibodies against porcine CD4 and CD8 antigens

    DEFF Research Database (Denmark)

    Lohse, Louise; Nielsen, Jens; Eriksen, Lis

    2006-01-01

    In vivo depletion of lymphocyte subsets allows investigation of the role of specific subsets in protective immunity. In the present study we evaluated the effects of long-term, low-dose treatment with murine monoclonal antibodies (mAbs) against porcine CD4 and CD8 surface antigens on lymphocyte....... Treatment with the anti-CD4 mAb almost completely eliminated the CD4(+) T-cell subset from the circulation after 2 weeks of therapy. This depletion persisted until the end of the experimental period 5 weeks after initiated therapy. Treatment with the anti-CD8 mAb was less effective, reducing the CD8(+) T......-cell subset in peripheral blood by approximately 50% of the initial level after 3 weeks of therapy. Further, the anti-CD8 mAb-treated pigs showed a parallel increase in the CD4(+) T-cell subset from day 7. Two-colour FCM analysis indicated that a shift in phenotype from single-positive CD4(+)/CD8(-) to double...

  3. Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis.

    Science.gov (United States)

    Marchetti, Roberta; Dillon, Michael J; Burtnick, Mary N; Hubbard, Mark A; Kenfack, Marielle Tamigney; Blériot, Yves; Gauthier, Charles; Brett, Paul J; AuCoin, David P; Lanzetta, Rosa; Silipo, Alba; Molinaro, Antonio

    2015-10-16

    Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections. PMID:26198038

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

    Directory of Open Access Journals (Sweden)

    Kazuko Kobayashi

    2015-01-01

    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.

  5. Pharmacokinetics of the monoclonal antibody B72.3 and its fragments labeled with either 125I or 111In

    International Nuclear Information System (INIS)

    A comparison of the pharmacokinetics of intact B72.3 (a murine monoclonal antibody specific for human breast and colon carcinoma) with F(ab')2 and Fab fragments labeled with 111In and 125I was done in athymic mice bearing target (LS174T) and non-target (HCT-15) tumors. IgG B72.3 labeled with either isotype imaged LS174T. Biodistributions of both labels were similar in all organs except liver. F(ab')2 also imaged the LS174T tumor, while Fab bearing either isotype did not. The blood clearance was Fab greater than F(ab')2 greater than immunoglobulin G B72.3 for both isotopes. 111In-labeled fragments yielded large accumulations in the kidneys which persisted for 2 days. The different patterns of biodistribution for the various forms of B72.3 labeled with the two isotopes suggest that the most desirable combination of fragment and isotope will depend on the intended use

  6. Microelectrochemical radioiodination of monoclonal antibody: a preliminary study

    International Nuclear Information System (INIS)

    The optimal reaction conditions for the microelectrochemical iodination of immunoglobulins were determined with non-specific human serum immunoglobulins. These conditions were used for the efficient radioiodination of a monoclonal antibody, 140.240, in submilligram quantities. An approximately five-fold decrease in the titre of the antibody against melanoma cells, as determined by the miniaturized mixed hemadsorption assay, was observed after iodination with an average of 0.85 atoms of iodine per molecule of antibody. (author)

  7. T-cell detection with monoclonal antibody T101 kits.

    OpenAIRE

    Pollack, S M; Cimino, E F; Robbins, D S; Hoffman, P M

    1986-01-01

    A solid-phase immunoadsorption procedure (Quantigen T&B cell kit; Bio-Rad Laboratories, Richmond, Calif.) employing monoclonal antibody T101 detected mean percentages of peripheral blood T cells comparable to those obtained by rosetting with sheep erythrocytes, while lower values were obtained with an indirect immunofluorescence procedure (Cytotag T&B cell kit; Hybritech, Inc., San Diego, Calif.) employing the same antibody. Therefore, T101 binding appears to be more easily detected by solid-...

  8. Guidelines to cell engineering for monoclonal antibody production

    OpenAIRE

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mino,Yasuaki

    1984-10-01

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

  10. Development of Monoclonal Antibodies in China: Overview and Prospects

    OpenAIRE

    2015-01-01

    Monoclonal antibodies (mAbs) have become increasingly important as human therapeutic agents. Yet, current research concentrates on technology itself and pays attention to developed countries. This paper aims to provide a comprehensive review of mAbs development in China through systematic analysis of drug registry, patent applications, clinical trials, academic publication, and ongoing R&D projects. The trends in therapeutic areas and industrialization process are also highlighted. Developmen...

  11. Transformation-related antigens identified by monoclonal antibodies.

    OpenAIRE

    Strand, M

    1980-01-01

    Tumor-cell proteins that were antigenic in a syngeneic animal were identified by immunoprecipitation with monoclonal antibodies. Spleen cells of BALB/c mice immunized with plasma membranes of Kirsten RNA sarcoma virus-transformed BALB/3T3 cells were fused with NS-1 myeloma cells. Antibodies secreted into the culture fluid from these hybridomas were distinguished by their reactivity against proteins of different target cells. A total of 191 cultures were established; 143 produced antibodies th...

  12. Sources of DNA for detecting B cell monoclonality using PCR.

    OpenAIRE

    Diss, T. C.; Pan, L.; Peng, H; Wotherspoon, A C; Isaacson, P G

    1994-01-01

    AIMS--To evaluate the polymerase chain reaction (PCR) demonstration of clonal immunoglobulin heavy chain gene rearrangements using routinely prepared, unstained, and stained formalin fixed, paraffin wax embedded tissue samples. METHODS--Extracts from (a) fresh frozen tissue samples, (b) unstained, and (c) haematoxylin and eosin stained formalin fixed, paraffin wax embedded 5 microns tissue sections from 42 cases of low grade B cell lymphoma, all shown to be monoclonal by Southern blot analysi...

  13. Screening Panels for Monoclonal Gammopathies: Time to Change

    OpenAIRE

    Katzmann, Jerry A.

    2009-01-01

    The introduction of quantitative assays for serum free light chains (FLC) has changed the approach to screening for monoclonal gammopathies. Recent guidelines from the International Myeloma Working Group have recommended the use of serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE) and FLC as the screening panel unless primary amyloidosis (AL) is suspected. If screening for AL, then urine IFE should also be performed. We discuss the background for these recommendations...

  14. Axonal neuropathy associated with monoclonal gammopathy of undetermined significance

    OpenAIRE

    GORSON, K.; Ropper, A.

    1997-01-01

    OBJECTIVE—The neuropathy associated with monoclonal gammopathy of undetermined significance (MGUS) is typically a predominantly demyelinating process that may have additional features of axonal degeneration. Sixteen patients with MGUS and a pure or predominantly axonal neuropathy are reported and compared with 20 consecutive patients with demyelinating neuropathy and MGUS who were seen during the same period.
METHODS—Retrospective review of a consecutive series of patients w...

  15. Monoclonal gammopathy of undetermined significance associated with blue finger syndrome

    OpenAIRE

    Alaa M. Ali; Aibek E. Mirrakhimov

    2013-01-01

    An 86-year-old Caucasian woman with a medical history of monoclonal gammopathy of undetermined significance (MGUS) was admitted to the hospital with a chief complaint of sudden onset of bluish discolouration of the fifth left hand digit. On a physical examination, cyanosis of the fifth digit of the left hand was noticed with decreased capillary fill but no ulcers. The patient had no tenderness on palpation. Pulses were palpable over the radial arteries bilaterally. Patients with MGUS may be a...

  16. Prevalence of monoclonal gammopathy in HIV patients in 2014

    OpenAIRE

    Philippe Genet; Laurent Sutton; Driss Chaoui; Ahmad Al Jijakli; Juliette Gerbe; Virginie Masse; Bouchra Wifaq

    2014-01-01

    Introduction: In non-HIV patients, Monoclonal Gammopathy of Undetermined Significance (MGUS) is associated with an increased risk of subsequent development of haematologic malignancies, especially multiple myeloma (MM) and it has been recently demonstrated that MM is always preceded by a MGUS phase. A higher prevalence of MGUS and MM has been observed in HIV patients compared to the general population. Nevertheless, it has been shown that MGUS in the context of HIV can disappear with antiretr...

  17. Production of Bartonella Genus-Specific Monoclonal Antibodies

    OpenAIRE

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

    2001-01-01

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

  18. Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy

    OpenAIRE

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

    2015-01-01

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

  19. Monoclonal antibody radioimmunodetection of human-derived colon cancer

    International Nuclear Information System (INIS)

    This study was designed to determine whether monoclonal antibody directed against carcinoembryonic antigen could successfully be used in the scintigraphic localization of a human-derived colon carcinoma in a hamster model. An immunoglobulin G (IgG)-1 kappa monoclonal antibody, prepared in this laboratory, against carcinoembryonic antigen (CEA) was radiolabeled with iodine-131 (131I). Four Syrian hamsters bearing GW-39 human colon cancers received intracardiac injections of 50 mu Ci of 131I (14 micrograms of antibody). Gamma camera images were obtained at 24-hour intervals. Animals were sacrificed at 11 days, and the tumors and entire animals were counted. A double-label antibody experiment was conducted with 131I anti-CEA and nonspecific MOPC 21 IgG iodine-125 (125I) to assess localization specificity. The scintiphotos clearly showed the tumor at 24 hours, but there was significant background (blood-pool activity). Later images at six and 11 days showed a gradual decrease in background activity and more clear definition of the tumor. Animals sacrificed at 11 days showed 48-80% of residual whole body radioactivity to be present in the tumor. However, these tumors were large at sacrifice, weighing 8.9 to 12.4 g. Specific localization was confirmed by the double-label experiments where specific localization was twice nonspecific accretion of IgG in the tumor. This study has shown that a specific monoclonal antibody can successfully be used to scintigraphically localize a colon tumor of human origin. Although clearance of background activity is a gradual process, eventually most radioactivity left in the animal is localized in the tumor. This study illustrates that the potential radiolabeled monoclonal antibodies hold as immunodiagnostic agents

  20. [Hashimoto's thyroiditis and monoclonal gammapathy (two cases) (author's transl)].

    Science.gov (United States)

    Groussin, P; Fine, J M; Leroux, E; Lebranchu, Y; Lecomte, P; Bertrand, G

    1979-01-01

    The authors describe two cases of Hashimoto's thyroiditis with a monoclonal dysproteinemia (IgG lambda in one case and IgG kappa in the other). They show that the antithyroid activity (anti-microsomial in the first case, anti thyroglobulin in the second case) is not carried by the abnormal gammaglobulin. The two diseases, therefore, appear to be separate but can both be considered as the consequence of a particular immunological state. PMID:117737

  1. [Renal involvement in benign monoclonal gammopathies: an underdiagnosed condition].

    Science.gov (United States)

    Ramos, R; Poveda, R; Bernís, C; Ara, J; Sunyer, M; Arrizabalaga, P; Grinyó, J M

    2008-01-01

    Renal involvement is observed frequently in association with malignant gammopathies, mainly those related to light chain deposition, although has also been described in non-malignant monoclonal gammopathy. This study reports the clinicopathological findings and outcome in 9 patients with nephropaty secondary to monoclonal immunoglobulin deposit in absence of malignancy. They were three men and six women and they were 59.2+/-12 years old. All patients presented proteinuria and different levels of renal insufficiency (mean creatinin = 315+/-187 micromol/L) at the moment of diagnostic. Two patients required dialysis at the time of renal biopsy. The pathology studies revealed a nodular sclerosing glomerulopathy in four cases, mesangiocapilary glomerulonephritis in three cases, only tubular lesions in one and mesangial lesions in the other one. The treatment applied was: Prednisone alone (two cases), with chemotherapy associated (melfalan in two, clorambucil in one and ciclophosphamide in another one). One patient received plasmapheresis and mycophenolate and another patient undergone a bone marrow authotransplant associated to mycophenolate and prednisone. One of the two patients who required dialysis at the moment of presentation was not treated. After a follow-up of more than 4 years (4.89 +/-DE: 3.69) renal function improved or remained stable in three patients and proteinuria was disappeared in more than 50% of patients. Four patients had a worsening of renal function and they required dialysis during the time of follow-up (in 2,4 years +/- DE: 4,3). In any case malignitation was observed. Chemotherapy stabilized or improved renal function in 3 of nine patients (33%) with non-malignant monoclonal gammopathy. Non-malignant monoclonal gammopathy could go unnoticed. Appearance of abnormalities in renal routine tests deserves more in-depth diagnostic procedures, including renal biopsy. Evolution to end stage renal disease could probably be avoided or reduced in

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

    International Nuclear Information System (INIS)

    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 A1, C7, D3, D7 and H4. As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D3 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 CPK1 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

  3. The Use of Monoclonal Antibodies in Human Prion Disease

    Science.gov (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.

  4. 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.

  5. A Rapid Murine Coma and Behavior Scale for Quantitative Assessment of Murine Cerebral Malaria

    OpenAIRE

    Carroll, Ryan W.; Mark S Wainwright; KIM, KWANG-YOUN; Kidambi, Trilokesh; Gómez, Noé D.; Taylor, Terrie; Haldar, Kasturi

    2010-01-01

    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 m...

  6. Murine Typhus in Southern Taiwan during 1992–2009

    OpenAIRE

    Chang, Ko; Chen, Yen-Hsu; Lee, Nan-Yao; Lee, Hsin-Chun; Lin, Chun-Yu; Tsai, Jih-Jin; Lu, Po-Liang; Chen, Tun-Chieh; Hsieh, Hsiao-Chen; Lin, Wei-Ru; Lai, Ping-Chang; Chang, Chia-Ming; Wu, Chi-Jung; Lai, Chung-Hsu; Ko, Wen-Chien

    2012-01-01

    Clinical information regarding murine typhus in Taiwan is limited. In this study, 81 cases of serologically documented murine typhus during 1992–2009 at four referral hospitals in southern Taiwan were analyzed. There was a significant correlation between average environmental temperature and case numbers of murine typhus (r = 0.747, P = 0.005). Acute hepatitis was found in 67% of cases, and hyperbilirubinemia (serum total bilirubin ≥ 23.9 μmol/L) was found in 38%. The intervals between the in...

  7. Proliferative capacity of murine hematopoietic stem cells

    International Nuclear Information System (INIS)

    The present study demonstrates a decrease in self-renewal capacity with serial transfer of murine hematopoietic stem cells. Production of differentiated cell progeny is maintained longer than stem cell self-renewal. In normal animals the capacity for self-renewal is not decreased with increasing donor age. The stem cell compartment in normal animals, both young and old, appears to be proliferatively quiescent. After apparent recovery from the alkylating agent busulfan, the probability of stem cell self-renewal is decreased, there is a permanent defect in the capacity of the bone marrow for serial transplantation, and the stem cells are proliferatively active. These findings support a model of the hematopoietic stem cell compartment as a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cells progress in the continuum in one direction and such progression is not reversible

  8. Analysis of T-cell-dependent and -independent antigens of Rickettsia conorii with monoclonal antibodies.

    OpenAIRE

    Feng, H M; Walker, D H; Wang, J. G.

    1987-01-01

    Four monoclonal antibodies from euthymic mice and two monoclonal antibodies from athymic mice were directed against antigens of Rickettsia conorii, as shown by both indirect immunofluorescence and an enzyme immunoassay. There was extensive cross-reactivity with other spotted fever group rickettsiae. Euthymic monoclonal antibodies 3-2 and 9-2 (immunoglobulin G2a [IgG2a]) and 27-10 (IgG1) distinctly outlined the acetone-fixed rickettsial surface, as determined by indirect immunofluorescence; on...

  9. Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry.

    OpenAIRE

    Flores, B M; Garcia, C A; Stamm, W E; Torian, B E

    1990-01-01

    Monoclonal antibodies to Naegleria fowleri and Acanthamoeba polyphaga were analyzed by enzyme-linked immunosorbent assay, indirect immunofluorescence microscopy, and fluorescence flow cytometry to assess specificity and cross-reactivity with axenically cultured N. fowleri and Acanthamoeba spp. Four monoclonal antibodies to N. fowleri were specific for N. fowleri and had no reactivity to A. polyphaga. Similarly, four monoclonal antibodies to A. polyphaga did not react with N. fowleri. Two of t...

  10. Schistosoma mansoni. Anti-egg monoclonal antibodies protect against cercarial challenge in vivo

    OpenAIRE

    1984-01-01

    Monoclonal antibodies that bind to surface membranes of developing schistosomula and/or cercarial tails were generated from mice immunized with living schistosome eggs or soluble egg antigen. These monoclonal antibodies detected at least three different surface epitopes. One surface antigen detected by anti-egg monoclonal antibody EG1C4B1 (E.1) persisted on the surface of developing schistosomula for 96 h posttransformation . The same or a cross-reactive antigen was also detected on the surfa...

  11. Anti-idiotypic immunity in multiple myeloma and monoclonal gammopathy of undetermined significance

    OpenAIRE

    Bergenbrant Glas, Susanne

    1997-01-01

    Monoclonal gammopathies such as multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are lymphoproliferative B cell diseases. B cells/plasma cells produce the monoclonal immunoglobulin (Ig) which can be detected in plasma and/or urine. Such Ig have unique variable regions of the heavy and light chains and may be regarded as tumour-specific antigens and as such potential targets for immune regulation of the tumour cell clone, by anti-idiotype (I...

  12. Laboratory investigation of monoclonal gammopathy during 10 years of screening in a general hospital.

    OpenAIRE

    V. Malacrida; De Francesco, D.; Banfi, G; Porta, F A; Riches, P G

    1987-01-01

    Protein electrophoresis was carried out on 102,000 samples from the patients of a district general hospital over 10 years, and a monoclonal protein was detected in 730 cases; of these, 114 could be classified as B cell malignancies and 261 as monoclonal gammopathy of undefined significance (MGUS). The various clinical and laboratory features of monoclonal gammopathy were examined with respect to distinguishing the malignant conditions from MGUS at first presentation.

  13. Population-based studies on familiality and prognosis in patients with monoclonal gammopathies

    OpenAIRE

    Kristinsson, Sigurður Yngvi

    2009-01-01

    Monoclonal gammopathies constitute a group of diseases which have in common an overproduction of monoclonal immunoglobulins, M-proteins. Clues to their etiology have been found in studies showing familial aggregation of these diseases. We included 2,144 patients with lymphoplasmacytic lymphoma/Waldenström s macroglobulinemia (LPL/WM) and 4,458 patients with monoclonal gammopathy of undetermined significance (MGUS), their 6,177 and 14,621 first-degree relatives respectively,...

  14. Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas.

    OpenAIRE

    Spieker-Polet, H; Sethupathi, P; Yam, P C; Knight, K L

    1995-01-01

    During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic...

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

    International Nuclear Information System (INIS)

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

  16. Monoclonal antibodies to coagulation factor IX define a high-frequency polymorphism by immunoassays.

    OpenAIRE

    Smith, K. J.

    1985-01-01

    Monoclonal antibodies have been used to demonstrate a polymorphism of human plasma coagulation factor IX antigen in double antibody solid-phase immunoradiometric assays. This polymorphism is detected in an assay where a monoclonal antibody (A-1) adsorbed to microtiter wells is used to bind factor IX from diluted plasma samples. Plasma samples with the factor IX polymorphism have less than 0.2 U/ml of apparent antigen when tested with the A-1 antibody, while assays with other monoclonal antibo...

  17. Claudin 7 expression and localization in the normal murine mammary gland and murine mammary tumors

    International Nuclear Information System (INIS)

    Claudins, membrane-associated tetraspanin proteins, are normally associated with the tight junctions of epithelial cells where they confer a variety of permeability properties to the transepithelial barrier. One member of this family, claudin 7, has been shown to be expressed in the human mammary epithelium and some breast tumors. To set the stage for functional experiments on this molecule, we examined the developmental expression and localization of claudin 7 in the murine mammary epithelium and in a selection of murine mammary tumors. We used real-time polymerase chain reaction, in situ mRNA localization, and immunohistochemistry (IHC) to examine the expression and localization of claudin 7. Frozen sections were examined by digital confocal microscopy for colocalization with the tight-junction protein ZO1. Claudin 7 was expressed constitutively in the mammary epithelium at all developmental stages, and the ratio of its mRNA to that of keratin 19 was nearly constant through development. By IHC, claudin 7 was located in the basolateral part of the cell where it seemed to be localized to discrete vesicles. Scant colocalization with the tight-junction scaffolding protein ZO1 was observed. Similar results were obtained from IHC of the airway epithelium and some renal tubules; however, claudin 7 did partly colocalize with ZO1 in EPH4 cells, a normal murine mammary cell line, and in the epididymis. The molecule was localized in the cytoplasm of MMTV-neu and the transplantable murine tumor cell lines TM4, TM10, and TM40A, in which its ratio to cytokeratin was higher than in the normal mammary epithelium. Claudin 7 is expressed constitutively in the mammary epithelium at approximately equal levels throughout development as well as in the murine tumors examined. Although it is capable of localizing to tight junctions, in the epithelia of mammary gland, airway, and kidney it is mostly or entirely confined to punctate cytoplasmic structures, often near the basolateral

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  19. Imaging thrombus with radiolabelled monoclonal antibody to platelets

    International Nuclear Information System (INIS)

    Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111In labelled monoclonal antibody, P256, directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P256 was radiolabelled with 111In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo - that is, by direct intravenous injection of P256 - in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111In kinetics recorded after intravenous P256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P256, three had documented thrombus, tow of whom gave positive results on P256 platelet scintigraphy. The third subject had chromic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis. 3 refs. (Author)

  20. 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.

  1. Partial characterization of murine migration inhibitory factor (MIF).

    Science.gov (United States)

    Kühner, A L; David, J R

    1976-01-01

    These studies describe the production of murine migration inhibitory factor (MIF)3 in sufficient quantities to allow its partial characterization by physiochemical and enzymatic methods. MIF was obtained from murine spleen cell cultures (C57BL/6 strain) stimulated with concanavalin A (Con A). Characterization of murine MIF was performed using Sephadex G-100 gel chromatography, isopycnic centrifugation in a CsCl density gradient, polyacrylamide disc electrophoresis, heat stability, and enzymatic treatment. MIF-containing and control fractions were assayed on normal C57BL/6 peritoneal exudate cells by using a microcapillary tube assay. Peak MIF activity was found in a Sephadex G-100 fraction containing molecules the size of albumin and slightly smaller, molecular weight 67,000 to 48,000. Murine MIF was stable to heating at 56 degrees C for 30 min but lost its activity at 80 degrees C for 30 min. Incubation of G-100 fractions containing MIF with water insoluble chymotrypsin destroyed the activity of MIF, indicating its protein nature. CsCl density gradient centrifugation revealed that murine MIF had a buoyand density greater than protein, consistent with its being a glycoprotein. Further, when subjected to disc electrophoresis on polyacylamide gels, murine MIF migrated in a region cathodal to albumin. Thus, mitogen stimulation of murine spleen cells produced MIF in quantities which allowed its partial characterization and purification, and its comparison with human and guinea pig MIF; this makes it feasible to analyze the role of murine MIF in cellular immunity and in its relationship to lymphocyte mediators which regulate humoral immune responses. PMID:1107423

  2. Human recombinant erythropoietin promotes differentiation of murine megakaryocytes in vitro.

    OpenAIRE

    Ishibashi, T.; Koziol, J A; Burstein, S A

    1987-01-01

    To determine if erythropoietin affects megakaryocytopoiesis, we measured acetylcholinesterase (AchE) activity, a marker of the murine megakaryocytic lineage, after the addition of human recombinant erythropoietin to serumless murine bone marrow cultures. Erythropoietin increased AchE activity substantially. Moreover, when the hormone was added to serumless cultures of 426 isolated single megakaryocytes derived from megakaryocytic colonies, erythropoietin induced a significant increase in the ...

  3. Dynamic Determination of Oxygenation and Lung Compliance in Murine Pneumonectomy

    OpenAIRE

    Gibney, Barry; Lee, Grace S; Houdek, Jan; Lin, Miao; Miele, Lino; Chamoto, Kenji; Konerding, Moritz A; Tsuda, Akira; Mentzer, Steven J.

    2011-01-01

    Thoracic surgical procedures in mice have been applied to a wide range of investigations, but little is known about the murine physiologic response to pulmonary surgery. Using continuous arterial oximetry monitoring and the FlexiVent murine ventilator, we investigated the effect of anesthesia and pneumonectomy on mouse oxygen saturation and lung mechanics. Sedation resulted in a dose-dependent decline of oxygen saturation that ranged from 55–82%. Oxygen saturation was restored by mechanical v...

  4. Surface Contaminants Inhibit Osseointegration in a Novel Murine Model

    OpenAIRE

    Bonsignore, Lindsay A.; Colbrunn, Robb W.; Tatro, Joscelyn M.; Messerschmitt, Patrick J.; Hernandez, Christopher J.; Goldberg, Victor M.; Stewart, Matthew C.; Greenfield, Edward M.

    2011-01-01

    Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopaedic implants after sterilization procedures and affect osseointegration. The goals of this study were to develop a murine model of osseointegration in order to determine whether removing surface contaminants enhances osseointegration. To develop the murine model, titanium alloy implants were implanted into a unicortical pilot hole in the mid-diaphysis of the femur and osseointegration was measured...

  5. Monoclonal Antibody Shows Promise as Potential Therapeutic for MERS | Poster

    Science.gov (United States)

    A monoclonal antibody has proven effective in preventing Middle Eastern Respiratory Syndrome (MERS) in lab animals, suggesting further development as a potential intervention for the deadly disease in humans, according to new research. MERS is a newly emerged coronavirus first detected in humans in 2012. Most cases have occurred in the Middle East, but the disease has appeared elsewhere. In all, MERS has infected more than 1,700 individuals and killed more than 600, according to the World Health Organization. No vaccines or antiviral therapies currently exist. Several candidate vaccines are being developed, and some have been tested in animal models, a prerequisite to human clinical trials.

  6. Preparation of monoclonal antibody against crocin and its characterization

    OpenAIRE

    Xuan, Lijiang; Tanaka, Hiroyuki; Xu, Yaming; Shoyama, Yukihiro

    1999-01-01

    Three crocin-carrier protein conjugates were synthesized and their hapten numbers were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Three monoclonal antibodies against crocin were produced by hybridomas fused with the splenocytes immunized with crocin hemisuccinate-bovine serum albumin conjugate and HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. They were identified as IgG2a and IgG2b possessing λ light chain, respectively. Their wide rea...

  7. Production and Characterization of Monoclonal Antibodies Against Thytoxine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  8. Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues

    DEFF Research Database (Denmark)

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

    1984-01-01

    glycosaminoglycan (GAG), particularly with respect to self-association and interactions with other extracellular matrix components. Interactions with specific molecules from different connective tissue types, such as the collagens and their associated glycoproteins, could be favoured by particular charge...... organizations on the GAG molecule endowed by the sulphate groups. So far, it has not been possible to identify and map chondroitins of differing sulphation in tissues, but we have now raised three monoclonal antibodies which specifically recognize unsulphated, 4-sulphated and 6-sulphated chondroitin and...

  9. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

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

    2007-01-01

    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...

  10. A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes.

    OpenAIRE

    Lee, J. S.; Burkholder, G D; Latimer, L J; Haug, B L; Braun, R P

    1987-01-01

    A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was as...

  11. Prevalence of monoclonal gammopathy in HIV patients in 2014

    Directory of Open Access Journals (Sweden)

    Philippe Genet

    2014-11-01

    Full Text Available Introduction: In non-HIV patients, Monoclonal Gammopathy of Undetermined Significance (MGUS is associated with an increased risk of subsequent development of haematologic malignancies, especially multiple myeloma (MM and it has been recently demonstrated that MM is always preceded by a MGUS phase. A higher prevalence of MGUS and MM has been observed in HIV patients compared to the general population. Nevertheless, it has been shown that MGUS in the context of HIV can disappear with antiretroviral therapy (ART. So, measuring MGUS prevalence in HIV patients in the recent period appears of special interest. Materials and Methods: From January to June 2014, in each out-patient seen in our unit, a serum protein electrophoresis was performed. Results: A total of 393 patients were screened. Eight patients with HIV2 and one patient with HIV1+HIV2 infection were excluded. Finally, 383 patients (173 female, 210 male with HIV1 infection were analyzed. Characteristics of patients were as follows: median age 42.2 years (19.1–79.1, hepatitis B virus (HBV and/or hepatitis C virus (HCV co-infection 47 (18.8%, median CD4 610 (2–1758, CD8 793 (113–4010, presence of a past AIDS event for 88 patients (23%. Median time with HIV infection was 11 years (0–30. Three hundred fifty-nine patients (93.7% were on ART for a median duration of 105 months (0–287. For 320 patients (83.6%, viral load was below 50 viral copies/ml. Twelve cases of MGUS (3.1% were observed: IgG Kappa (five cases, IgG Lambda (five cases, biclonal with two IgG Kappa (one case and in one case, three monoclonal immunoglobulins were observed (IgG Kappa×2+IgG Lambda. The monoclonal immunoglobulin's level was low and below 1 g/l in all cases except two (2.1 and 11.6 g/l. No factor was found to be predictive of the presence of MGUS in particular age, CD4, HBV/HCV co-infection, viral load or ART. Conclusions: In the context of modern ART, the prevalence of MGUS remains above those observed in the

  12. Structural identification and characterization of monoclonal antibodies to rat angiotensinogen

    International Nuclear Information System (INIS)

    Balb/c mice were immunised in vivo using angiotensinogen obtained from rats. In order to confirm that an immunoreaction had taken place, the concentration of specific antibodies was determined in selected sera on the basis of a radioimmunological method. In view of the fact that the affinity of the antibodies of the three monoclonal lines isolated here was calculated to be in the order of 107 l/mol it appears that their main field of use in affinity chromatography would be the purification of angiotensinogen from rats. (orig./MG)

  13. Immunosuppression associated with novel chemotherapy agents and monoclonal antibodies.

    Science.gov (United States)

    Morrison, Vicki A

    2014-11-15

    The introduction of novel agents to the therapeutic armamentarium for oncologic, rheumatologic, and neurologic disorders has resulted in major clinical advances. These agents impact immune function, resulting in a discrete spectrum of infectious complications. Purine analogues and alemtuzumab alter cell-mediated immunity, resulting in opportunistic viral/fungal infections. Herpes zoster incidence increases with bortezomib. Hepatitis B reactivation may occur with rituximab. Cases of progressive multifocal leukoencephalopathy have occurred following monoclonal antibody therapy. Tumor necrosis factor-α inhibitor therapy is complicated by tuberculosis reactivation and fungal infections. We summarize the impact of these therapies on pathogenesis and spectrum of infection complicating their usage. PMID:25352632

  14. Monoclonal antibody imaging of human melanoma. Radioimmunodetection by subcutaneous or systemic injection

    International Nuclear Information System (INIS)

    Fab fragments of monoclonal antibodies (MoAb) to melanoma, radiolabeled with 131I, were evaluated as diagnostic reagents to determine their ability to localize systemic--MoAb injected intravenously (IV)--or nodal metastatic disease--injected subcutaneously (SQ) at a site proximal to draining lymph nodes. Sixty-one scans were performed (40 IV, 21 SQ) in 59 patients who had injections of 0.2-50 mg of 131I coupled (0.2-12 mCi) antibody. These included 48.7, which identifies a high molecular weight antigen (HMW), or 96.5, which identifies a transferrin like molecule, p97. 125I coupled nonspecific Fab 1.4, reacting with murine leukemia virus, or the whole antibody BL3, reactive with a human B cell idiotypic determinant, was generally used in tandem with the patients injected SQ as a nonspecific control. All patients had immunohistochemical studies performed and demonstrated binding to the antibodies injected. Of the IV patients, 22/38 (58%) had (+) scans, 13 at SQ or nodal sites, four at visceral sites, and five at visceral and SQ sites. Patients with clinical stage II disease had SQ injection of MoAb, including 11 additional patients injected with the whole antibody 9.2.27 (anti-HMW) labeled with 111In (6 patients) or 131I (5 patients). Nodal dissection was performed 2-4 days later. All 111In coupled antibodies demonstrated excellent nodal delineation without specific identification of tumor deposits. Of the 21 patients injected SQ with MoAb, 17 had confirmed tumor in nodes. Of patients injected with Fab fragments, 50% had specific uptake of MoAb, although only two were successfully imaged. Increased uptake of antimelanoma antibodies was observed in some patients in lymph nodes not containing tumor. Clearance of labeled antibody from the injection site occurred with a half life of 16-50 hours. Toxicity was limited to local discomfort at the site of SQ injection

  15. 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

    2002-01-01

    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.

  16. Isolation of human monoclonal antibodies from peripheral blood B cells.

    Science.gov (United States)

    Huang, Jinghe; Doria-Rose, Nicole A; Longo, Nancy S; Laub, Leo; Lin, Chien-Li; Turk, Ellen; Kang, Byong H; Migueles, Stephen A; Bailer, Robert T; Mascola, John R; Connors, Mark

    2013-10-01

    Isolation of monoclonal antibodies is an important technique for understanding the specificities and characteristics of antibodies that underlie the humoral immune response to a given antigen. Here we describe a technique for isolating monoclonal antibodies from human peripheral blood mononuclear cells. The protocol includes strategies for the isolation of switch-memory B cells from peripheral blood, the culture of B cells, the removal of the supernatant for screening and the lysis of B cells in preparation for immunoglobulin heavy-chain and light-chain amplification and cloning. We have observed that the addition of cytokines IL-2, IL-21 and irradiated 3T3-msCD40L feeder cells can successfully stimulate switch-memory B cells to produce high concentrations of IgG in the supernatant. The supernatant may then be screened by appropriate assays for binding or for other functions. This protocol can be completed in 2 weeks. It is adaptable to use in other species and enables the efficient isolation of antibodies with a desired functional characteristic without prior knowledge of specificity. PMID:24030440

  17. Development of Biodegradable Nanocarriers Loaded with a Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Andrew Gdowski

    2015-02-01

    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.

  18. Monkey-derived monoclonal antibodies against Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Stanley, H.A.; Reese, R.T.

    1985-09-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a M/sub r/ 95,000 antigen. Radioimmunoprecipitation assays using /sup 125/T-antibodies were done.

  19. Treating multiple sclerosis with monoclonal antibodies: a 2013 update.

    Science.gov (United States)

    Deiß, Annika; Brecht, Isabel; Haarmann, Axel; Buttmann, Mathias

    2013-03-01

    The third part of this in-depth review series on the treatment of multiple sclerosis (MS) with monoclonal antibodies covers the years 2010-2012. The natalizumab section gives a progressive multifocal leukoencephalopathy update, focusing on clinically relevant aspects. Furthermore, it outlines problems around natalizumab cessation and current evidence on therapeutic strategies thereafter. Finally, it reviews evidence on Janus-faced modes of natalizumab action besides anti-inflammatory effects, including proinflammatory effects. The section on alemtuzumab critically analyzes recent Phase III results and discusses which patients might be best suited for alemtuzumab treatment, and reviews the long-term immunological impact of this anti-CD52 antibody. The daclizumab section critically summarizes results from the Phase IIb SELECT/SELECTION trial and introduces the Phase III program. The section on anti-CD20 antibodies reviews Phase II results on ocrelizumab and ofatumumab, and discusses current perspectives of these antibodies for MS therapy. Promising recent Phase II results on the anti-IL-17A antibody secukinumab (AIN457) are outlined and a short update on tabalumab (LY2127399) is given. Other highlighted antibodies currently being tested in MS patients include GNbAC1, BIIB033, MOR103 and MEDI-551. Finally, the authors give an update on the role monoclonal antibodies could play in the therapeutic armamentarium for MS in the medium term. PMID:23448220

  20. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Hiroyuki Tanaka

    2012-01-01

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

  2. Immunolocalization of neuroblastoma using radiolabeled monoclonal antibody UJ13A

    International Nuclear Information System (INIS)

    The monoclonal antibody UJ13A, raised after immunization of mice with human fetal brain, recognized an antigen expressed on human neuroblastoma cell lines and fresh tumors. Antibody was purified and radiolabeled with iodine isotopes using chloramine-T. In preclinical studies, 125I-labeled UJ13A was injected intravenously into nude mice bearing xenografts of human neuroblastoma. Radiolabeled UJ13A uptake by the tumors was four to 23 times greater than that by blood. In control animals, injected with a similar quantity of a monoclonal antibody known not to bind to neuroblastoma cells in vitro (FD44), there was no selective tumor uptake. Nine patients with histologically confirmed neuroblastoma each received 100 to 300 micrograms UJ13A radiolabeled with 1 to 2.8 mCi 123I or 131I. Sixteen positive sites were visible on gamma scans 1 to 7 days after injection: 15 were primary or secondary tumor sites, and one was a false positive; there were two false negatives. In two of the 15 positive sites, tumor had not been demonstrated by other imaging techniques; these were later confirmed as areas of malignant infiltration. No toxicity was encountered

  3. Detection of grass carp reovirus (GCRV) with monoclonal antibodies.

    Science.gov (United States)

    Hongli, Jing; Lifeng, Zhang; Zhenzhen, Fang; Lipu, Xu; Min, Zhang; Na, Wang; Yulin, Jiang; Xiangmei, Lin

    2014-04-01

    Grass carp reovirus (GCRV) is a pathogen that causes hemorrhagic disease of grass carp. It is the most serious infectious disease of carp and causes serious losses of fingerlings of grass carp and black carp. In this study, a recombinant VP4, one of the viral core proteins, was constructed with a histidine tag and expressed at a high level in E. coli, and the expressed protein was mainly found in the form of inclusion bodies. The expressed VP4 protein was recognized by an anti-His-tag monoclonal antibody and goat anti-GCRV serum. Four monoclonal antibodies (16B7, 39E12, 13C3 and 14D1) against the recombinant VP4 protein were produced. These MAbs did not react with any of the tested viruses or fish cells lines in the ELISA tests except GCRV. In western blotting analysis, a protein band was observed when the recombinant VP4 protein of GCRV was used as an antigen, but a 68-kDa band was observed when natural capsid proteins of GCRV were used as antigens. Furthermore, a sandwich ELISA was developed for detection of GCRV. The detection limit of the test was 105 TCID50 of GCRV per mL. PMID:24122108

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

    Science.gov (United States)

    Meagher, Richard B.; Laterza, Vince

    2006-12-12

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

  5. 76 FR 63317 - Prospective Grant of Exclusive License: The Development of Human Anti-Mesothelin Monoclonal...

    Science.gov (United States)

    2011-10-12

    ... monoclonal antibody m912 (SM-101) as an antibody therapy for the treatment of pancreatic cancer, ovarian... Human Anti-Mesothelin Monoclonal Antibodies for the Treatment of Human Cancers AGENCY: National... the antibody for the treatment of mesothelin-expressing cancers, including mesothelioma, lung...

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

    NARCIS (Netherlands)

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

    1992-01-01

    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 retaine

  7. 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

    2010-10-01

    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

  8. Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

    Science.gov (United States)

    Shaw, D M; Embleton, M J; Westwater, C; Ryan, M G; Myers, K A; Kingsman, S M; Carroll, M W; Stern, P L

    2000-12-15

    The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs. PMID:11113573

  9. Remodeling of alveolar septa after murine pneumonectomy.

    Science.gov (United States)

    Ysasi, Alexandra B; Wagner, Willi L; Bennett, Robert D; Ackermann, Maximilian; Valenzuela, Cristian D; Belle, Janeil; Tsuda, Akira; Konerding, Moritz A; Mentzer, Steven J

    2015-06-15

    In most mammals, removing one lung (pneumonectomy) results in the compensatory growth of the remaining lung. In mice, stereological observations have demonstrated an increase in the number of mature alveoli; however, anatomic evidence of the early phases of alveolar growth has remained elusive. To identify changes in the lung microstructure associated with neoalveolarization, we used tissue histology, electron microscopy, and synchrotron imaging to examine the configuration of the alveolar duct after murine pneumonectomy. Systematic histological examination of the cardiac lobe demonstrated no change in the relative frequency of dihedral angle components (Ends, Bends, and Junctions) (P > 0.05), but a significant decrease in the length of a subset of septal ends ("E"). Septal retraction, observed in 20-30% of the alveolar ducts, was maximal on day 3 after pneumonectomy (P alveolar duct diameter ratio (Dout:Din) was significantly lower 3 days after pneumonectomy compared to all controls except for the detergent-treated lung (P surface tension within the alveolar duct, resulting in a new equilibrium at a higher total energy and lower surface area. The spatial and temporal association of these microstructural changes with postpneumonectomy lung growth suggests that these changes represent an early phase of alveolar duct remodeling. PMID:26078396

  10. Isolation of Murine Embryonic Hemogenic Endothelial Cells.

    Science.gov (United States)

    Fang, Jennifer S; Gritz, Emily C; Marcelo, Kathrina L; Hirschi, Karen K

    2016-01-01

    The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level. PMID:27341393

  11. ESCRT Requirements for Murine Leukemia Virus Release

    Science.gov (United States)

    Bartusch, Christina; Prange, Reinhild

    2016-01-01

    The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion release was profoundly inhibited in CHMP2A- and CHMP4B-knockdown cells. In contrast, neither the CHMP2B and CHMP4A isoforms nor CHMP3, CHMP5, and CHMP6 were found to be essential. In case of CHMP1, we unexpectedly observed that the CHMP1A isoform was specifically required for virus budding, but dispensable for VLP release. Hence, MLV utilizes only a subset of ESCRT factors, and viral and viral-like particles differ in ESCRT-III factor requirements. PMID:27096867

  12. 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.

  13. Radiolocalization of human small cell lung cancer and antigen-positive normal tissues using monoclonal antibody LS2D617

    International Nuclear Information System (INIS)

    The murine monoclonal antibody LS2D617, which reacts with an antigen associated with human small cell lung carcinoma (SCLC), was tested in preclinical models to assess its potential for specific targeting of tumors in human SCLC cancer patients. LS2D617 detects a cell antigen on the surface of cultured SCLC and neuroblastoma cell lines. Scatchard analysis of the binding of LS2D617 to NCIH69 SCLC cells indicates an affinity constant of about 1 x 10(8) M-1 and an epitope expression level of approximately 2 x 10(6) antigenic sites/cell. Molecular weight analysis of the target antigen and antibody competition experiments showed that LS2D617 should be classified as a SCLC Cluster 1 antibody. LS2D617 was labeled with 111In and tested for biodistribution (4, 24, 48, 72, and 96 h postinjection) in nude mice bearing the human SCLC NCIH69 tumor. Tumor values peaked at about 35% injected dose/g (Day 3) compared with about 8% injected dose/g for an irrelevant IgG1 antibody while normal tissue accumulation for both antibodies was about 2-8% injected dose/g. Immunohistochemical studies demonstrated that LS2D617 reacts with the central nervous system, peripheral nerves, endocrine tissues, and heart tissue of rabbits as it does in human tissues. The ability of LS2D617 to accumulate in vivo in normal tissues that express the specific target antigen was tested in rabbits. Rabbits given i.v. injections of 111In-LS2D617 or control labeled antibody were sacrificed at 48 h and tissues were examined by gamma well counting, autoradiography, and immunohistochemical staining for murine immunoglobulin. Specific uptake was seen in all sites defined as antigen positive by immunohistology (i.e., heart, liver bile duct, peripheral nerves, pituitary, adrenal), except the central nervous system (brain and spinal cord) which was inaccessible to antibody because of the blood brain barrier

  14. Isoform-specific upregulation of palladin in human and murine pancreas tumors.

    Directory of Open Access Journals (Sweden)

    Silvia M Goicoechea

    Full Text Available Pancreatic ductal adenocarcinoma (PDA is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA. We performed immunohistochemistry and immunoblot analyses on patient samples and tumor-derived cells using an isoform-selective monoclonal antibody and a pan-palladin polyclonal antibody. Immunoblot and real-time quantitative reverse transcription-PCR were used to quantify palladin mRNA levels in human samples. We show that there are two major palladin isoforms expressed in pancreas: 65 and 85-90 kDa. The 65 kDa isoform is expressed in both normal and neoplastic ductal epithelial cells. The 85-90 kDa palladin isoform is highly overexpressed in tumor-associated fibroblasts (TAFs in both primary and metastatic tumors compared to normal pancreas, in samples obtained from either human patients or genetically engineered mice. In tumor-derived cultured cells, expression of palladin isoforms follows cell-type specific patterns, with the 85-90 kDa isoform in TAFs, and the 65 kDa isoform predominating in normal and neoplastic epithelial cells. These results suggest that upregulation of 85-90 kDa palladin isoform may play a role in the establishment of the TAF phenotype, and thus in the formation of a desmoplastic tumor microenvironment. Thus, palladin may have a potential use in the early diagnosis of PDA and may have much broader significance in understanding metastatic behavior.

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

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    A. Sasikalaveni

    2015-05-01

    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.

  16. A murine model of coxsackievirus A16 infection for anti-viral evaluation.

    Science.gov (United States)

    Liu, Qingwei; Shi, Jinping; Huang, Xulin; Liu, Fei; Cai, Yicun; Lan, Ke; Huang, Zhong

    2014-05-01

    Coxsackievirus A16 (CA16) is one of the main causative agents of hand, foot and mouth disease (HFMD), which is a common infectious disease in children. CA16 infection may lead to severe nervous system damage and even death in humans. However, study of the pathogenesis of CA16 infection and development of vaccines and anti-viral agents are hindered partly by the lack of an appropriate small animal model. In the present study, we developed and characterized a murine model of CA16 infection. We show that neonatal mice are susceptible to CA16 infection via intraperitoneal inoculation. One-day-old mice infected with 2×10(6)TCID50 of CA16/SZ05 strain consistently exhibited clinical signs, including reduced mobility, and limb weakness and paralysis. About 57% of the mice died within 14days after infection. Significant damage in the brainstem, limb muscles and intestines of the infected mice in the moribund state was observed by histological examination, and the presence of CA16 in neurons of the brainstem was demonstrated by immunohistochemical staining with a CA16-specific polyclonal antibody, strongly suggesting the involvement of the central nervous system in CA16 infection. Analysis of virus titers in various organs/tissues collected at 3, 6 and 9days post-infection, showed that skeletal muscle was the major site of virus replication at the early stage of infection, while the virus mainly accumulated in the brain at the late stage. In addition, susceptibility of mice to CA16 infection was found to be age dependent. Moreover, different CA16 strains could exhibit varied virulence in vivo. Importantly, we demonstrated that post-exposure treatment with an anti-CA16 monoclonal antibody fully protected mice against lethal CA16 infection. Collectively, these results indicate the successful development of a CA16 infection mouse model for anti-viral evaluation. PMID:24583030

  17. 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;

    1999-01-01

    -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...

  18. 99mTc direct labeling of a new anti-MUC1 monoclonal antibody, PR81, as a potential agent for imaging of human breast cancer in nuclear medicine

    International Nuclear Information System (INIS)

    Radioimmunoscintigraphy (RIS) is a technique which uses radiolabeled antibodies to visualize tumors, taking advantage of antigens preferentially expressed by malignant tissues. Human epithelial mucin, MUC1, is commonly over expressed in adenocarcinomas including 80% of breast cancers and represents a useful target for RIS. The PR81 is a new murine anti-MUC1 monoclonal antibody that was found to react with the membrane extracts of several human breast cancer tissues and the cell surface of MUC1 positive cell lines. In this study we have developed a direct method for labeling of this MAb with 99mTc which is simple, rapid, efficient and particularly suitable for development of a 'kit'. The quality control of new radiopharmaceutical and immunoscintigraphy studies in BALB/c mice bearing breast tumor xenografts were also performed. (author)

  19. 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: yuasa@a8.keio.jp [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)

    2015-09-04

    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.

  20. Analysis of cardiomyocyte movement in the developing murine heart

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    Gurjot Basra

    2012-01-01

    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.

  2. 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

    2012-02-01

    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.

  3. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples.

    Science.gov (United States)

    Brooks, B W; Devenish, J; Lutze-Wallace, C L; Milnes, D; Robertson, R H; Berlie-Surujballi, G

    2004-10-01

    A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples. PMID:15381269

  4. Structures of A[beta]-Related Peptide−Monoclonal Antibody Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Gardberg, Anna; Dice, Lezlee; Pridgen, Kathleen; Ko, Jan; Patterson, Paul; Ou, Susan; Wetzel, Ronald; Dealwis, Chris; (Case Western); (CIT); (Tennessee-K)

    2009-06-15

    Passive immunotherapy (PI) is being explored as a potential therapeutic against Alzheimer's disease. The most promising antibodies (Abs) used in PI target the EFRH motif of the A{beta} N-terminus. The monoclonal anti-A{beta} Ab PFA1 recognizes the EFRH epitope of A{beta}. PFA1 has a high affinity for A{beta} fibrils and protofibrils (0.1 nM), as well as good affinity for A{beta} monomers (20 nM). However, PFA1 binds the toxic N-terminally modified pyroglutamate peptide pyro-Glu3-A{beta} with a 77-fold loss in affinity compared to the WT A{beta}(1-8). Furthermore, our earlier work illustrated PFA1's potential for cross-reactivity. The receptor tyrosine kinase Ror2, which plays a role in skeletal and bone formation, possesses the EFRH sequence. PFA1 Fab binds the Ror2(518-525) peptide sequence REEFRHEA with a 3-fold enhancement over WT A{beta}(1-8). In this work, the crystal structures of the hybridoma-derived PFA1 Fab in complex with pyro-Glu3-A{beta} peptide and with a cross-reacting peptide from Ror2 have been determined at resolutions of 1.95 and 2.7 {angstrom}, respectively. As with wild-type A{beta}, these peptides bind to the Fab via a combination of charge- and shape-complementarity, hydrogen-bonding, and hydrophobic interactions. Comparison of the structures of the four peptides A{beta}(1-8), Grip1, pyro-Glu3-A{beta}(3-8), and Ror2 in complex with PFA1 shows that the greatest conformational flexibility occurs at residues 2 to 3 and 8 of the peptide. These structures provide a molecular basis of the specificity tolerance of PFA1 and its ability to recognize A{beta} N-terminal heterogeneity. The structures provide clues to improving mAb specificity and affinity for pyroglutamate A{beta}.

  5. Positron emission tomography and optical imaging of tumor CD105 expression with a dual-labeled monoclonal antibody.

    Science.gov (United States)

    Zhang, Yin; Hong, Hao; Engle, Jonathan W; Yang, Yunan; Theuer, Charles P; Barnhart, Todd E; Cai, Weibo

    2012-03-01

    CD105 (endoglin) is an independent prognostic marker for poor prognosis in >10 solid tumor types, including breast cancer. The goal of this study was to develop a CD105-specific agent for both positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging, which can have potential clinical applications in diagnosis and imaged-guided surgery of breast cancer. TRC105, a chimeric anti-CD105 monoclonal antibody, was labeled with both a NIRF dye (i.e., 800CW) and (64)Cu to yield (64)Cu-NOTA-TRC105-800CW. Flow cytometry analysis revealed no difference in CD105 binding affinity/specificity between TRC105 and NOTA-TRC105-800CW. Serial PET imaging revealed that the 4T1 murine breast tumor uptake of (64)Cu-NOTA-TRC105-800CW was 5.2 ± 2.7, 11.0 ± 1.4, and 13.0 ± 0.4% ID/g at 4, 24, and 48 h postinjection respectively. Tumor uptake as measured by ex vivo NIRF imaging exhibited a good linear correlation with the % ID/g values obtained from PET (R = 0.74). Biodistribution data were consistent with the PET/NIRF findings. Blocking experiments, control studies with dual-labeled cetuximab (an isotype-matched control antibody), and histology confirmed the CD105 specificity of (64)Cu-NOTA-TRC105-800CW. Successful PET/NIRF imaging of CD105 expression warrants further investigation and clinical translation of dual-labeled TRC105-based imaging agents. PMID:22292418

  6. Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci.

    Science.gov (United States)

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

    2016-07-01

    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. PMID:26993086

  7. 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

    2006-03-01

    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.

  8. Internal radiation dosimetry, pharmacokinetics and biodistribution of the 99mTc labeled ior egf/r3 monoclonal antibody

    International Nuclear Information System (INIS)

    The aim of this work was to assess the internal radiation dosimetry, human pharmacokinetics and biodistribution of the 99mTc-labeled murine monoclonal antibody (MAb) ior egf/r3, used for diagnosis of epithelial tumors. Five patients were included in this study. Multiple blood and urine samples were collected and sequential anterior and posterior whole-body scintigraphies up to 24 hr post-injection were acquired from all patients. The internal radiation dosimetry was estimated using the methods developed by the Medical Internal Radiation Dosimetry (MIRD) committee. Raw data were computed from operations between scintigraphic images and regions of interest (ROI). The residence times of the activity on the source organs were computed to assess the absorbed dose by 24 target organs. The dosimetric results showed that liver, gallbladder and spleen received the higher absorbed dose. The computed mean values were 0.69 mGy/MBq, 0.19mGy/MBq and 0.37 mGy/MBq, respectively. The mean value of effective dose was 1,2E-01 mSv/MBq and the effective equivalent dose was 9,2E-02 mSv/MBq. The pharmacokinetics and biodistribution results showed that this compound has a biexponential plasmatic and blood clearance with a rapid biodistribution phase and a slower elimination phase. This compound was excreted by the urinary and hepatobiliary systems. Liver was the principal target organ of this product showing a great retention of the MAb. These dosimetric results have allowed to use the ior egf/r3 kit in a safe and controlled way. (author)

  9. 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.

  10. Classification method for heterogeneity in monoclonal cell population

    Science.gov (United States)

    Aburatani, S.; Tashiro, K.; Kuhara, S.

    2015-09-01

    Monoclonal cell populations are known to be composed of heterogeneous subpopulations, thus complicating the data analysis. To gain clear insights into the mechanisms of cellular systems, biological data from a homogeneous cell population should be obtained. In this study, we developed a method based on Latent Profile Analysis (LPA) combined with Confirmatory Factor Analysis (CFA) to divide mixed data into classes, depending on their heterogeneity. In general cluster analysis, the number of measured points is a constraint, and thereby the data must be classified into fewer groups than the number of samples. By our newly developed method, the measured data can be divided into groups depending on their latent effects, without constraints. Our method is useful to clarify all types of omics data, including transcriptome, proteome and metabolic information.

  11. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

    Skottrup, Peter; Frøkiær, Hanne; Hearty, Stephen; O'Kennedy, Richard; Hejgaard, Jørn; Nicolaisen, Mogens; Justesen, Annemarie Fejer

    2007-01-01

    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...... curves for both assays had good intra- and interday reproducibility. The subtractive inhibition ELISA had greater sensitivity with a detection limit of 1.5 105 spores ml1. Cross-reactivity studies of Pst mAb8 in the subtractive inhibition ELISA, showed reaction with other Puccinia spores only, suggesting...

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

    Science.gov (United States)

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

    2002-05-01

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

  13. Parenteral Treatment of Multiple Sclerosis: The Advent of Monoclonal Antibodies.

    Science.gov (United States)

    Singer, Barry A

    2016-04-01

    Improved disease control is critical for enhancing the lives of those living with multiple sclerosis. With specific immunologic targets, monoclonal antibody (mAb) treatments are highly effective options for relapsing forms of multiple sclerosis. The mechanism, efficacy, and current safety profiles are detailed for the two mAb therapies, natalizumab and alemtuzumab, with regulatory approval in multiple countries. Daclizumab, which targets the interleukin-2 receptor, and ocrelizumab, which depletes B cells, have convincing phase 3 clinical trial data and may very well provide new options in the near future. Trial results of other B-cell-directed therapies, ofatumumab and rituximab, are reviewed. Less-frequent dosing of glatiramer acetate and interferon β-1a highlight developments in the first generation of parenteral immunomodulatory therapy. Remyelination using mAbs has moved into clinical trials with the first agents, anti-LINGO-1, rHIgM22, and anti-SEMA 4D. PMID:27116720

  14. Design and manufacture of monoclonal antibodies for radioimmunotherapy

    International Nuclear Information System (INIS)

    Appropriate design and manufacture of monoclonal antibodies is fundamental to their use for radioimmunotherapy. Besides the right selection of antibody specificity and affinity, recombinant antibodies can be designed to simplify manufacture and minimise unwanted side effects. Although many innovative new technologies have been developed in recent years, antibodies are still most commonly produced from mammalian cells and purified by column chromatography. Purification methods have to be designed and validated to remove potential contaminants, especially retroviruses which in principle might be present in mammalian cell lines. Adherence to relevant Good Manufacturing Practice is mandatory in the production of any medicinal product and there are numerous guidelines regarding the manufacture of antibodies. This article outlines some methods used for fermentation, purification and quality control of antibodies intended for radiolabelling

  15. A monoclonal antibody to pestviruses in bovine and ovine sera

    International Nuclear Information System (INIS)

    An enzyme-linked immunoabsorbent assay (ELISA) has been developed to defeat antibodies to pestviruses in bovine and ovine sera. Single sera from 211 cattle and 22 sheep from 7 different farms were tested using ELISA and Serum Neutralisation Test (SNT). 17 Monoclonal antibodies (Mabs) directed against P80, gp48 and gp53 were tested for ability to coat ELISA plates and capture the bovine viral diarrhea antigen. 5 mabs(WB 103, WB, 105, WB 112 against P80 kDa protein, WB 210 and WB 214 directed against gp48 and gp 53 kDa protein. Specific antibody to BVDV was detected by rabbit anti-bovine and anti-ovine IgG antisera. The quantitative correlation between two tests was good

  16. Preparation and Characteristic Identification of Monoclonal Antibody Against Sulfamethazine

    Institute of Scientific and Technical Information of China (English)

    DING Liangjun; LI Jichang; FU Rui; ZHOU Yanjun; HUO Guicheng

    2006-01-01

    Two artificial antigens were synthesized successfully by diazotizing method, sulfamethazine(SM2)-human serum albumin (HSA) was used for the immunogen, and SM2-ovalbumin(OVA) was used for the coating antigen.The coupled reaction was successful by confirmation of the ultraviolet scanning spectrometer, and the conjugation ratio of SM2 with HSA and OVA was 9:1 and 15:1, respectively. Using cell-fusion and limiting dilution method to reclone 5times to get 3 hybridoma strains, which could stably secret monoclonal antibody (Mab), named CB7, BC4 and BB12. The subtype of BC4 Mab was IgG1 and chain, the molecular weight was 162 ku, the numbers of chromosomal were about 90,the affinity constant was 6.1 × 1012 M-1. No cross reactivity was seen between the Mab and the other 4 sulfonamides, as well as the 2 carries proteins. The Mab antibody had excellent stability.

  17. Iodination of monoclonal antibodies, proteins and peptide using iodogen

    International Nuclear Information System (INIS)

    The use of the iodinating reagent 1,3,4,6-tetrachloro-3α, 6α-diphenylglycholuril (Iodogen) to label monoclonal antibodies (McAbs). Proteins and peptides was invesrigated with McAbs identified as mouse IgG and IgM, arginine-vasopressin (AVP), glucagon (Glu), human insulin(hI) and albumin(Alb). The labeled products were purified by gel chromatography and their immunoreactivity were detected by RIA or IRMA> Comparison of the Iodogen method with the lactoperoxides and chloramine-T methods showed that the Iodogen method had a number of advantages: 1) technically simpler ; 2) a high labeling efficiency could be obtained; 3) the immunoreactivity of the products was minimally affected; 4) the products were stable for up to 4 months

  18. Characterization of monoclonal antibodies directed against human thyroid stimulating hormone

    International Nuclear Information System (INIS)

    Monoclonal antibodies directed against human thyroid stimulating hormone (TSH) were obtained from hybrid myelomas, following fusion of mouse NSI myeloma cells with mouse spleen cells. Ten different antibodies were obtained from 4 separate fusions. Eight antibodies were of the IgG1 subclass. Affinities of antibodies for TSH were in the range 2 x 108-5 x 1010 M-1. Five of the antibodies were specific for TSH and did not react with LH, FSH or hCG. The remaining antibodies reacted with all these hormones and were assumed to recognise their common (α) subunit. The 5 specific antibodies fell into 3 subgroups recognising distinct antigenic determinants, whereas the 5 non-specific antibodies recognised a single determinant or closely related set of sites. It is concluded that these antibodies should be valuable reagents for use in sensitive and specific two-site immunoradiometric assays. (Auth.)

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

    International Nuclear Information System (INIS)

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

  20. Characteristics of Monoclonal Antibody Against Infectious Bursal Disease Virus

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    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.

  1. Characteristics of Monoclonal Antibody Against Infectious Bursal Disease Virus

    Institute of Scientific and Technical Information of China (English)

    LiYan-Fei; WangWei; 等

    1999-01-01

    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.

  2. Novel CD20 monoclonal antibodies for lymphoma therapy

    Directory of Open Access Journals (Sweden)

    Cang Shundong

    2012-10-01

    Full Text Available Abstract Rituximab (RTX, a monoclonal antibody (mAb against CD20, has been widely used for lymphoma therapy. RTX in combination with cyclophosphamide /doxorubicin /vincristine /prednisone (R-CHOP remains the standard frontline regimen for diffuse large B-cell lymphoma. However, suboptimal response and /or resistance to rituximab have remained a challenge in the therapy of B-cell non-Hodgkin’s lymphoma (NHL. Novel agents are under active clinical trials. This review will summarize the latest development in new mAbs against CD20, which include second-generation mAbs, ofatumumab, veltuzumab (IMMU-106, ocrelizumab (PRO70769, and third-generation mAbs, AME-133v (ocaratuzumab, PRO131921 and GA101 (obinutumumab.

  3. Localization of melanoma with radiolabelled monoclonal antibody fragments and iodoamphetamine

    Energy Technology Data Exchange (ETDEWEB)

    Liewendahl, K.; Kairento, A.L.; Lindroth, L.; Pyroenen, S.; Franssila, K.; Virkkunen, P.; Asko-Seljavaara, S.; Launes, J.

    1986-10-01

    In two melanoma patients, metastases accumulated both /sup 99m/Tc-labelled monoclonal anti-tumor F(ab')/sub 2/ fragments and N-isopropyl-p-(/sup 123/I)-iodoamphetamine. Small metastatic deposits were localized only by labelled antibody, for which a higher target-to-nontarget ratio was observed than for radioiodoamphetamine, indicating that immunoscintigraphy may be the more sensitive method. In these two patients positive immunohistochemical staining for the antibody used was observed, whereas in a third patient, with no concentration of labelled antibody, the staining result was negative showing the specificity of the immunoscintigraphy findings. It is possible that the accumulation of radio-iodoamphetamine is due to binding to melanin but this is not certain as tissue samples from one of the two patients with positive scintigrams did not contain stainable melanin.

  4. 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

    2004-01-01

    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.

  5. Discovery and characterization of hydroxylysine in recombinant monoclonal antibodies.

    Science.gov (United States)

    Xie, Qing; Moore, Benjamin; Beardsley, Richard L

    2016-01-01

    Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MS(n) experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified. PMID:26651858

  6. Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

    Science.gov (United States)

    Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

    Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

  7. Monoclonal Antibodies as Treatment Modalities in Head and Neck Cancers

    Directory of Open Access Journals (Sweden)

    Vivek Radhakrishnan

    2015-11-01

    Full Text Available The standard treatments of surgery, radiation, and chemotherapy in head and neck squamous cell carcinomas (HNSCC causes disturbance to normal surrounding tissues, systemic toxicities and functional problems with eating, speaking, and breathing. With early detection, many of these cancers can be effectively treated, but treatment should also focus on retaining the function of the proximal nerves, tissues and vasculature surrounding the tumor. With current research focused on understanding pathogenesis of these cancers in a molecular level, targeted therapy using monoclonal antibodies (MoAbs, can be modified and directed towards tumor genes, proteins and signal pathways with the potential to reduce unfavorable side effects of current treatments. This review will highlight the current MoAb therapies used in HNSCC, and discuss ongoing research efforts to develop novel treatment agents with potential to improve efficacy, increase overall survival (OS rates and reduce toxicities.

  8. Radioimmunoassay of human growth hormone: characterization of a monoclonal antibody

    International Nuclear Information System (INIS)

    The introduction of cell-hybridization techniques by Koehler and Milstein in 1975 to produce monoclonal antibodies (MA) was a definite improvement in methodological tools of radioimmunoassay, quite apart from all other applications of this technique in immunohistochemistry, affinity chromatography, target-directed drug delivery systems etc. MAs would ideally be suited in RIAs, when the specificity is the crucial aspect of the determination. However, for reasons which are not completely understood, assays with MAs very often lack the attribute of being highly sensitive. Despite several reports in the literature on MAs against human growth hormone (HGH), none of these seem sensitive enough to be of use in clinical chemistry, where a too strongly marked specificity may even be unwanted. However, from the scientific point of view, MAs against polypeptide hormones are of great interest. An MA to HGH was developed with a sensitivity limit of 0.2 ng. The titre of the ascites fluid is higher than 1:106 and the specificity against human placental lactogen, human prolactin and rat growth hormone is nearly complete. A critical step of the RIA procedure is the separation of bound and free hormone. A combination of human immunoglobulin (Sandoglobulin) with polyethylene glycol gives optimal results. A Scatchard plot reveals an affinity constant of 4x10-11M-bar and a maximal binding capacity of 2x108cpm/μL. In conclusion, our monoclonal antibody represents an excellent investigational tool for endocrine research and it seems to be the most sensitive and specific MA to HGH described to date. However, for practical clinical applications, there seems to be little advantage of an MA over a conventional polyclonal antiserum. (author)

  9. Immunophenotypic studies of monoclonal gammopathy of undetermined significance

    Directory of Open Access Journals (Sweden)

    McKenna Robert W

    2008-11-01

    Full Text Available Abstract Background Monoclonal gammopathy of undetermined significance (MGUS is a common plasma cell dyscrasia, comprising the most indolent form of monoclonal gammopathy. However, approximately 25% of MGUS cases ultimately progress to plasma cell myeloma (PCM or related diseases. It is difficult to predict which subset of patients will transform. In this study, we examined the immunophenotypic differences of plasma cells in MGUS and PCM. Methods Bone marrow specimens from 32 MGUS patients and 32 PCM patients were analyzed by 4-color flow cytometry, using cluster analysis of ungated data, for the expression of several markers, including CD10, CD19, CD20, CD38, CD45, CD56 and surface and intracellular immunoglobulin light chains. Results All MGUS patients had two subpopulations of plasma cells, one with a "normal" phenotype [CD19(+, CD56(-, CD38(bright +] and one with an aberrant phenotype [either CD19(-/CD56(+ or CD19(-/CD56(-]. The normal subpopulation ranged from 4.4 to 86% (mean 27% of total plasma cells. Only 20 of 32 PCM cases showed an identifiable normal subpopulation at significantly lower frequency [range 0–32%, mean 3.3%, p 90% CD19(- plasma cells showed progression of disease, whereas none of the cases with >10% CD19(+ plasma cells evolved to PCM. Conclusion MGUS cases with potential for disease progression appeared to lack CD19 expression on >90% of their plasma cells, displaying an immunophenotypic profile similar to PCM plasma cells. A higher relative proportion of CD19(+ plasma cells in MGUS may be associated with a lower potential for disease progression.

  10. Laboratory testing in monoclonal gammopathy of renal significance (MGRS).

    Science.gov (United States)

    Leung, Nelson; Barnidge, David R; Hutchison, Colin A

    2016-06-01

    Recently, monoclonal gammopathy of renal significance (MGRS) reclassified all monoclonal (M) gammopathies that are associated with the development of a kidney disease but do not meet the definition of symptomatic multiple myeloma (MM) or malignant lymphoma. The purpose was to distinguish the M gammopathy as the nephrotoxic agent independent from the clonal mass. The diagnosis of MGRS obviously depends on the detection of the M-protein. More importantly, the success of treatment is correlated with the reduction of the M-protein. Therefore, familiarity with the M-protein tests is a must. Protein electrophoresis performed in serum or urine is inexpensive and rapid due to automation. However, poor sensitivity especially with the urine is an issue particularly with the low-level M gammopathy often encountered with MGRS. Immunofixation adds to the sensitivity and specificity but also the cost. Serum free light chain (sFLC) assays have significantly increased the sensitivity of M-protein detection and is relatively inexpensive. It is important to recognize that there is more than one assay on the market and their results are not interchangeable. In addition, in certain diseases, immunofixation is more sensitive than sFLC. Finally, novel techniques with promising results are adding to the ability to identify M-proteins. Using the time of flight method, the use of mass spectrometry of serum samples has been shown to dramatically increase the sensitivity of M-protein detection. In another technique, oligomeric LCs are identified on urinary exosomes amplifying the specificity for the nephrotoxic M-protein. PMID:27107835

  11. Cell line profiling to improve monoclonal antibody production.

    Science.gov (United States)

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

    2014-04-01

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

  12. Preparation of monoclonal antibodies against radiation-induced protein

    International Nuclear Information System (INIS)

    We obtained the 6 monoclonal antibodies against gamma-induced proteins of Deinococcus radiodurans, and these antibodies were designated as Mab-3F, 4B, 4D, 4F, 4G and 12G. Using these antibodies, we investigated the relations between gamma-induced proteins and other stress protein in strain R1, and the induction of proteins were compared among strain R1, resistant mutant (rec1) and radiosensitive mutant (rec30). We found new 6 proteins recognized by these monoclonal antibodies which were induced after gamma-irradiation especially in strain R1 and rec 1, but not induced in strain rec30. We suppose that these proteins participate in repair of DNA damages including double strand breaks caused by gamma-irradiation. One of them was around 46kDa protein band recognized by Mab-12G, and this protein was so induced in a large quantity after irradiation that the protein could detect by gold staining. In addition to this observation, we found some proteins which were induced in R1 and rec 1 by gamma-irradiation and other stress, but not in strain rec30, such as 31kDa protein band recognized by Mab-3F, 4B and 4G, and other 11 proteins which were especially induced in irradiated strain R1. The latter proteins might be reinforcement factor to radioresistance such as GroE and DnaK, or participant in repair of damage by gamma-irradiation in strain R1. (author)

  13. A competitive solid-phase radioimmunoassay for translational factors employing monoclonal antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibodies produced by the hybridoma techniques were purified by chromatography on DEAE Affi-Gel blue, and covalently coupled to Affi-Gel 10 to purify their antigens. The purified components were used to develop a sensitive competitive radioimmune assay for the quantitative determination of translational factors, as described here with a monoclonal antibody directed against yeast elongation factor 3. Antigen was adsorbed to polyvinyl chloride plastic surfaces and a limiting concentration of monoclonal antibody necessary to bind to the adsorbed antigen was determined. Varying concentrations of purified antigen and of samples containing unknown amounts of antigen were then mixed with the limiting concentration of monoclonal antibody, prior to or at the same time as the reaction of the antibody with the surface-adsorbed antigen. The amount of monoclonal antibody that bound to the surface-adsorbed antigen was determined with a second antibody, radioactive goat anti-mouse antibody. The addition of the free antigen preparations to the monoclonal antibody served to compete for the antibody with the antigen adsorbed to the plastic surfaces. The concentration of antigen in the unknown samples was estimated from the titration curves obtained with varying concentrations of pure antigen. This technique did not require isotopic labeling, modification or derivatization of the monoclonal antibody or its antigen. (Auth.)

  14. Murine interleukin 2 receptor. IV. Biochemical characterization

    International Nuclear Information System (INIS)

    The IL 2 receptor isolated from the IL 2-dependent CTL-L cell line was subjected to biochemical analysis. Pulse-chase and tunicamycin studies, as well as digestion with the endoglycosidases, Endo-F and Endo-H, of 35S-methionine-labeled IL 2 receptors suggested a single protein pecursor of 32,000 (p32) daltons. The p32 precursor was rapidly processed by addition of high-mannose-containing core N-linked sugars to intracytoplasmic precursor intermediates of 38,000 (p38) and 40,000 (p40) daltons, which undergo further processing to yield a mature surface receptor with heterogeneous apparent m.w. of 52,000 to 65,000 (p58). Two-dimensional gel studies indicated that p58 exhibited broad charge heterogeneity between pH 4.6 and 6.3. Endo-F digestions of p58 shifted the isoelectric focus point to a more basic 5.5 to 7.4. This considerable charge heterogeneity is consistent with the possibility that other post-translational modifications to the mouse IL 2 receptor occur besides addition of complex N-linked glycans. Immunoprecipitations of the IL 2 receptor from surface iodinated cells also revealed an additional band at 110,000 (p110) daltons. IEF vs SDS-PAGE two-dimensional gel studies demonstrated that p110 also had an isoelectric focus point identical to p58. Western blot studies with an anti-IL 2 receptor monoclonal antibody (7D4) demonstrates that p38, p40, p58, and p110 each expressed the epitope recognized by this antibody. Thus, it is likely that p110 is not a unique molecule that coprecipitates with IL 2 receptor. Western blot analysis of mitogen-stimulated T and B lymphocytes also revealed bands similar to p58 and p110, although these bands had an average apparent m.w. 3000 and 6000 less than those seen for CTL-L cells

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

    OpenAIRE

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

    1984-01-01

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

  16. A cytotoxic humanized anti-ganglioside antibody produced in a murine cell line defective of N-glycolylated-glycoconjugates.

    Science.gov (United States)

    Fernández-Marrero, Yuniel; Roque-Navarro, Lourdes; Hernández, Tays; Dorvignit, Denise; Molina-Pérez, Marively; González, Addys; Sosa, Katya; López-Requena, Alejandro; Pérez, Rolando; de Acosta, Cristina Mateo

    2011-12-01

    Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors. PMID:21802167

  17. 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.

  18. Development of an enzyme-linked immunosorbent assay based on the murine leukemia virus p30 capsid protein.

    Science.gov (United States)

    Wu, Dai-Tze; Aiyer, Sriram; Villanueva, Rodrigo A; Roth, Monica J

    2013-11-01

    Retroviral vectors derived from the murine leukemia virus (MuLV) are widely used as the starting material in the development of vectors for gene therapy and critical in answering questions relating to viral pathogenesis. The p30 capsid (CA) is the major viral core protein and an internal group antigen in MuLV. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of MuLV infectious particles with p30 CA core antigen protein. The ELISA was developed using several goat-polyclonal serum against MuLV p30 generated by the NCI as primary antibody and a rat-monoclonal antibody to CA available from ATCC. The MuLV p30 CA antigen was standardized against recombinant MuLV p30 CA expressed from bacteria. The assay is sensitive, accurate and linear within a defined concentration range of CA. Comparison with different MuLV quantitative methods including reporter gene transfer, reverse transcriptase activity assay, and viral RNA quantitative PCR, showed this ELISA protocol to be highly quantifiable within defined ranges, which can be correlated with infectious viral titer. PMID:23810854

  19. 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.

  20. Mutational dynamics of murine angiogenin duplicates

    Directory of Open Access Journals (Sweden)

    Fares Mario A

    2010-10-01

    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

  1. Nanoelectroablation therapy for murine basal cell carcinoma

    International Nuclear Information System (INIS)

    Highlights: ► Nanoelectroablation is a new, non-thermal therapy that triggers apoptosis in tumors. ► Low energy, ultrashort, high voltage pulses ablate the tumor with little or no scar. ► Nanoelectroablation eliminates 99.8% of the BCC but may leave a few remnants behind. ► Pilot clinical trials on human BCCs are ongoing and leave no remnants in most cases. -- Abstract: When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1+/−K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology and in response to drug therapy . We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5–7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 ± 5 (SEM) mm3 shrunk by 76 ± 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

  2. Nanoelectroablation therapy for murine basal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nuccitelli, Richard, E-mail: rich@bioelectromed.com [BioElectroMed Corp., 849 Mitten Rd., Suite 104, Burlingame, CA 94010 (United States); Tran, Kevin; Athos, Brian; Kreis, Mark; Nuccitelli, Pamela [BioElectroMed Corp., 849 Mitten Rd., Suite 104, Burlingame, CA 94010 (United States); Chang, Kris S.; Epstein, Ervin H. [The Children' s Hospital Oakland Research Institute, Oakland, CA 94609 (United States); Tang, Jean Y. [The Children' s Hospital Oakland Research Institute, Oakland, CA 94609 (United States); Stanford University, Stanford, CA 94305 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nanoelectroablation is a new, non-thermal therapy that triggers apoptosis in tumors. Black-Right-Pointing-Pointer Low energy, ultrashort, high voltage pulses ablate the tumor with little or no scar. Black-Right-Pointing-Pointer Nanoelectroablation eliminates 99.8% of the BCC but may leave a few remnants behind. Black-Right-Pointing-Pointer Pilot clinical trials on human BCCs are ongoing and leave no remnants in most cases. -- Abstract: When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1{sup +/-}K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology and in response to drug therapy . We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 {+-} 5 (SEM) mm{sup 3} shrunk by 76 {+-} 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4 mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

  3. 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

    2010-12-01

    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

  4. 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

    2010-12-13

    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

  5. 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

    2005-07-01

    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

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

    Science.gov (United States)

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

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

    DEFF Research Database (Denmark)

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

    1990-01-01

    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...... silver nitrate and toluidine blue O. Immunofluorescence using the monoclonal antibodies from the NIH was significantly more sensitive than any other single staining method and than the combination of Giemsa and methenamine silver nitrate staining. The study also showed that the cytospin centrifuge was...

  8. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    Science.gov (United States)

    Wilkinson, Trevor C I

    2016-06-15

    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. PMID:27284048

  9. Anti-Mesothelin Monoclonal Antibodies for the Treatment of Cancer | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute, Laboratory of Molecular Biology is seeking parties interested in collaborative research to further co-develop monoclonal antibodies for the treatment of mesothelin-expressing cancers.

  10. Metal chelate conjugated monoclonal antibodies, wherein the metal is an α emitter

    International Nuclear Information System (INIS)

    Methods of manufacturing and purifying metal chelate conjugated monoclonal antibodies are described, wherein the chelated metal emits alpha radiation. The conjugates are suited for therapeutic uses being substantially free of nonchelated radiometal. (author)

  11. CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST BOTH PIG AND RABBIT ZONA PELLUCIDA

    Institute of Scientific and Technical Information of China (English)

    OURU-QIANG

    1989-01-01

    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

  12. Use of monoclonal antibodies against avian retroviral protein p19 for competitive radioimmunoassay and immunodiffusion

    International Nuclear Information System (INIS)

    Monoclonal antibodies were used in competitive binding assays to investigate the arrangement of three epitopes on protein p19 of the avian myeloblastosis virus (AMV). It is reasoned that if the epitopes recognized by two monoclonal antibodies are physically close, the binding of one antibody will sterically block the binding of the other; conversely no blocking will occur if the epitopes are sufficiently distant. The results of these competitive binding assays demonstrated the presence of two distinct antigenic sites on protein p19. The monoclonal antibodies against protein p19 of AMV were also tested in gel double immunodiffusion. Since p19 protein shows strong tendency to aggregate, it was not surprising that clear precipitin lines with these monoclonal antibodies were obtained. (author)

  13. 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

    1999-01-01

    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

  14. DEMONSTRATION OF MULTIPLE ANTIGENIC DETERMINANTS ON 'MYCOPLASMA PNEUMONIAE' ATTACHMENT PROTEIN BY MONOCLONAL ANTIBODIES

    Science.gov (United States)

    Distinct multiple antigenic determinants of the attachment protein of Mycoplasma pneumoniae have been identified by limited proteolytic cleavage using specific monoclonal antibodies. Western blots prepared from the gels containing the cleaved fragments were probed with antiserum ...

  15. Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.

    Science.gov (United States)

    Spanier, Justin A; Frederick, Daniel R; Taylor, Justin J; Heffernan, James R; Kotov, Dmitri I; Martinov, Tijana; Osum, Kevin C; Ruggiero, Jenna L; Rust, Blake J; Landry, Samuel J; Jenkins, Marc K; McLachlan, James B; Fife, Brian T

    2016-01-01

    Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes. PMID:27292946

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

    Science.gov (United States)

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

    2002-09-01

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

  17. Turnover of T cells in murine gammaherpesvirus 68-infected mice

    DEFF Research Database (Denmark)

    Hamilton-Easton, A M; Christensen, Jan Pravsgaard; Doherty, P C

    1999-01-01

    Respiratory challenge of C57BL/6 mice with murine gammaherpesvirus 68 induces proliferation of T lymphocytes early after infection, as evidenced by incorporation of the DNA precursor bromodeoxyuridine. Using pulse-chase analysis, splenic and peripheral blood activated T lymphocytes were found to ...

  18. Murine myocardium OCT imaging with a blood substitute

    Science.gov (United States)

    Kim, Jeehyun; Villard, Joseph W.; Lee, Ho; Feldman, Marc D.; Milner, Thomas E.

    2002-06-01

    Imaging of the in vivo murine myocardium using optical coherence tomography (OCT) is described. Application of conventional techniques (e.g. MRI, Ultrasound imaging) for imaging the murine myocardium is problematic because the wall thickness is less than 1.5mm (20g mouse), and the heart rate can be as high as six-hundred beats per minute. To acquire a real-time image of the murine myocardium, OCT can provide sufficient spatial resolution (10 micrometers ) and imaging speed (1000 A-Scans/s). Strong light scattering by blood in the heart causes significant light attenuation making delineation of the endocardium-chamber boundary problematic. By replacing whole blood in the mouse with an artificial blood substitute we demonstrate significant reduction of light scattering in the murine myocardium. The results indicate a significant reduction in light scattering as whole blood hematocrit is diminished below 5%. To measure thickness change of the myocardium during one cycle, a myocardium edge detection algorithm is developed and demonstrated.

  19. Pharmacodynamics of Doxycycline in a Murine Malaria Model▿

    OpenAIRE

    Batty, Kevin T.; Law, Angela S. F.; Stirling, Verity; Moore, Brioni R.

    2007-01-01

    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.

  20. Failure of a diagnostic monoclonal immunofluorescent reagent to detect Legionella pneumophila in environmental samples.

    OpenAIRE

    Vickers, R M; Stout, J E; Yu, V L

    1990-01-01

    Three commercial diagnostic fluorescein-labeled antibodies, one monoclonal and two polyclonal, were compared to evaluate their abilities to detect Legionella pneumophila in environmental samples. The monoclonal conjugate failed to detect L. pneumophila in the 12 environmental samples studied by direct immunofluorescence. In contrast, the two polyclonal conjugates detected L. pneumophila in all 12 samples by both direct and indirect immunofluorescence. However, isolates recovered by culture fr...

  1. Monoclonal antibodies to the apical chloride channel in Necturus gallbladder inhibit the chloride conductance.

    OpenAIRE

    Finn, A L; Tsai, L M; Falk, R J

    1989-01-01

    Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The...

  2. Gel Electrophoresis as Quality Control Method of the Radiolabeled Monoclonal Antibodies

    Czech Academy of Sciences Publication Activity Database

    Kocurová, Veronika

    Rijeka : InTech, 2012 - (Magdeldin, S.), s. 447-462 ISBN 978-953-51-0457-5 R&D Projects: GA AV ČR IBS1048301 Institutional research plan: CEZ:AV0Z10480505 Keywords : monoclonal antibodies * gel electrophoresis * radiodiagnostic Subject RIV: FR - Pharmacology ; Medidal Chemistry http://www.intechopen.com/books/ gel - electrophoresis -advanced-techniques/ gel - electrophoresis -as-quality-control-method-of-the-radiolabeled-monoclonal-antibodies

  3. In silico design, construction and cloning of Trastuzumab humanized monoclonal antibody: A possible biosimilar for Herceptin

    OpenAIRE

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

    2012-01-01

    Background: There is a novel hypothesis in that antibodies may have specificity for two distinct antigens that have been named "dual specificity." This hypothesis was evaluated for some defined therapeutic monoclonal antibodies (mAbs) such as Trastuzumab, Pertuzumab, Bevacizumab, and Cetuximab. In silico design and construction of expression vectors for trastuzumab monoclonal antibody also in this work were performed. Materials and Methods: First, in bioinformatics studies the 3D structur...

  4. Development of an antigen microarray for high throughput monoclonal antibody selection

    OpenAIRE

    Staudt, Nicole; Müller-Sienerth, Nicole; Wright, Gavin J.

    2014-01-01

    Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five differ...

  5. High-Efficiency Screening of Monoclonal Antibodies for Membrane Protein Crystallography

    OpenAIRE

    Lim, Hyun-Ho; Fang, Yiling; Williams, Carole

    2011-01-01

    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 mon...

  6. Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses.

    OpenAIRE

    Walls, H H; Harmon, M W; Slagle, J J; Stocksdale, C; Kendal, A P

    1986-01-01

    Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein. The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected H1N1, H2N2, and H3N2 influenza A virus strains collected between 1934 and 1984. Each of the inf...

  7. Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma.

    Science.gov (United States)

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Krejci, Eric; Blake, Thomas A; Johnson, Rudolph C; Masson, Patrick; Lockridge, Oksana

    2016-01-01

    Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals. PMID:26585590

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

    OpenAIRE

    Gyorffy, S; Clarke, A J

    1992-01-01

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

  9. Study of Leishmania major-infected macrophages by use of lipophosphoglycan-specific monoclonal antibodies.

    OpenAIRE

    Handman, E

    1990-01-01

    Leishmania major infection of macrophages is followed by a time-dependent appearance of lipophosphoglycan (LPG) that can be detected on the surface of infected cells by monoclonal antibodies. The origin of these LPG epitopes is probably the intracellular amastigote. LPG epitopes could be detected on the amastigote and the infected macrophage by a number of monoclonal antibodies directed to several distinct determinants on the phosphoglycan moiety. The macrophage-expressed LPG may be modified ...

  10. Immunohistochemical study of human pulmonary surfactant apoproteins with monoclonal antibodies. Pathologic application for hyaline membrane disease.

    OpenAIRE

    Kuroki, Y.; Dempo, K.; Akino, T

    1986-01-01

    Three monoclonal antibodies, PC6, PE10, and PE12, were used for immunohistochemical studies of human lungs by immunoperoxidase staining. Monoclonal antibodies PC6 and PE10 against pulmonary surfactant apoproteins stained faint granules in the cytoplasm of some alveolar wall cells in adult lung. These stained cells appeared to be alveolar Type II cells. A fetal lung of 20 weeks' gestation had no any positive staining. However, a few scattered positive cells were observed in a newborn lung of 3...

  11. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

    OpenAIRE

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

    2014-01-01

    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...

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

    OpenAIRE

    Sanjeev Goswami

    2013-01-01

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

  13. Staining characteristics of six commercially available monoclonal immunofluorescence reagents for direct diagnosis of Chlamydia trachomatis infections.

    OpenAIRE

    Cles, L D; Bruch, K; Stamm, W. E.

    1988-01-01

    Using purified elementary bodies of 14 Chlamydia trachomatis serovars in an in vitro assay, we compared the staining characteristics of six commercially available monoclonal antibody reagents used for direct immunofluorescent staining of patient smears. Considerable variation in the degree of brightness, consistency of staining, and specificity of the six reagents was found. Monoclonal antibodies against the major outer membrane proteins of C. trachomatis produced brighter fluorescence, more ...

  14. Specificities of monoclonal antibodies against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis.

    OpenAIRE

    Huber-Lukac, M; Lüthy, P; Braun, D G

    1983-01-01

    Eight hybrid cell lines secreting monoclonal antibodies directed against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis were grown in BALB/c mice. Ascites fluids were collected, and the antibodies were purified by antigen-affinity chromatography. The specificity of each monoclonal antibody for the toxin and protoxin was established by the indirect enzyme-linked immunosorbent assay. All the antibodies consisted of gamma 1 heavy and kappa light chains. They were reac...

  15. Labelling, quality control and clinical evaluation of monoclonal antibodies for scintigraphy. Final report of a co-ordinated research programme 1991-1996

    International Nuclear Information System (INIS)

    Realizing the potential of labelled monoclonal antibodies for in vivo diagnosis and therapy and the interest in many developing Member States for acquiring expertise in this field the IAEA initiated a co-ordinated research programme in 1991 focusing on 99Tcm labelling of antibodies, their quality control and scintigraphic evaluation. Twelve laboratories from Asia, Latin America, Europe and North America participated in this programme which was concluded in 1996. During this programme the participants investigated the 99Tcm labelling of a murine anti-CEA antibody using the method of chelating 99Tcm with the free sulfhydryl groups generated by reaction with reducing agents such as mercapto ethanol. During the later part of the programme this method was also extended to 99Tcm labelling of hIgG. All the participating laboratories could gain valuable experience in 99Tcm antibody labelling techniques and formulation of kits. Many of them have been use in patients by collaborating nuclear medicine specialists with satisfactory results. This report is a compilation of the detailed results obtained by the participating laboratories and includes a summary and assessment of the achievement of the CRP

  16. Radioimmunoimaging of experimental thrombi in dogs using Tc-99m labeled monoclonal antibody fragments [MAPab-F(ab')/sub 2/] reactive with human platelets

    International Nuclear Information System (INIS)

    Radioimmunoimaging of thrombi could have great clinical value in the management of coronary artery and thromboembolic disease. In-111-oxine-labeled platelets currently used require platelet isolation, delayed imaging, background subtraction and there is also potential for damaging or contaminating platelets during labeling. Murine monoclonal antibody (IgG/sub 2/a) fragments directed against human platelet membrane components (cross-reactive with dog platelets) were labeled with Tc-99m and repurified from ''kits''. After radiolabeling, 91.5-93.3% of the Tc-99m was antibody-associated. The preparations retained immunoreactivity, as determined by the ratio of cell to plasma-associated radioactivity (ratios 54.7-63.8). Tc-99m-MAPAb-F(ab')/sub 2/ were injected i.v. into dogs with thrombi produced in peripheral and pulmonary veins and arteries. About 50% of the radioactivity was cleared from the blood in 3-6 min. and 18-24% was excreted in the urine within 3 hrs. The thrombi were consistently and easily visible within 1-3 hrs. with no need for blood pool subtraction. In some cases, intimal damage along the path of catheters was seen. No adverse side effects were observed. The advantages of this method are: short and simple preparation, no need for blood pool subtraction and early visualization of thrombi. Human studies are warranted to determine its clinical efficacy

  17. Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: An evaluation of their neutralizing activity in a cell culture system.

    Science.gov (United States)

    Kobayashi, Tominari; Takahashi, Masaharu; Tanggis; Mulyanto; Jirintai, Suljid; Nagashima, Shigeo; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2016-07-01

    Hepatitis E virus (HEV) is the causative agent of acute hepatitis. Rat HEV is a recently discovered virus related to, but distinct from, human HEV. Since laboratory rats can be reproducibly infected with rat HEV and a cell culture system has been established for rat HEV, this virus may be used as a surrogate virus for human HEV, enabling studies on virus replication and mechanism of infection. However, monoclonal antibodies (MAbs) against rat HEV capsid (ORF2) protein are not available. In this study, 12 murine MAbs were generated against a recombinant ORF2 protein of rat HEV (rRatHEV-ORF2: amino acids 101-644) and were classified into at least six distinct groups by epitope mapping and a cross-reactivity analysis with human HEV ORF2 proteins. Two non-cross-reactive MAbs recognizing the protruding (P) domain detected both non-denatured and denatured rRatHEV-ORF2 protein and efficiently captured cell culture-produced rat HEV particles that had been treated with deoxycholate and trypsin, but not those without prior treatment. In addition, these two MAbs were able to efficiently neutralize replication of cell culture-generated rat HEV particles without lipid membranes (but not those with lipid membranes) in a cell culture system, similar to human HEV. PMID:26992654

  18. Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica.

    Science.gov (United States)

    Tachibana, H; Kobayashi, S; Nagakura, K; Kaneda, Y; Takeuchi, T

    1991-01-01

    Twenty monoclonal antibodies were produced against trophozoites of Entamoeba histolytica strains HK-9 and HM-1: IMSS. When reactivity to various enteric protozoa was examined by an indirect fluorescence antibody test, 15 of the monoclonal antibodies were strongly reactive with E. histolytica trophozoites. Species-specific antigens recognized by these monoclonal antibodies were located on the plasma membrane, nucleus, cytoplasm, and cytoskeletal structures of the trophozoites. Two of the remaining five monoclonals reacted strongly with trophozoites of the E. histolytica-like Laredo strain. The determinant antigen was located in the cytoplasm. The three remaining monoclonal antibodies were found to recognize cross-reactive antigens between E. histolytica and E. histolytica-like Laredo, E. hartmanni, E. coli, Dientamoeba fragilis, Giardia lamblia, and Trichomonas hominis. These three antibodies were also reactive with T. vaginalis and mammalian cells such as HeLa cells. Thus, the combined use of monoclonal antibodies seems capable of distinguishing E. histolytica and/or E. histolytica-like Laredo from other enteric protozoa. PMID:1724012

  19. Extracorporeal adsorption therapy: A Method to improve targeted radiation delivered by radiometal-labeled monoclonal antibodies

    International Nuclear Information System (INIS)

    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

  20. Potent neutralizing monoclonal antibodies against Ebola virus infection.

    Science.gov (United States)

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  1. Radioiodination of monoclonal antibody intact anti-CEA

    International Nuclear Information System (INIS)

    The purpose of this study is to examine a convenient system that can be used to iodinate monoclonal antibodies which is rapid, simple, efficient and reproducible, and which can be accomplished in radiopharmaceutical laboratories. It is important to remember that antibodies are sensitive biochemicals, subject to losses of the activity that is essential to their mode of action, namely the ability to bind specific antigen. The advent of solid phase iodination agents has greatly expanded the range of gentle iodination techniques available for iodinating sensitive biological materials. The agent most widely used is the Iodogen (1,3,4,6 tetrachloro-3a-6a diphenylglycoluril) method. Anti-CEA 4C sub(11) IgG sub(2a,k) (prepared in the Ludwig Institute-Sao Paulo-Brazil ) is used as model to evaluate the Iodogen methodology. The miniature chromatographic system, also rapid, accurate, simple, efficient was elaborated to determine the labelling efficiency incorporation of iodine into immunoglobulin, and the radiochemical purity of sup(131)I-anti-CEA. (author)

  2. Treating multiple sclerosis with monoclonal antibodies: a 2010 update.

    Science.gov (United States)

    Buttmann, Mathias

    2010-05-01

    Treating multiple sclerosis (MS) with monoclonal antibodies (mAbs) has been marked by both progress and setbacks in the past 2 years, which are reviewed here. The natalizumab section of the article centers around progressive multifocal leukoencephalopathy (PML), and discusses PML risk in relation to treatment duration, bioassays for individual risk prediction, the concept of drug holidays, clinical course and treatment of PML, as well as safety-related regulatory actions. The rituximab section critically analyzes recent clinical trial results, discusses the clinical relevance of anti-idiotypic mAbs and makes a short excursion to neuromyelitis optica. Following this, the newer anti-CD20 mAbs ocrelizumab and ofatumumab, which are currently being tested in Phase II for MS, are reviewed and compared. The alemtuzumab section highlights novel data on mechanisms of action, potentially allowing individual risk prediction, and new results from the CAMMS223 trial, as well as the current status of the pivotal MS studies. The daclizumab section summarizes new open-label data, shedding more light on the adverse-effect profile of the drug in MS patients, and reports on its Phase III status. Subsequently, a failed ustekinumab trial and LY2127399 are reviewed. Taking into account late Phase II and III data on novel oral agents, the final section attempts to provide a detailed perspective on disease-modifying MS therapy in the medium term. PMID:20420497

  3. Gamma radiations an effective way of monoclonal antibodies sterilization

    International Nuclear Information System (INIS)

    The sterilization for radiations of pharmaceutical products is an effective, sure and reliable procedure; that it have been proving technically and grateful for different pharmacopoeia. The Monoclonal Antibodies (Acm) produced in the Center of Molecular Immunology (CIM) are products parenteral for the one which results indispensable that they complete the requirements of established sterility. The radio sterilization result the method more recommend for the sterilization of the Acm deep drying, due to the contained first floor of humidity remnant that minimizes the formation of sub-product that they affect their properties. With the objective of proposing a good dose of irradiation for the sterilization, we were carried out a study of the radius sensibility so much of the product like of the polluting of greater frequency of isolation of the clean area of the CIM. The characterization of the radius sensibility of the different micro- organisms was determined by D10 characteristic of each isolated strains. From the developed studies the Gram-positive rods endospore-forming were the most resistant strains at the deep drying, the radiations and they were of the greater frequency of apparition in the carried out isolations. We could conclude that utilizing a dose of 10 kGy it is possible to eliminate of the pollution more radio resistant, assuring the sterility required in the product, and without inducing effects under desire radiolytic in the same

  4. ANTITUMOR EFFECTS OF MONOCLONAL ANTIBODY FAB′ FRAGMENT CONTAINING IMMUNOCONJUGATES

    Institute of Scientific and Technical Information of China (English)

    刘小云; 甄永苏

    2002-01-01

    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.

  5. Production of monoclonal antibodies reactive with ovine eosinophils

    Directory of Open Access Journals (Sweden)

    Meeusen Els NT

    2007-09-01

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

  6. Clinical application of antibody monoclonal humanized anti-EGFrnimotuzumab labeled

    International Nuclear Information System (INIS)

    Most malignant tumors are of epithelial origin. These are characterized by overexpression of the receptor of epidermal growth factor (EGFR), which the neoplastic cells escape the regulatory mechanisms are allowed, so its high concentration of membrane is generally associated with a poor prognosis . By binding an antibody specifically to this receptor, preventing binding of EGF latter and activation mechanism tyrosine kinase inhibiting cell mitosis and apoptosis causing tumor cell. For this reason, the EGFr has been considered as an attractive target for anti-tumor therapy. The humanized monoclonal antibody anti-EGFr nimotuzumab was developed by the Center of Molecular Immunology (Havana, Cuba). Numerous clinical trials have been developed in the Department of Clinical Research Center Isotopes (Cuba), in which it has been applied this antibody, both labeled with 99mTc for immuno gammagraphic detection of tumors, as labeled with 188Re for radioimmunotherapy of gliomas high degree of malignancy. The aim of this paper is to show the experience of the Department of Clinical Research of CENTIS in various clinical trials with marking for both immuno gammagraphics detection of tumors, such as for radioimmunotherapy nimotuzumab. (author)

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  8. DNA immunization as a technology platform for monoclonal antibody induction.

    Science.gov (United States)

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-01-01

    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. PMID:27048742

  9. Application of monoclonal antibodies in diagnostics of the colorectal cancer

    International Nuclear Information System (INIS)

    Immunoscintigraphy with CEA monoclonal antibodies (MoA) in patients with colorectal cancer has been applied since 1987 by the authors. MoA from the hybridoma F023C5 are used (IgG1-class) and their fragments labelled with 131I and 111In. The labelled MoA are introduced intravenously in the course of 30 min, the total activity is 2.5 - 3.5 mCi. The scanning is made 48 and 96 hours on gamma camera. An additional activity on 99mTc-sulfocolloid and 99mTc-DTPA is applied for outlining the liver and kidney contours. Digital substraction technique is applied for image processing with contrast and background reduction. The thyroid is blocked with Lugol solution in a course of 5-6 days. Among all of the 18 investigated patients a positive result has been observed in 16. Metastases bigger than 1 cm have a positive scan. No initial invasion in the regional lymph nodes has been established. 3 figs., 4 refs

  10. Potent neutralizing monoclonal antibodies against Ebola virus infection

    Science.gov (United States)

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F.; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  11. Cysteinylation of a monoclonal antibody leads to its inactivation.

    Science.gov (United States)

    McSherry, Troy; McSherry, Jennifer; Ozaeta, Panfilo; Longenecker, Kenton; Ramsay, Carol; Fishpaugh, Jeffrey; Allen, Steven

    2016-01-01

    Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism. PMID:27050640

  12. Epitope Mapping of Fc gamma RIIa Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Caroline Tan Sardjono

    2015-11-01

    Full Text Available FcγRIIa (CD32 is an IgG receptor which has been shown to be important in autoimmune disease pathology. IV.3, 8.7, and 7.30 are anti-FcγRIIa monoclonal antibodies (mAbs, which block the interaction between FcγRIIa and complex IgG. In this study, the three mAbs were demonstrated to inhibit FcγRIIa function. The determination of the precise epitopes of the IV.3, 8.7, and 7.30 mAbs may become a potential approach for designing inhibitors for FcγRIIa. The epitope of IV.3, 8.7, and 7.30 were determined using chimeric receptors based on the extracellular domains of FcγRIIa and the FcεRI a chain. The epitopes for IV.3 was found to be mapped on amino acid residues 132-137, while 8.7 and 7.30 were on amino acid residues 112-119 and 157-162. Based on the crystal 3D model of FcγRIIa molecule, these amino acid sequences are clustered together forming a contiguous region within the ligand binding site of the receptor.

  13. Reversible cluster formation in concentrated monoclonal antibody solutions

    Science.gov (United States)

    Godfrin, P. Douglas; Porcar, Lionel; Falus, Peter; Zarraga, Isidro; Wagner, Norm; Liu, Yun

    2015-03-01

    Protein cluster formation in solution is of fundamental interest for both academic research and industrial applications. Recently, industrial scientists are also exploring the effect of reversible cluster formation on biopharmaceutical processing and delivery. However, despite of its importance, the understanding of protein clusters at concentrated solutions remains scientifically very challenging. Using the neutron spin echo technique to study the short time dynamics of proteins in solutions, we have recently systematically studied cluster formation in a few monoclonal antibody (mAb) solutions and their relation with solution viscosity. We show that the existence of anisotropic attraction can cause the formation of finite sized clusters, which increases the solution viscosity. Interestingly, once clusters form at relatively low concentrations, the average size of clusters in solutions remains almost constant over a wide range of concentrations similar to that of micelle formation. For a different mAb we have also investigated, the attraction is mostly induced by hydrophobic patches. As a result, these mAbs form large clusters with loosely linked proteins. In both cases, the formation of clusters all increases the solution viscosity substantially. However, due to different physics origins of cluster formation, solutions viscosities for these two different types of mAbs need to be controlled by different ways.

  14. Production of radiolabeled monoclonal antibody conjugates by photoaffinity labeling

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-06-01

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

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

    Science.gov (United States)

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

    1999-07-20

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

  16. Warfarin improves neuropathy in monoclonal gammopathy of undetermined significance.

    Science.gov (United States)

    Henry Gomez, Teny; Holkova, Beata; Noreika, Danielle; Del Fabbro, Egidio

    2016-01-01

    We report a case of a 60-year-old man who was referred to a palliative care clinic with monoclonal gammopathy of undetermined significance (MGUS)-associated neuropathy, responding to a therapeutic trial of warfarin. Electromyography showed distal symmetric sensory axonal neuropathy. The patient reported having had improvement of his neuropathic symptoms while taking warfarin postoperatively for thromboprophylaxis 1 year prior, and recurrence of his symptoms after the warfarin was discontinued. The patient was rechallenged with a trial of warfarin, targeting an international normalised ratio of 1.5-2.0. His pain scores decreased from 5/10 to 3/10 at 1 month and symptom improvement was maintained through 24 months of follow-up. Warfarin had a remarkable impact on our patient's symptoms and quality of life. The mechanisms mediating the symptomatic benefit with warfarin are unclear; however, a placebo effect is unlikely. Further studies may help guide the use of warfarin for MGUS-associated neuropathy. PMID:27317760

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

    Directory of Open Access Journals (Sweden)

    Caleb D. Marceau

    2014-01-01

    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.

  18. DNA immunization as a technology platform for monoclonal antibody induction

    Science.gov (United States)

    Liu, Shuying; Wang, Shixia; Lu, Shan

    2016-01-01

    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. PMID:27048742

  19. Development of a monoclonal antibody specific to cooked mammalian meats.

    Science.gov (United States)

    Hsieh, Y H; Sheu, S C; Bridgman, R C

    1998-04-01

    Detection of species adulteration in ground meat products is important for consumer protection and food-labeling law enforcement. This study was conducted to develop monoclonal antibodies (MAbs) that can be used in an enzyme-linked immunosorbent assay (ELISA) for rapid detection of any cooked mammalian meats in cooked poultry products. Soluble muscle proteins extracted from cooked pork (heated at 100 degrees C for 15 min) were used as the antigen to immunized mice for developing the MAb. One that was developed, MAb 2F8 (IgG2b class), strongly reacted with cooked meat of five mammalian species (beef cattle, hogs, sheep, horse, and deer) but did not react with any cooked poultry (chicken, turkey, and duck) or raw meats. At least 0.5% by weight of pork, beef, lamb, and horse meats in a chicken-based mixture could not detect using the indirect ELISA with MAb 2F8. The MAb 2F8 is useful in a single initial screening test to detect the presence of five nonpoultry meat adulterants in cooked poultry products. PMID:9709213

  20. Novel monoclonal autoantibody specificity associated with ribonucleoprotein complexes

    International Nuclear Information System (INIS)

    The authors describe an IgG/sub 2a/, kappa monoclonal autoantibody (mAb) F78 derived from a 6-month old MRL-Mp lpr/lpr mouse that recognizes a novel epitope associated with small nuclear ribonuclear protein complexes (snRNP). Indirect immunofluorescent staining of HEp-2 cells with F78 showed a nonnucleolar speckled nuclear pattern characteristic of anti-RNP and anti-Sm mAbs which could be abrogated by pretreating fixed cells with 0.1M HCl prior to staining. Immunoblots of whole cell extracts (dissociated in SDS, urea and mercaptan at 40C then subjected to SDS-PAGE) showed that F78 selectively bound to a component of M/sub r/ = 100,000 clearly distinct from components recognized by two mAbs described by Billings et al that detected, respectively, proteins of M/sub r/ = 70,000 associated with RNP and M/sub r/ = 13,000 associated with Sm. Incubation of extracts at 1000C prior to SDS-PAGE eliminated subsequent binding of F78 but not of the other nAbs. F78 as well as the other mAbs selectively immunoprecipitated characteristic patterns of small nuclear RNAs (U1, U2, U4, U5, U6) from extracts of 32P-phosphate labeled HeLa cells. These results suggest a new specificity associated with snRNP that is recognized in the MRL autoimmune response

  1. Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum.

    Science.gov (United States)

    Wiens, G D; Kaattari, S L

    1991-02-01

    Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57. PMID:1987079

  2. Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Ramos Franco Antonia Maria

    1997-01-01

    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

  3. 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

    2010-01-01

    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.

  4. Single Amino Acid Insertion in Loop 4 Confers Amphotropic Murine Leukemia Virus Receptor Function upon Murine Pit1

    DEFF Research Database (Denmark)

    Lundorf, Mikkel D.; Pedersen, Finn Skou; O'Hara, Bryan; Pedersen, Lene

    1998-01-01

    Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch of...

  5. Monoclonal antibodies to the thyrotropin receptor raised by an autoantiidiotypic protocol and their relationship to monoclonal autoantibodies from Graves' patients

    International Nuclear Information System (INIS)

    Monoclonal antibodies that bind to the TSH receptor were obtained by an autoantiidiotypic approach in which immunization of BALB/c mice was performed with mixtures of bovine (b) and human (h) TSH. Two of 28 positive wells were selected for cloning and characterization: D2 and 4G11. Their antiidiotypic character was evidenced by TSH-inhibitable binding to affinity-purified polyclonal anti-TSH. The specificity of D2 and 4G11 for the hormone-binding region of the TSH receptor was demonstrated by several findings: 1) they inhibited the binding of [125I]iodo-bTSH to receptor in a dose-dependent manner; 2) their binding to partially purified thyroid plasma membranes could be completely inhibited by bTSH and hTSH; and 3) they inhibited the TSH-dependent growth and adenylate cyclase stimulation in FRTL-5 cells in a dose-dependent manner. By Western blot analysis of bovine thyroid membranes, D2 bound to a polypeptide of 188,000-195,000 mol wt under nonreducing conditions and 54,000-59,000 mol wt after treatment of membranes with beta-mercaptoethanol; the 4G11 epitope was undetectable. Scatchard analysis of the binding of 125I-labeled antibodies to receptor showed that 4G11 bound to a single site with a Kd of 5.7 X 10(-9) M, whereas D2 showed complex binding characterized by high affinity (Kd = 1.74 X 10(-11) M) and low affinity (Kd = 1.3 X 10(-8) M) sites. Binding studies in which D2 and 4G11 competed with each other for the TSH receptor showed mutual but unequal inhibition. The data suggest that portions of the D2 and 4G11 epitopes overlap, but that there is a high affinity binding site(s) for D2 for which 4G11 competes less effectively. The binding of D2 and 4G11 to TSH receptor was inhibited by monoclonal antibodies secreted by Graves' heterohybridomas, showing that D2 and 4G11 share characteristics with autoantibodies of Graves' disease

  6. Antibody discovery: sourcing of monoclonal antibody variable domains.

    Science.gov (United States)

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described. PMID:24168292

  7. 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

    2012-01-01

    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.

  8. 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

    1999-01-01

    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 the......, 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....

  9. Distribution of Murine Tissue Specific γ-Glutamyltransferase: Comparison of six monoclonal antibody applications in Enzyme linked immunosorbent assay, radioimmunoassay, immunohistochemistry, and autoradiography

    International Nuclear Information System (INIS)

    γ-Glutamyltransferase (GGT: E.C. 2.3.2.2.) is a glycoprotein enzyme which is involved in glutathione metabolism and amino acid transport through the plasma membrane. It is distributed widely in several organs including liver and kidney. Several isozymes of GGT have been reported and some of the isozymes may be associated with hepatocarcinogenesis. We have produced six monoclnal antibodies (mAbs) against GGT purified from the liver of 2-acetamidofluorene (AAF) treated rats. All of the six mAbs were obtained by immunizing mice with liver GGT. Six hybridomas which produced anti-GGT Abs were extensively subcloned and injected into the peritoneal cavity of BALB/c mice to obtain large quantities of Abs. These mAbs were purified from ascites by ammonium sulfate precipitation and protein A sepharose CL-4B column chromatography. Using these mAbs we performed enzyme-linked immunosorbent assay (ELISA), radioimmvnoassay (RIA), immunohistochemistry (IHC), and autoradiography (ARG) to study the distribution of GGT isozyme in tissue. The results indicate that GGT-mAb 1 is specific for the AAF treated liver GGT, GGT-mAb 5 for the normal liver GGT, and GGT-mAb 6 for the normal kidney GGT. These mAbs may be used to evaluate the distribution of GGT isozymes in different tissues.

  10. Suppression of Murine Allergic Airway Disease by IL-2:Anti-IL-2 Monoclonal Antibody-Induced Regulatory T Cells1

    OpenAIRE

    Wilson, Mark S; Pesce, John T; Thirumalai R Ramalingam; Thompson, Robert W.; Cheever, Allen; Wynn, Thomas A

    2008-01-01

    Regulatory T cells (Treg) play a decisive role in many diseases including asthma and allergen-induced lung inflammation. However, little progress has been made developing new therapeutic strategies for pulmonary disorders. In the current study we demonstrate that cytokine:antibody complexes of IL-2 and anti-IL-2 mAb reduce the severity of allergen-induced inflammation in the lung by expanding Tregs in vivo. Unlike rIL-2 or anti-IL-2 mAb treatment alone, IL-2:anti-IL-2 complexes dampened airwa...

  11. Distinct antigenic characteristics of murine parietal yolk sac laminin

    DEFF Research Database (Denmark)

    Wewer, U M; Tichy, D; Damjanov, A; Paulsson, M; Damjanov, I

    1987-01-01

    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 pu...... derived from normal or malignant PYS cells has distinct antigenic sites that are immunochemically not apparent in most other basement membranes....

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

    OpenAIRE

    Leili Aghebati; Jalal Abdolalizadeh; Jafar Majidi; Behzad Baradaran; Koushan Sineh Sepehr; Fatemeh Zare Shahneh

    2013-01-01

    Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously ...

  13. Should a Study of Monoclonal Gammopathy of Undetermined Significance Be of Clinical Interest to Predict Myeloma Overcome?

    OpenAIRE

    J Véronique-Baudin; Pierre-Louis, O.; Smith-Ravin, J; M Vestris; Rose, M.; J-C Méniane; J Macni; Joachim, C.; P Escarmant; M-S Pierre-Louis

    2016-01-01

    The majority of monoclonal gammopathy of undetermined significance (MGUS) are benign forms that require no treatment but can be considered as a pre-cancerous state. Monoclonal gammopathy of undetermined significance is quite frequent, and related to age. The risk of progression to multiple myeloma or another type of malignant lymphoproliferation is estimated at 1% per year. Monoclonal gammopathy of undetermined significance is twice as frequent in patients of African descent, and prevalence ...

  14. Monoclonal antibodies specific for Escherichia coli J5 lipopolysaccharide: cross-reaction with other gram-negative bacterial species.

    OpenAIRE

    Mutharia, L M; Crockford, G; Bogard, W C; Hancock, R E

    1984-01-01

    Four monoclonal antibodies against Escherichia coli J5 were studied. Each of these monoclonal antibodies reacted with purified lipopolysaccharides from E. coli J5, the deep rough mutant Salmonella minnesota Re595, Agrobacterium tumefaciens, and Pseudomonas aeruginosa PAO1 as well as with the purified lipid A of P. aeruginosa. Enzyme-linked immunosorbent assays using the outer membranes from a variety of gram-negative bacteria demonstrated that these lipid A-specific monoclonal antibodies inte...

  15. Targeting of colorectal cancer using radiolabelled monoclonal antibody

    International Nuclear Information System (INIS)

    Full text: Radioimmune targeting of tumours using monoclonal antibody (Mab) has been shown to have a clinical role in detecting and treating tumours. The aim of this study was to evaluate a Mab (c.30.6, CRC Biopharmaceuticals, Sydney) for targeting colorectal cancer by labelling with I-123 for diagnosis. I-123 labelling of the Mab was achieved using Iodogen and purification of the labelled Mab was standardised with FPLC gel filtration. The process was validated for production of sterile, pyrogen free and immunoreactive product, Ten patients studies have been evaluated so far. Whole body and SPECT imaging were performed at 0, 4, 24 and 48 hours post injection. Pharmacokinetics was assessed by serial blood sampling and dosimetry calculated using serial imaging. The mean labelling efficiency was 68.0+/-7.0% and the labelled product found to be stable > 24 hours with the mean immunoreactive fraction of 60.7+/-8.6%. The pharmacokinetics of the labelled Mab has an alpha T1/2 of 2.7+/-0.5 minutes and beta T1/2 of 58.4+/-6.9 hours and the radiation dosimetry was 3.2+/0.3 E -02 mSv/MBq. The labelled antibody was found to localise in both primary and metastatic tumour. In conclusion we have successfully developed the process and infrastructure for radioimmune tumour targeting within St Vincent's Campus. This may enable us to investigate various MAbs for diagnosis and therapy. Copyright (2000) The Australian and New Zealand Society of Nuclear Medicine Inc

  16. Structural Characterization of a Monoclonal Antibody-Maytansinoid Immunoconjugate.

    Science.gov (United States)

    Luo, Quanzhou; Chung, Hyo Helen; Borths, Christopher; Janson, Matthew; Wen, Jie; Joubert, Marisa K; Wypych, Jette

    2016-01-01

    Structural characterization was performed on an antibody-drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0-8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the C(H)2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C(H)2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C(H)2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody. PMID:26629796

  17. Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

    Directory of Open Access Journals (Sweden)

    Yin Xiuchen

    2012-11-01

    Full Text Available Abstract Background The VP3 protein of goose parvovirus (GPV or Muscovy duck parvovirus (MDPV, a major structural protein, can induce neutralizing antibodies in geese and ducks, but monoclonal antibodies (MAbs against VP3 protein has never been characterized. Results Three hybridoma cell lines secreting anti-GPV VP3 MAbs were obtained and designated 4A8, 4E2, and 2D5. Immunoglobulin subclass tests differentiated them as IgG2b (4A8 and 4E2 and IgG2a (2D5. Dot blotting assays showed that three MAbs reacted with His-VP3 protein in a conformation-independent manner. A competitive binding assay indicated that the MAbs delineated two epitopes, A and B of VP3. Immunofluorescence assay showed that MAbs 4A8, 4E2, and 2D5 could specifically bind to goose embryo fibroblast cells (GEF or duck fibroblast cells (DEF infected with GPV and MDPV. Dot blotting also showed that the MAbs recognized both nature GPV and MDPV antigen. Western blotting confirmed that the MAbs recognized VP3 proteins derived from purified GPV and MDPV particles. The MAbs 4A8 and 2D5 had universal reactivity to heterologous GPV and MDPV tested in an antigen-capture enzyme-linked immunosorbent assay. Conclusions Preparation and characterization of these the MAbs suggests that they may be useful for the development of a MAb-capture ELISA for rapid detection of both GPV and MDPV. Virus isolation and PCR are reliable for detecting GPV and MDPV infection, but these procedures are laborious, time-consuming, and requiring instruments. These diagnosis problems highlight the ongoing demand for rapid, reproducible, and automatic methods for the sensitive detection of both GPV and MDPV infection.

  18. Reappraisal of risk factors for monoclonal gammopathy of undetermined significance.

    Science.gov (United States)

    Boursi, Ben; Weiss, Brendan M; Haynes, Kevin; Mamtani, Ronac; Yang, Yu-Xiao

    2016-06-01

    A number of epidemiologic studies have demonstrated associations between obesity and diabetes and the risk of monoclonal gammopathy of undetermined significance (MGUS). However, since MGUS is an asymptomatic condition we evaluated whether these are true associations or the result of detection-bias. We conducted a nested case-control study using a large primary-care database. Cases were defined as those with incident diagnosis of MGUS. For every case, four eligible controls matched on age, sex, practice site, and duration of follow-up were selected. Exposure variables included obesity and diabetes (including antidiabetic therapies) as well as other metabolic risk factors. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using conditional logistic regression. The study included 2363 MGUS patients and 9193 matched controls. In the primary analysis, obesity and diabetes were associated with higher MGUS risk with an adjusted ORs of 1.15 (95% CI 1.02-1.29) and 1.30 (95% CI 1.13-1.50), respectively. However, after adjustment to the number of laboratory tests prior to the MGUS diagnosis, there was no association between obesity and diabetes and MGUS risk (ORs of 1.08, 95% CI 0.96-1.22 and 1.08, 95% CI 0.93-1.25, respectively). In an additional analysis of antidiabetic therapies and MGUS risk, there was a nonsignificant decrease in MGUS risk among diabetes patients treated with metformin alone compared to subjects without diabetes (OR 0.77, 95% CI 0.56-1.05). In summary, while previously described risk factors for MGUS might be the result of detection bias, metformin should be further evaluated as a possible chemoprevention modality. Am. J. Hematol. 91:581-584, 2016. © 2016 Wiley Periodicals, Inc. PMID:26953904

  19. Identification of Aeromonas hydrophila infection with specific monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Paisarn Sithigorngul

    2007-08-01

    Full Text Available Whole cell of Aeromonas hydrophila 1234 was used for immunization to produce monoclonal antibodies (MAbs. Three different groups of MAbs specific to Aeromonas were obtained. The first group of MAbs demonstrated high specificity and bound to the A. hydrophila 1234 only but did not bind to the other two A. hydrophila isolates. This group of MAbs bound to a series of lipo-polysaccharides (LPS with molecular masses range from 10 to190 kDa. The second group of antibodies recognized A. hydrophila 1234 and 2798 isolates, and bound to a series of LPS with molecular masses range from 5-200 kDa. The third group of MAbs recognized all three isolates of A. hydrophila and two isolates of A. sobria, and lightly bound to A. caviae. This group of MAbs also bound to an unknown protein with molecular mass of 20 kDa. The MAbs in group 1 and group 2 can be used to detect the bacteria in tissues by immunohistochemistry. Both groups of MAbs bound to LPS at different sites in which the MAbs in group 2 bound to the side chain of O antigen while the MAbs in group 1 bound to the polymerization site at the core of oligosaccharide. All of the MAbs can be used to identify Aeromonas by dot blotting with the sensitivity range from 103-104 CFU/l. This study demonstrated a convenient immunological tool that can be used for simple and accurate identification of A. hydrophila, as well as for diagnosis of the A. hydrophila infection in animals. This immunological tool can replace costly and laborious biochemical tests.

  20. Immunodot blot assay to detect Helicobacter pylori using monoclonal antibodies against the 26 kDa protein.

    Science.gov (United States)

    Amini Najafabadi, Hossein; Paknejad, Maliheh; Farshad, Shohreh; Mohammadian, Taher; Seyyed Ebrahimi, Shadi Sadat; Amini Najafabadi, Azadeh

    2012-12-01

    Development of a specific immunoassay to detect Helicobacter pylori infection in stool samples requires monoclonal antibody against the specific antigen. The aims of this study were to establish monoclonal antibodies against the 26 kDa protein of H. pylori and develop an immunodot blot for their application to recognize H. pylori infection using stool samples. Mice were immunized intraperitoneally with homogenized gel containing the 26 kDa band of cell surface proteins of H. pylori in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The monoclonal antibodies were produced using the hybridoma technique. Reactivity of monoclonal antibodies was tested with the purified 26 kDa antigen and cell surface proteins from cultured H. pylori by ELISA. Furthermore reactivity of monoclonal antibodies was tested on negative and positive stool samples for H. pylori and suspensions of several major bacteria in stool by immunodot blot assay. Five stable hybridoma monoclones were obtained. The concordant reactivity of the monoclonal antibodies with H. pylori present in the stool samples, which had been tested previously using an ACON ELISA kit for H. pylori stool antigen testing, and unreactivity with several different major fecal bacteria in immunodot blotting indicates high specificity of the immunodot blot based on the reaction of produced monoclonal antibodies with the H. pylori antigen in stools. The findings indicate that the novel immunodot blot developed based on new monoclonal antibodies for stool antigens would be useful as a noninvasive method of diagnosing H. pylori infection. PMID:23244318

  1. Resveratrol-loaded nanocapsules inhibit murine melanoma tumor growth.

    Science.gov (United States)

    Carletto, Bruna; Berton, Juliana; Ferreira, Tamara Nascimento; Dalmolin, Luciana Facco; Paludo, Katia Sabrina; Mainardes, Rubiana Mara; Farago, Paulo Vitor; Favero, Giovani Marino

    2016-08-01

    In this study, resveratrol-loaded nanocapsules were developed and its antitumor activity tested on a melanoma mice model. These nanocapsules were spherically-shaped and presented suitable size, negative charge and high encapsulation efficiency for their use as a modified-release system of resveratrol. Nanoencapsulation leads to the drug amorphization. Resveratrol-loaded nanoparticles reduced cell viability of murine melanoma cells. There was a decrease in tumor volume, an increase in the necrotic area and inflammatory infiltrate of melanoma when resveratrol-loaded nanocapsules were compared to free resveratrol in treated mice. Nanoencapsulation of resveratrol also prevented metastasis and pulmonary hemorrhage. This modified-release technology containing resveratrol can be used as a feasible approach in order to inhibit murine melanoma tumor growth. PMID:27070053

  2. 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

    2007-01-01

    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

  3. Quantitative immunocytochemical characterization of four murine macrophage-like cell lines.

    Science.gov (United States)

    Nibbering, P H; van Furth, R

    1988-03-01

    The aim of the present study was to obtain quantitative data on the expression of seven cell-surface antigens by the murine macrophage-like cell lines WEHI-3, P388-D1, J774.1, and PU5-1.8, and to compare these data with those obtained for various mononuclear phagocytes. Binding of monoclonal antibodies F4/80, M1/70, 2.4.G.2., 30.G.12, M3/38, M3/84, and 59AD2.2 to cells from these four cell lines was detected by the biotin-avidin immunoperoxidase staining method and quantified by cytophotometry. The results are expressed as the percentage cells expressing a given antigen and the mean specific integrated absorbance per 0.25 micron2 cell-surface area. The results revealed that the phenotypes of the four macrophage-like cell lines differ markedly. Expression of the cell antigens by the cells of the various cell lines did not follow a normal distribution. Although the cell lines divide continuously and have certain characteristics in common with mature mononuclear phagocytes, none of the macrophage-like cell lines accurately resembles any of the mononuclear phagocyte populations. The strongest correlations for membrane were found between on the one hand WEHI-3 and P388-D1 cells and monoblasts and promonocytes (P greater than 0.011) on the other. P388-D1, J774.1 and PU5-1.8 cells expressed four of the six antigens to the same extent as skin macrophages (P greater than 0.012). With respect to expression of antigens recognized by antibodies 2.4.G.2., 30.G.12, M3/38, and M3/84 PU5-1.8 cells resembled activated peritoneal macrophages (P greater than 0.031). It is concluded that WEHI-3 is the most immature cell line, followed by the P388-D1 cell line, while J774.1, and PU5-1.8 are the most mature cell lines. PMID:3292404

  4. 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.

  5. Cytomegalovirus infection of murine testicular interstitial Leydig cells.

    OpenAIRE

    Baskar, J F; Stanat, S C; Huang, E S

    1983-01-01

    We studied the susceptibility of mouse testicular interstitial Leydig cells to cytomegalovirus both in vivo and in vitro. The in vivo studies included intratesticular and intraperitoneal infection of 6-week-old mice with murine cytomegalovirus (MCMV); the in vitro studies involved an MCMV-Leydig cell interaction using a Leydig tumor cell line (I-10). MCMV-specific antigens were detected in interstitial Leydig cells in sections of MCMV-inoculated testes by an indirect immunofluorescence test. ...

  6. Molecular and physiological alterations in murine ventricular dysfunction.

    OpenAIRE

    Rockman, H A; Ono, S.; Ross, R.S.; Jones, L R; Karimi, M.; Bhargava, V.; Ross, J; Chien, K R

    1994-01-01

    The present study reports the development and characterization of a murine model of right ventricular dysfunction following graded constriction in the pulmonary artery via microsurgical approaches. To analyze in vivo ventricular function, a technique of x-ray contrast microangiography was developed to allow the quantitative analysis of ventricular volumes and of ejection fraction in normal and pressure-overloaded right ventricle. Severe, chronic pulmonary arterial banding for 14 days resulted...

  7. Lactobacillus rhamnosus GG as an Effective Probiotic for Murine Giardiasis

    OpenAIRE

    Nisha Goyal; Ram Prakash Tiwari; Geeta Shukla

    2011-01-01

    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. r...

  8. Pharmacodynamics of Fluconazole in a Murine Model of Systemic Candidiasis

    OpenAIRE

    Louie, Arnold; Drusano, George L.; Banerjee, Partha; Liu, Qing-Feng; Liu, Weiguo; Kaw, Pamela; Shayegani, Mehdi; Taber, Harry; Miller, Michael H.

    1998-01-01

    In this study we defined the pharmacodynamic parameter that optimizes outcome in deep-seated Candida albicans infections treated with fluconazole. Using a murine model of systemic candidiasis, we conducted single-dose dose-ranging studies with fluconazole to determine the dosage of this drug that resulted in a 50% reduction in fungal densities (50% effective dose [ED50]) in kidneys versus the fungal densities in the kidneys of untreated controls. We found that the ED50 of fluconazole given in...

  9. Characterization of murine macrophages from bone marrow, spleen and peritoneum

    OpenAIRE

    Wang Changqi; Yu Xiao; Cao Qi; Wang Ya; Zheng Guoping; Tan Thian Kui; Zhao Hong; Zhao Ye; Wang Yiping; Harris David CH

    2013-01-01

    Abstract Background Macrophages have heterogeneous phenotypes and complex functions within both innate and adaptive immune responses. To date, most experimental studies have been performed on macrophages derived from bone marrow, spleen and peritoneum. However, differences among macrophages from these particular sources remain unclear. In this study, the features of murine macrophages from bone marrow, spleen and peritoneum were compared. Results We found that peritoneal macrophages (PMs) app...

  10. Murine immunization by cesium-137 irradiation attenuated Schistosoma mansoni cercariae

    International Nuclear Information System (INIS)

    Cesium-137, becoming a more readily available ionizing gamma radiation source for laboratory use, was shown to effectively attenuate Schistosoma mansoni cercariae for vaccine production. In parallel comparison studies with the murine model, cesium-137 attenuated cercariae consistently afforded better protection than did the cobalt-60 prepared vaccine. Dose-response data indicated that the optimal total irradiation with cesium-137 was between 45 and 50 Krad

  11. Murine immunization by cesium-137 irradiation attenuated Schistosoma mansoni cercariae

    Energy Technology Data Exchange (ETDEWEB)

    Stek, M. Jr.; Minard, P.; Cruess, D.F.

    1984-06-01

    Cesium-137, becoming a more readily available ionizing gamma radiation source for laboratory use, was shown to effectively attenuate Schistosoma mansoni cercariae for vaccine production. In parallel comparison studies with the murine model, cesium-137 attenuated cercariae consistently afforded better protection than did the cobalt-60 prepared vaccine. Dose-response data indicated that the optimal total irradiation with cesium-137 was between 45 and 50 Krad.

  12. Integration of murine leukemia virus DNA depends on mitosis.

    OpenAIRE

    Roe, T.; Reynolds, T. C.; Yu, G.; Brown, P O

    1993-01-01

    In synchronized rat or mouse cells infected with Moloney murine leukemia virus (MLV), integration of viral DNA and production of viral proteins occur only after the cells traverse mitosis. Integration is blocked when cells are prevented from progressing through mitosis. Viral nucleoprotein complexes isolated from arrested cells contain full-length viral DNA and can integrate this viral DNA in vitro, showing that the block to integration in arrested cells is not due to a lack of mature integra...

  13. Specific receptor binding of staphylococcal enterotoxins by murine splenic lymphocytes.

    OpenAIRE

    Buxser, S; Bonventre, P F; Archer, D L

    1981-01-01

    We describe a reliable assay to measure the specific binding of 125I-labeled staphylococcal enterotoxin A (SEA) by murine spleen cells. Toxin binding by lymphocytes was specific in that it was inhibited by unlabeled SEA but not by unrelated proteins. The biological activity of SEA (T-lymphocyte mitogenesis) correlated with toxin binding to splenic lymphocytes. In the presence of high concentrations of [125I]SEA, specific binding increased rapidly and approached saturation after 2 h. Toxin bin...

  14. Factors Influencing RBC Alloimmunization: Lessons Learned from Murine Models

    OpenAIRE

    Ryder, Alex B.; Zimring, James C.; Hendrickson, Jeanne E

    2014-01-01

    Red blood cell (RBC) alloimmunization may occur following transfusion or pregnancy/delivery. Although observational human studies have described the immunogenicity of RBC antigens and the clinical significance of RBC alloantibodies, studies of factors influencing RBC alloimmunization in humans are inherently limited by the large number of independent variables involved. This manuscript reviews data generated in murine models that utilize transgenic donor mice, which express RBC-specific model...

  15. A Murine Model of Contact Lens–Associated Fusarium Keratitis

    OpenAIRE

    Sun, Yan; Chandra, Jyotsna; Mukherjee, Pranab; Szczotka-Flynn, Loretta; Ghannoum, Mahmoud A.; Pearlman, Eric

    2010-01-01

    The 2006 outbreak of contact lens–associated Fusarium keratitis resulted in more than 300 cases in the United States in which a commercial lens care product was implicated. In the current study, Fusarium grown as biofilm on silicone hydrogel lenses induced keratitis in a murine model and severity of disease and survival of the organisms were dependent on MyD88, IL-1R1, and TLR4.

  16. Embryonic expression and cloning of the murine GATA-3 gene.

    OpenAIRE

    K. M. George; Leonard, M W; Roth, M. E.; Lieuw, Ken; Kioussis, D; Grosveld, Frank; Engel, Douglas

    1994-01-01

    textabstractWe describe the embryonic expression pattern as well as the cloning and initial transcriptional regulatory analysis of the murine (m) GATA-3 gene. In situ hybridization shows that mGATA-3 mRNA accumulation is temporally and spatially regulated during early development: although found most abundantly in the placenta prior to 10 days of embryogenesis, mGATA-3 expression becomes restricted to specific cells within the embryonic central nervous system (in the mesencephalon, diencephal...

  17. 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)

    2011-01-15

    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.)

  18. Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

    Science.gov (United States)

    Ferguson, Carly N.; Gucinski-Ruth, Ashley C.

    2016-05-01

    Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

  19. Neuropathies associated with IgG and IgA monoclonal gammopathy.

    Science.gov (United States)

    Nobile-Orazio, E; Casellato, C; Di Troia, A

    2002-10-01

    Neuropathy has been frequently reported in patients with monoclonal gammopathy, particularly those with monoclonal gammopathy of undetermined significance (MGUS). While the neuropathy associated with IgM-MGUS is well characterized and is often associated with a reactivity of the monoclonal protein with neural antigens, the relationship between the neuropathy and IgG and IgA MGUS is less clear. We review here the clinical, electrophysiological and pathogenetic features of neuropathies associated with IgG and IgA M-proteins in order to determine whether they represent distinct clinical entities and, most importantly, whether the finding of an IgG or IgA monoclonal gammopathy in a patient with neuropathy should led to different diagnostic or therapeutical approaches. This review will mainly focus on neuropathies associated with MGUS since the disclosure of a malignant monoclonal gammopathy, including multiple or osteosclerotic myeloma, lymphoma or primary amyloidosis, in a patient with neuropathy usually divert the therapeutical decisions to the hematologist for an appropriate therapy of the underlying life threatening condition. PMID:12407307

  20. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    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