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  1. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

    Science.gov (United States)

    Choy, Milly M; Zhang, Summer L; Costa, Vivian V; Tan, Hwee Cheng; Horrevorts, Sophie; Ooi, Eng Eong

    2015-11-01

    The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue. PMID:26565697

  2. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

    Directory of Open Access Journals (Sweden)

    Milly M Choy

    2015-11-01

    Full Text Available The mosquito-borne dengue virus (DENV is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.

  3. Sclerostin antibody inhibits skeletal deterioration due to reduced mechanical loading.

    Science.gov (United States)

    Spatz, Jordan M; Ellman, Rachel; Cloutier, Alison M; Louis, Leeann; van Vliet, Miranda; Suva, Larry J; Dwyer, Denise; Stolina, Marina; Ke, Hua Zhu; Bouxsein, Mary L

    2013-04-01

    Sclerostin, a product of the SOST gene produced mainly by osteocytes, is a potent negative regulator of bone formation that appears to be responsive to mechanical loading, with SOST expression increasing following mechanical unloading. We tested the ability of a murine sclerostin antibody (SclAbII) to prevent bone loss in adult mice subjected to hindlimb unloading (HLU) via tail suspension for 21 days. Mice (n = 11-17/group) were assigned to control (CON, normal weight bearing) or HLU and injected with either SclAbII (subcutaneously, 25 mg/kg) or vehicle (VEH) twice weekly. SclAbII completely inhibited the bone deterioration due to disuse, and induced bone formation such that bone properties in HLU-SclAbII were at or above values of CON-VEH mice. For example, hindlimb bone mineral density (BMD) decreased -9.2% ± 1.0% in HLU-VEH, whereas it increased 4.2% ± 0.7%, 13.1% ± 1.0%, and 30.6% ± 3.0% in CON-VEH, HLU-SclAbII, and CON-SclAbII, respectively (p bone volume, assessed by micro-computed tomography (µCT) imaging of the distal femur, was lower in HLU-VEH versus CON-VEH (p bone outcomes appeared to be enhanced by normal mechanical loading. Altogether, these results confirm the ability of SclAbII to abrogate disuse-induced bone loss and demonstrate that sclerostin antibody treatment increases bone mass by increasing bone formation in both normally loaded and underloaded environments.

  4. Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection.

    Science.gov (United States)

    Chan, Kuan Rong; Zhang, Summer Li-Xin; Tan, Hwee Cheng; Chan, Ying Kai; Chow, Angelia; Lim, Angeline Pei Chiew; Vasudevan, Subhash G; Hanson, Brendon J; Ooi, Eng Eong

    2011-07-26

    The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes. PMID:21746897

  5. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Directory of Open Access Journals (Sweden)

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  6. Antibody to a molecular marker of cell position inhibits synapse formation in retina.

    OpenAIRE

    Trisler, D.; Bekenstein, J; Daniels, M P

    1986-01-01

    A topographic gradient of TOP molecules in retina can be used to identify neuron position. Antibody to TOP from hybridoma cells that were injected into in vivo embryo eyes diffused into the retina and bound in a topographic gradient of [antibody.TOP] ([Ab.TOP]) complexes. Synapse formation in retina was inhibited in the presence of anti-TOP antibody. This suggests that TOP is involved in synapse formation and that recognition of position by neurons is necessary for normal synapse formation.

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

    OpenAIRE

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

    2002-01-01

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

  8. Fully human antagonistic antibodies against CCR4 potently inhibit cell signaling and chemotaxis.

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    Urs B Hagemann

    Full Text Available CC chemokine receptor 4 (CCR4 represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs and on tumor cells in several cancer types and its role in metastasis.Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies.For the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing. The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer.

  9. Insulin action is blocked by a monoclonal antibody that inhibits insulin receptor kinase

    International Nuclear Information System (INIS)

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor β subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor β subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin

  10. Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice

    Directory of Open Access Journals (Sweden)

    D.M. Grosso

    2008-12-01

    Full Text Available In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(- on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/- mice (6 per group were immunized with the following antibodies (100 µg each: mouse anti-LDL(- monoclonal IgG2b, rabbit anti-LDL(- polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25% cholesterol and then every week for 21 days. The passive immunization with anti-LDL(- monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD, respectively compared to control (124.9 ± 13.2 µm². Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(- monoclonal antibody (3.5 ± 0.70 per field x 10 compared to controls (21.5 ± 3.5 per field x 10. Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(- in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU, the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol, and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein. In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

  11. Antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity

    International Nuclear Information System (INIS)

    Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the β subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the β subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the β subunit suggest that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor

  12. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

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    Xiaocong Yu

    2016-01-01

    Full Text Available Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89 on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells.

  13. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition.

    Science.gov (United States)

    Yu, Xiaocong; Duval, Mark; Gawron, Melissa; Posner, Marshall R; Cavacini, Lisa A

    2016-01-01

    Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells. PMID:27419146

  14. Streptococcus pneumoniae-Induced Inhibition of Rat Ependymal Cilia Is Attenuated by Antipneumolysin Antibody

    OpenAIRE

    Hirst, Robert A; Mohammed, Bashir J.; Mitchell, Timothy J.; Andrew, Peter W.; O'Callaghan, Christopher

    2004-01-01

    Ciliated ependymal cells line the ventricular surfaces and aqueducts of the brain. In ex vivo experiments, pneumolysin caused rapid inhibition of the ependymal ciliary beat frequency and caused ependymal cell disruption. Wild-type pneumococci and pneumococci deficient in pneumolysin caused ciliary slowing, but penicillin lysis of wild-type, not pneumolysin-deficient, pneumococci increased the extent of ciliary inhibition. This effect was abolished by antipneumolysin antibody. Ependymal ciliar...

  15. Structural basis for EGF receptor inhibition by the therapeutic antibody IMC-11F8.

    Science.gov (United States)

    Li, Shiqing; Kussie, Paul; Ferguson, Kathryn M

    2008-02-01

    Therapeutic anticancer strategies that target and inactivate the epidermal growth factor receptor (EGFR) are under intense study in the clinic. Here we describe the mechanism of EGFR inhibition by an antibody drug IMC-11F8. IMC-11F8 is a fully human antibody that has similar antitumor potency as the chimeric cetuximab/Erbitux and might represent a safer therapeutic alternative. We report the X-ray crystal structure of the Fab fragment of IMC-11F8 (Fab11F8) in complex with the entire extracellular region and with isolated domain III of EGFR. We compare this to our previous study of the cetuximab/EGFR interaction. Fab11F8 interacts with a remarkably similar epitope, but through a completely different set of interactions. Both the similarities and differences in binding of these two antibodies have important implications for the development of inhibitors that could exploit this same mechanism of EGFR inhibition.

  16. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies

    DEFF Research Database (Denmark)

    Hansen, J E; Clausen, H; Nielsen, C;

    1990-01-01

    Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N...

  17. Inhibition of middle east respiratory syndrome coronavirus infection by anti-CD26 monoclonal antibody

    NARCIS (Netherlands)

    K. Ohnuma (Kei); B.L. Haagmans (Bart); R. Hatano (Ryo); V.S. Raj (Stalin); H. Mou (Huihui); S. Iwata (Satoshi); R.L. Dang (Rong); B.J. Bosch (Berend Jan); C. Morimoto (Chikao)

    2013-01-01

    textabstractWe identified the domains of CD26 involved in the binding of Middle East respiratory syndrome coronavirus (MERS-CoV) using distinct clones of anti-CD26 monoclonal antibodies (MAbs). One clone, named 2F9, almost completely inhibited viral entry. The humanized anti-CD26 MAb YS110 also sign

  18. Antibody-dependent enhancement of dengue virus infection is inhibited by SA-17, a doxorubicin derivative.

    Science.gov (United States)

    Ayala-Nuñez, Nilda V; Jarupathirun, Patsaporn; Kaptein, Suzanne J F; Neyts, Johan; Smit, Jolanda M

    2013-10-01

    Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus (DENV)-induced disease during a heterologous re-infection. Despite ADE's clinical impact, only a few antiviral compounds have been assessed for their anti-ADE activity. We reported earlier that SA-17, a doxorubicin derivative, efficiently inhibits the in vitro infection of DENV and yellow fever virus. Here we explored SA-17's mechanism of inhibition and investigated if the compound is active against ADE of DENV infection. Since enhanced infectivity stimulated by antibodies has been observed with standard and immature DENV, both types of virions were included in the study. We observed that SA-17 (i) inhibits DENV infection by preventing binding/entry to the cell and (ii) interferes with antibody-mediated infection of both standard and immature DENV2. SA-17 markedly reduced the infectivity of DENV2 in ADE conditions, with IC50s ranging from 0.26 to 2.89μM. The compound exerted its activity when added before, during, and after antibody-opsonization of standard and immature virus. Thus, molecules with the characteristics of SA-17 may be attractive antiviral agents since they can be used both to block DENV2 entry during primary and secondary infection and to inhibit ADE of standard and immature virus. PMID:23994499

  19. Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

    DEFF Research Database (Denmark)

    List, K; Høyer-Hansen, G; Rønne, E;

    1999-01-01

    Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or...

  20. Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies

    Science.gov (United States)

    Zarling, Joyce M.; Moran, Patricia A.; Haffar, Omar; Sias, Joan; Richman, Douglas D.; Spina, Celsa A.; Myers, Dorothea E.; Kuebelbeck, Virginia; Ledbetter, Jeffrey A.; Uckun, Fatih M.

    1990-09-01

    FUNCTIONAL impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells1,2. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA3,4. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000 (ref. 5), which inhibits replication of certain plant RNA viruses6-8, and of herpes simplex virus, poliovirus and influenza virus9-11. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CDS, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.

  1. Inhibition of HCV 3a genotype entry through Host CD81 and HCV E2 antibodies

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    Ashfaq Usman A

    2011-11-01

    Full Text Available Abstract Background HCV causes acute and chronic hepatitis which can eventually lead to permanent liver damage hepatocellular carcinoma and death. HCV glycoproteins play an important role in HCV entry by binding with CD81 receptors. Hence inhibition of virus at entry step is an important target to identify antiviral drugs against HCV. Methods and result The present study elaborated the role of CD81 and HCV glycoprotein E2 in HCV entry using retroviral pseudo-particles of 3a local genotype. Our results demonstrated that HCV specific antibody E2 and host antibody CD81 showed dose- dependent inhibition of HCV entry. HCV E2 antibody showed 50% reduction at a concentration of 1.5 ± 1 μg while CD81 exhibited 50% reduction at a concentration of 0.8 ± 1 μg. In addition, data obtained with HCVpp were also confirmed with the infection of whole virus of HCV genotype 3a in liver cells. Conclusion Our data suggest that HCV specific E2 and host CD81 antibodies reduce HCVpp entry and full length viral particle and combination of host and HCV specific antibodies showed synergistic effect in reducing the viral titer.

  2. Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

    Science.gov (United States)

    Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

    2013-08-01

    In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  4. Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus.

    Science.gov (United States)

    Jacob, Christian L; Lamorte, Louie; Sepulveda, Eliud; Lorenz, Ivo C; Gauthier, Annick; Franti, Michael

    2013-09-01

    Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection. PMID:23849792

  5. Use of bacteriophage particles displaying influenza virus hemagglutinin for the detection of hemagglutination-inhibition antibodies.

    Science.gov (United States)

    Domm, William; Brewer, Matthew; Baker, Steven F; Feng, Changyong; Martínez-Sobrido, Luis; Treanor, John; Dewhurst, Stephen

    2014-03-01

    Bacteriophage lambda capsids provide a flexible molecular scaffold that can be engineered to display a wide range of exogenous proteins, including full-length viral glycoproteins produced in eukaryotic cells. One application for such particles lies in the detection of virus-specific antibodies, since they may obviate the need to work with infectious stocks of highly pathogenic or emerging viruses that can pose significant biosafety and biocontainment challenges. Bacteriophage lambda capsids were produced that displayed an insect-cell derived, recombinant H5 influenza virus hemagglutinin (HA) on their surface. The particles agglutinated red blood cells efficiently, in a manner that could be blocked using H5 HA-specific monoclonal antibodies. The particles were then used to develop a modified hemagglutinination-inhibition (HAI) assay, which successfully identified human sera with H5 HA-specific HAI activity. These results demonstrate the utility of HA-displaying bacteriophage capsids for the detection of influenza virus-specific HAI antibodies.

  6. Monoclonal Antibodies Recognizing HIV-1 gp41 Could Inhibit Env-Mediated Syncytium Formation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Geng; CHEN Yinghua

    2005-01-01

    Some monoclonal antibodies (mAbs) could inhibit infection by HIV-1. In this study, four mAbs against HIV-1 gp41 were prepared in mice. All four mAbs could bind to the recombinant soluble gp41 and recognize the native envelope glycoprotein gp160 expressed on the HIV-Env+ CHO-WT cell in flow cytometry analysis. Interestingly, the results show that all four mAbs purified by affinity chromatography could inhibit HIV-1 Env-mediated membrane fusion (syncytium formation) by 40%-60% at 10 μg/mL, which implies potential inhibitory activities against HIV-1.

  7. [Detection of influenza B virus antibodies in different age groups using hemagglutination inhibition tests].

    Science.gov (United States)

    Sonuvar, S; Kocabeyoğlu, O; Emekdaş

    1991-01-01

    Antibody levels against influenza B virus were investigated by using hemagglutination-inhibition (HA-I) tests in 402 sera obtained from different age groups. Hemagglutination antigens were obtained by production of influenza B virus (B/Singapur/LLC 6201) in trypsinized Madin Darby Bovine Kidney (MDBK) cell cultured and they were used in tests. In 355 out of 402 sera (88.3%) antibodies against influenza B virus were detected at titers varying between 1/20 and 1/1280. However in 47 sera (11.7%) no antibodies were detected at 1/20 titer. High titers of antibody (1/640-1/1280) were not detected in none of the sera obtained from an age group between 1 and 14. However high titer antibodies were detected in 15.6% of the sera from an age group between 26 and 35, in the 17.3% of the sera from a group above 50 years of age. Our findings suggest that the increase in the rates of seropositivity against influenza B virus depends on getting older and, that the infections by this virus may be widely seen in our country.

  8. Chronic inhibition of cyclooxygenase-2 attenuates antibody responses against vaccinia infection.

    Science.gov (United States)

    Bernard, Matthew P; Bancos, Simona; Chapman, Timothy J; Ryan, Elizabeth P; Treanor, John J; Rose, Robert C; Topham, David J; Phipps, Richard P

    2010-02-01

    Generation of optimal humoral immunity to vaccination is essential to protect against devastating infectious agents such as the variola virus that causes smallpox. Vaccinia virus (VV), employed as a vaccine against smallpox, provides an important model of infection. Herein, we evaluated the importance cyclooxygenase-2 (Cox-2) in immunity to VV using Cox-2 deficient mice and Cox-2 selective inhibitory drugs. The effects of Cox-2 inhibition on antibody responses to live viruses such as vaccinia have not been previously described. Here, we used VV infection in Cox-2 deficient mice and in mice chronically treated with Cox-2 selective inhibitors and show that the frequency of VV-specific B cells was reduced, as well as the production of neutralizing IgG. VV titers were approximately 70 times higher in mice treated with a Cox-2 selective inhibitor. Interestingly, Cox-2 inhibition also reduced the frequency of IFN-gamma producing CD4(+) T helper cells, important for class switching. The significance of these results is that the chronic use of non-steroidal anti-inflammatory drugs (NSAIDs), and other drugs that inhibit Cox-2 activity or expression, blunt the ability of B cells to produce anti-viral antibodies, thereby making vaccines less effective and possibly increasing susceptibility to viral infection. These new findings support an essential role for Cox-2 in regulating humoral immunity.

  9. A human monoclonal antibody which inhibits the coaggregation activity of Porphyromonas gingivalis.

    OpenAIRE

    Abiko, Y; Ogura, N; Matsuda, U; Yanagi, K.; Takiguchi, H

    1997-01-01

    A B-cell line producing a human monoclonal antibody (HuMAb) against a recombinant 40-kDa outer membrane protein (OMP) of Porphyromonas gingivalis was constructed by in vivo immunization of a severe combined immunodeficiency C.B.-17/Icr mouse, which had been injected with human peripheral blood lymphocytes, with recombinant 40-kDa OMP and subsequent Epstein-Barr virus immortalization of B cells isolated from the spleen of the mouse. This HuMAb inhibited coaggregation between P. gingivalis vesi...

  10. Involvement of lymphocyte function-associated antigen-1 (LFA-1) in HIV infection: inhibition by monoclonal antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Mathiesen, Lars Reinhardt;

    1991-01-01

    Monoclonal antibodies (MAbs) against the alpha- and beta-chain of lymphocyte-associated antigen-1 (LFA-1) were examined for inhibition of HIV-1 infection in vitro. Infection of the T cell line MT4 and the monocytic cell line U937 by isolates HTLVIIIB and SSI-002, respectively was inhibited...

  11. Ramucirumab (IMC-1121B): Monoclonal antibody inhibition of vascular endothelial growth factor receptor-2.

    Science.gov (United States)

    Spratlin, Jennifer

    2011-04-01

    Angiogenesis, a well-recognized characteristic of malignancy, has been exploited more than any other pathway targeted by biologic anti-neoplastic therapies. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the critical receptor involved in malignant angiogenesis with its activation inducing a number of other cellular modifications resulting in tumor growth and metastases. Ramucirumab (IMC-1121B; ImClone Systems Corporation, Branchburg, NJ) is a fully human monoclonal antibody developed to specifically inhibit VEGFR-2. Ramucirumab is currently being investigated in multiple clinical trials across a variety of tumor types. Herein, angiogenesis inhibition in cancer is reviewed and up-to-date information on the clinical development of ramucirumab is presented.

  12. A monoclonal antibody against KCNK9 K+ channel extracellular domain inhibits tumour growth and metastasis

    Science.gov (United States)

    Sun, Han; Luo, Liqun; Lal, Bachchu; Ma, Xinrong; Chen, Lieping; Hann, Christine L.; Fulton, Amy M.; Leahy, Daniel J.; Laterra, John

    2016-01-01

    Two-pore domain potassium (K2P) channels act to maintain cell resting membrane potential—a prerequisite for many biological processes. KCNK9, a member of K2P family, is implicated in cancer, owing to its overexpression in human tumours and its ability to promote neoplastic cell survival and growth. However, KCNK9's underlying contributions to malignancy remain elusive due to the absence of specific modulators. Here we describe the development of monoclonal antibodies against the KCNK9 extracellular domain and their functional effects. We show that one antibody (Y4) with the highest affinity binding induces channel internalization. The addition of Y4 to KCNK9-expressing carcinoma cells reduces cell viability and increases cell death. Systemic administration of Y4 effectively inhibits growth of human lung cancer xenografts and murine breast cancer metastasis in mice. Evidence for Y4-mediated carcinoma cell autonomous and immune-dependent cytotoxicity is presented. Our study reveals that antibody-based KCNK9 targeting is a promising therapeutic strategy in KCNK9-expressing malignancies. PMID:26842342

  13. Inhibition of Insulin Degradation by Hepatoma Cells after Microinjection of Monoclonal Antibodies to a Specific Cytosolic Protease

    Science.gov (United States)

    Shii, Kozui; Roth, Richard A.

    1986-06-01

    Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell.

  14. INHIBITION OF Acinetobacter baumannii ADHESION BY ANTI-FIMBRIAL ANTIBODY: THE FIMBRIAL ANTIGEN EFFECTIVENESS

    Directory of Open Access Journals (Sweden)

    Hadeel K. Musafer

    2013-01-01

    Full Text Available Collecting samples of Acinetobacter baumannii taken from different clinical cases of wounds, septicemia, and urinary tract infections. That was accomplished by taking (296 samples from Baghdad educational hospital and Ibn-al-Baladi hospital. Samples were cultured on solid media (McConkey and blood agars, and according to microscopical, cultural, and biochemical identification, in addition to using API 20-E system, (21 isolates of A. baumannii were identified and in percentage of 47.619, 9.523, 14.285, and 28.571 for wound, blood, sputum, and urine samples, respectively. Methods: detection of fimbriated bacterial isolates among 21 isolates, and all those isolated were fimbriae forming isolates; isolate number (9 was selected as an effective isolate in formation of fimbriae. Non-forming fimbriae isolate of Shigella flexneri is used as negative control. Results and Conclusion: the average of adherence of fimbriated bacterial cell with human epithelial cells was reached (50 adherent bacterial cell per epithelial cell compared with the average of adherence of control isolate (12 adherent bacterial cell per epithelial cell, the inhibition processes are performed: Inhibition of bacterial adherence by specific antibodies of fimbriae antigen showed inhibition effect of adherence in respect to fimbriated isolate A. baumannii 9 also the subminimum inhibitory concentration for four antibiotics (Gentamicin, Tobramycin, Cefepime, and Amikacin inhibit the adherence of fimbriated isolate. The isolates (used in the study have the ability to agglutinate Saccharomyces cerevisiae and human red blood corpuscles (RBCs. The study of effect of different fimbriae extract concentrations (25, 50, 100 μg/ml on immune cells; consequently, reached to the following results: Concentrations of (25, 50, 100 μg/ml showed a negative effect on lymphocyte and PMNs viability which increased significantly (P≤0.05 with increasing of fimbriae extract concentration. On the other hand

  15. Triosephosphate isomerase of Taenia solium (TTPI): phage display and antibodies as tools for finding target regions to inhibit catalytic activity.

    Science.gov (United States)

    Sanabria-Ayala, Víctor; Belmont, Iaraset; Abraham, Landa

    2015-01-01

    Previous studies demonstrated that antibodies against triosephosphate isomerase of Taenia solium (TTPI) can alter its enzymatic catalysis. In the present study, we used antibodies produced against the NH2-terminal region of TTPI (1/3NH2TTPI) and the phage display technology to find target regions to inhibit TTPI activity. As a first step, we obtained polyclonal antibodies against non-conserved regions from the 1/3NH2TTPI, which had an inhibitory effect of about 74 % on catalytic activity. Afterward, they were used to screen a library of phage-displayed dodecapeptides; as a result, 41 phage mimotope clones were isolated and grouped according to their amino acid sequence, finding the consensus A1 (VPTXPI), A2 (VPTXXI), B (LTPGQ), and D (DPLPR). Antibodies against selected phage mimotope clones were obtained by rabbit's immunization; these ones clearly recognized TTPI by both Western blot and ELISA. However, only the mimotope PDTS16 (DSVTPTSVMAVA) clone, which belongs to the VPTXXI consensus, raised antibodies capable of inhibiting the TTPI catalytic activity in 45 %. Anti-PDTS16 antibodies were confronted to several synthetic peptides that encompass the 1/3NH2TTPI, and they only recognized three, which share the motif FDTLQK belonging to the helix-α1 in TTPI. This suggests that this motif is the main part of the epitope recognized by anti-PDTS16 antibodies and revealed its importance for TTPI catalysis.

  16. Inhibition of Neisseria gonorrhoeae attachment to HeLa cells with monoclonal antibody directed against a protein II.

    OpenAIRE

    Sugasawara, R J; Cannon, J G; Black, W J; Nachamkin, I; Sweet, R L; Brooks, G F

    1983-01-01

    This study showed that a protein II (PII) of Neisseria gonorrhoeae FA1090 appeared to act as a mediator of attachment to HeLa cells. Two colony variants of FA1090 were selected. Both gonococcal variants were nonpiliated, but one contained a PII and the other did not. A monoclonal antibody (1090-10.1), which was directed against the PII, inhibited the apparent PII-mediated attachment to HeLa cells. Antibodies produced from clone 1035-4, which had no PII specificity, did not inhibit the attachm...

  17. Monoclonal antibody targeting chikungunya virus envelope 1 protein inhibits virus release.

    Science.gov (United States)

    Masrinoul, Promsin; Puiprom, Orapim; Tanaka, Atsushi; Kuwahara, Miwa; Chaichana, Panjaporn; Ikuta, Kazuyoshi; Ramasoota, Pongrama; Okabayashi, Tamaki

    2014-09-01

    Chikungunya virus (CHIKV) causes an acute clinical illness characterized by sudden high fever, intense joint pain, and skin rash. Recent outbreaks of chikungunya disease in Africa and Asia are a major public health concern; however, there is currently no effective licensed vaccine or specific treatment. This study reported the development of a mouse monoclonal antibody (MAb), CK47, which recognizes domain III within the viral envelope 1 protein and inhibited the viral release process, thereby preventing the production of progeny virus. The MAb had no effect on virus entry and replication processes. Thus, CK47 may be a useful tool for studying the mechanisms underlying CHIKV release and may show potential as a therapeutic agent.

  18. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B

    Directory of Open Access Journals (Sweden)

    Yongfeng Fan

    2015-08-01

    Full Text Available Existing antibodies (Abs used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B contains a zinc endopeptidase light chain (LC domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS. The equilibrium dissociation constants (KD of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM. Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

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

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

  20. Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor

    Energy Technology Data Exchange (ETDEWEB)

    Green, S.J.; Underhill, C.B. (Georgetown Univ. Medical Center, Washington, DC (USA)); Tarone, G. (Univ. of Turin (Italy))

    1988-10-01

    To examine the role of the hyaluronate receptor in cell to cell adhesion, the authors have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, they investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind ({sup 3})hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a M{sub r} of 99,500, which is considerably larger than the 85,000 M{sub r} for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.

  1. Inhibiting CD146 by its Monoclonal Antibody AA98 Improves Radiosensitivity of Cervical Cancer Cells.

    Science.gov (United States)

    Cheng, Huawen

    2016-01-01

    BACKGROUND Cervical cancer is one of the major causes of cancer death of females worldwide. Radiotherapy is considered effective for cervical cancer treatment, but the low radiosensitivity found in some cases severely affects therapeutic outcomes. This study aimed to reveal the role of CD146, an important adhesion molecule facilitating tumor angiogenesis, in regulating radiosensitivity of cervical cancer cells. MATERIAL AND METHODS CD146 protein expression was compared in normal cells, cervical cancer cells with lower radiosensitivity, and cervical cancer cells with higher sensitivity from cervical squamous cell carcinoma patients. Anti-CD146 monoclonal antibody AA98 was used to inhibit CD146 in human cervical cancer SiHa cells with relatively low radiosensitivity, and then the cell survival and apoptosis changes after radiation were detected by colony formation assay and flow cytometry. RESULTS CD146 protein was significantly up-regulated in cervical cancer cells (Pcancer cells with lower radiosensitivity. The SiHa cells treated with AA98 showed more obvious inhibition in cell survival (Papoptosis (Pcancer cells, which might allow improvement in treatment outcome in cervical cancer. Further studies are necessary for understanding the detailed mechanism of CD146 in regulating radiosensitivity. PMID:27647179

  2. A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth in vivo

    Directory of Open Access Journals (Sweden)

    Powers David

    2007-11-01

    Full Text Available Abstract Background Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin α5β1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200, inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine α5β1, precluding its use in standard mouse xenograft models. Methods We generated a panel of rat-anti-mouse α5β1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for α5β1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models. Results A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin α5β1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM. In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40–60% (p Conclusion The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-α5β1

  3. Antibodies against analogous heptad repeat peptide HR212 of Newcastle Disease Virus inhibit virus-cell membrane fusion

    Institute of Scientific and Technical Information of China (English)

    LI Ying; TIEN Po

    2007-01-01

    Membrane fusion is a key step in enveloped virus entry. Highly conserved heptad repeat regions (HR1 and HR2) of Newcastle disease virus (NDV) fusion protein (F) are critical functional domains for viral membrane fusion. They display different conformations in the membrane fusion states and are viewed as candidate targets for neutralizing antibody responses. We previously reported that an analog of heptad repeat peptides HR2-HR1-HR2(HR212) and HR2 could inhibit NDV induced cell-cell membrane fusion. Here, we show that HR212 can induce the production of highly potent antibody in immunized rabbits, which could recognize full length peptides of both HR1 and HR2, and inhibit NDV hemagglutination and NDV entry. These suggest that either HR212 or its antibody could be an inhibitor of virus-induced cell-cell membrane fusion.

  4. INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22

    Science.gov (United States)

    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC. SP22, a rat sperm membrane protein that is highly-correlated w...

  5. Molecular characterization of monoclonal antibodies that inhibit acetylcholinesterase by targeting the peripheral site and backdoor region.

    Directory of Open Access Journals (Sweden)

    Yves Bourne

    Full Text Available The inhibition properties and target sites of monoclonal antibodies (mAbs Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE, have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.

  6. Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth

    DEFF Research Database (Denmark)

    Pratt-Riccio, Lilian Rose; Bianco, Cesare; Totino, Paulo Renato Rivas;

    2011-01-01

    The glutamate-rich protein (GLURP) is an exoantigen expressed in all stages of the Plasmodium falciparum life cycle in humans. Anti-GLURP antibodies can inhibit parasite growth in the presence of monocytes via antibody-dependent cellular inhibition (ADCI), and a major parasite-inhibitory region h...

  7. High concentrations of therapeutic IgG1 antibodies are needed to compensate for inhibition of antibody-dependent cellular cytotoxicity by excess endogenous immunoglobulin G.

    Science.gov (United States)

    Preithner, Susanne; Elm, Stefanie; Lippold, Sandra; Locher, Mathias; Wolf, Andreas; da Silva, Antonio J; Baeuerle, Patrick A; Prang, Nadja S

    2006-03-01

    A common feature of human IgG1 antibodies used for cancer treatment is that their anti-tumour efficacy requires high serum trough levels and continued therapy for several months. Treatment cycles, thereby, consume several grams of IgG1 translating into significant drug needs and costs. The basis for the low in vivo efficacy, which is in contrast to high in vitro antibody-dependent cellular cytotoxicity (ADCC), is not well understood. Here, we have explored factors contributing to this discrepancy using adecatumumab (MT201), a fully human monoclonal IgG1 against epithelial cell adhesion molecule (Ep-CAM) and trastuzumab (Herceptin), a humanized IgG1 with specificity for the human epithelial growth factor receptor type 2 (HER-2) antigen. We found that physiological levels of human sera strongly inhibited ADCC of both IgG1 antibodies. Effects showed some dependence on the density of Ep-CAM and HER-2 targets, the tumour cell line tested and on effector cell and serum donors. Removal of IgG by affinity chromatography abolished the inhibitory effect of a serum pool. Inhibition of ADCC was fully restored by adding back the IgG fraction or by an equal amount of IgG from a commercial source. We further demonstrate that CD56-positive lymphocytes within human PBMC contributed >90% to ADCC and that normal serum levels of IgG effectively competed for in vitro binding of an IgG1 antibody to low-affinity Fcgamma receptor type III (CD16), as is present on natural killer (NK) cells. Competition of serum IgG for binding of therapeutic IgG1 to NK cell may be one important reason why high antibody doses are required in the clinic for treatment of cancer by an ADCC-based mechanism. PMID:16102830

  8. Inhibition of B cell growth factor (BCGF) by monoclonal antibodies directed against the C3d receptor (CR2).

    Science.gov (United States)

    Perri, R T; Wilson, B S; Kay, N E

    1986-04-01

    Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). Human BCGF provides the final proliferative signal to normal, activated B cells. We herein show that anti-CR2 monoclonal antibodies inhibit human B cell responsiveness to purified BCGF. Addition of anti-CR2 antibody, AB5, was capable of completely inhibiting BCGF-mediated enhancement of either anti-mu or staphylococcal protein A-activated human B cells (191 +/- 21 cpm vs. 3942 +/- 622 cpm, mean +/- SEM). Inhibition of B cell response to BCGF by AB5 occurred in a dose-dependent manner. Monoclonal antibody anti-B2, which recognizes the same 140-kDa glycoprotein as AB5, in comparable concentrations also inhibited B cell responsiveness to BCGF. Monoclonal antibodies of the same subclass (IgG1) showed no inhibitory effect on BCGF enhancement of B cell proliferation. The F(ab')2 fragment of AB5 generated by pepsin digestion was similarly inhibitory as was the intact Ig. AB5-mediated inhibition was independent of the target B cell state of activation. Both resting and activated B cells (anti-mu or staphylococcal protein A activated) incubated with similar concentrations of AB5 were unresponsive to BCGF. The ability of anti-CR2 antibodies to block BCGF-dependent B cell proliferation suggests that occupancy of C3d membrane receptors may result in modulation of B cell proliferation in physiologic or clinical disease states. PMID:2938967

  9. Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation.

    Directory of Open Access Journals (Sweden)

    Eva Zilian

    Full Text Available Antibody-mediated rejection (AMR is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA class I (HLA I antibodies (Abs play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs. The antioxidant enzyme heme oxygenase (HO-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]. Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.

  10. Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.

    Science.gov (United States)

    Gong, Yan; Bourhis, Eric; Chiu, Cecilia; Stawicki, Scott; DeAlmeida, Venita I; Liu, Bob Y; Phamluong, Khanhky; Cao, Tim C; Carano, Richard A D; Ernst, James A; Solloway, Mark; Rubinfeld, Bonnee; Hannoush, Rami N; Wu, Yan; Polakis, Paul; Costa, Mike

    2010-01-01

    β-Catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for

  11. Trimeresurus venom inhibition of anti-HPA-1a and anti-HPA-1b antibody binding to human platelets.

    Science.gov (United States)

    Wlodar, S J; Stone, D L; Sinor, L T

    1995-01-01

    A solid-phase red cell adherence assay was used to demonstrate the specific inhibitory effect of seven species of Trimeresurus snake venom on the binding of HPA-1a- and HPA-1b-specific platelet antibodies. Trimeresurus venom did not inhibit the binding of HLA-, HPA-3a-, HPA-3b-, HPA-4a-, HPA-5a-, and HPA-5b-specific platelet antibodies. Venom from other genera of snakes, including representatives from Agkistrodon, Ancistrodon, Bitis, Bothrops, Bungarus, Causus, Crotalus, Dendroaspis, Ecis, Micrurus, Naja, Notechis, Ophiophagus, Pseudechis, Sepedon (Hemachatus), and Vipera, all failed to specifically inhibit anti-HPA-1a and HPA-1b binding. These results may indicate that the component in Trimeresurus snake venom previously reported to bind to the platelet GPIIb-IIIa complex, inhibiting fibrinogen binding, binds close to the HPA-1a and HPA-1b epitopes.

  12. Structural and functional basis for inhibition of erythrocyte invasion by antibodies that target Plasmodium falciparum EBA-175.

    Directory of Open Access Journals (Sweden)

    Edwin Chen

    Full Text Available Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175 engages the host receptor Glycophorin A (GpA during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics.

  13. An antisperm monoclonal antibody inhibits sperm fusion with zona-free hamster eggs but not homologous eggs.

    Science.gov (United States)

    Primakoff, P; Hyatt, H

    1986-09-01

    The zona-free hamster egg penetration assay (HEPA) was evaluated as a test for identifying fertilization-blocking antibodies. A monoclonal antibody, AH-20, that binds to the surface of guinea pig sperm was used to test antibody inhibition of sperm-egg fusion. AH-20 strongly inhibited guinea pig sperm fusion with zona-free hamster eggs but had no effect on guinea pig sperm fusion with zona-free guinea pig eggs. No inhibition by AH-20 was found in the homologous fusion assay over a wide range of sperm concentration, fertilization rate, and fertilization index. The results suggest that although guinea pig sperm can fuse with both hamster and guinea pig eggs, some aspect of the fusion mechanism is different in the two cases. The findings also indicate that HEPA, which is frequently used to assess the fertility potential of human sperm, can identify as blockers of sperm-egg fusion antibodies that have no effect on homologous sperm-egg fusion. PMID:3527769

  14. Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation.

    Directory of Open Access Journals (Sweden)

    Nakamura M

    2002-02-01

    Full Text Available We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE. Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B, anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1, a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin, a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside, and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA, Ricinus communis Agglutinin I (RCA-I, and Concanavalin A (ConA showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains.

  15. INHIBITION OF HUMAN EXPERIMENTAL GASTRIC CARCINOMA METASTASIS IN VIVO BY P-SELECTIN MONOCLONAL ANTIBODY

    Institute of Scientific and Technical Information of China (English)

    陈金联; 陈维雄; 朱金水; 陈尼维; 姚明; 周同

    2001-01-01

    Objeetive To study the role of cell adhesion molecule P-selectin monoclonal antibody ( MAb ) in tumor metastasis of an orthotopic metastatic model. Methods SCID mice were implanted orthotopically SGC-7901 human gastric cancer tissue. 3d later, animals received i. v. injections of PBS or P-selectin MAb ( 100μg /injection ) twice weekly for 3 weeks. 42d after operation, all animals were sacrificed. Tissues from all organs were obtained for histopathological evaluation. Results 10 of the animals ( n = 11 ) treated with PBS were found to develop metastatic tumors in the regional lymph nodes, liver, and lung. In contrast, 2 of the animals ( n = 9 ) treated with P-selectin MAb developed metastatic tumors in the organs examined. The expression of P-selectin mRNA in gastric cancer tissue of SCID mice with tumor metastasis was higher than that without such metastasis. Conclusion P-selectin expression is associated with tumor metastasis, and the metastasis may be inhibited by the MAb.

  16. Potentiation of thrombin generation in hemophilia A plasma by coagulation factor VIII and characterization of antibody-specific inhibition.

    Directory of Open Access Journals (Sweden)

    Bhavya S Doshi

    Full Text Available Development of inhibitory antibodies to coagulation factor VIII (fVIII is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20-30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa. However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as "type I" kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as "type II" inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined f

  17. Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies.

    Science.gov (United States)

    Porcino, Gabriane Nascimento; Carvalho-Campos, Cristiane; Maia, Ana Carolina Ribeiro Gomes; Detoni, Michelle Lima; Faria-Pinto, Priscila; Coimbra, Elaine Soares; Marques, Marcos José; Juliano, Maria Aparecida; Juliano, Luiz; Diniz, Vanessa Álvaro; Corte-Real, Suzana; Vasconcelos, Eveline Gomes

    2012-10-01

    Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control. PMID:22921497

  18. IL-6 blockade by monoclonal antibodies inhibits apolipoprotein (a) expression and lipoprotein (a) synthesis in humans[S

    OpenAIRE

    Müller, Nike; Schulte, Dominik M.; Türk, Kathrin; Freitag-Wolf, Sandra; Hampe, Jochen; Zeuner, Rainald; Johann O Schröder; Gouni-Berthold, Ioanna; Heiner K Berthold; Krone, Wilhelm; Rose-John, Stefan; Schreiber, Stefan; Laudes, Matthias

    2015-01-01

    Lipoprotein (a) [Lp(a)] is a highly atherogenic lipid particle. Although earlier reports suggested that Lp(a) levels are mostly determined by genetic factors, several recent studies have revealed that Lp(a) induction is also caused by chronic inflammation. Therefore, we aimed to examine whether cytokine blockade by monoclonal antibodies may inhibit Lp(a) metabolism. We found that interleukin 6 (IL-6) blockade by tocilizumab (TCZ) reduced Lp(a) while TNF-α-inhibition by adalimumab in humans ha...

  19. Adenovirus-expressed preS2 antibody inhibits hepatitis B virus infection and hepatic carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Qian Zhang; Zhi-Qing Li; Hu Liu; Jia-He Yang

    2012-01-01

    AIM: To investigate the inhibitory effect of hepatitis B virus (HBV) preS2 antibody (preS2Ab) against HBV infection and HBV-associated hepatic carcinogenesis. METHODS: An adenoviral vector carrying the fulllength light and heavy chains of the HBV preS2Ab gene, Ad315-preS2Ab, was constructed. Enzyme linked immunosorbent assay (ELISA) and Western blotting analyses were used to determine the preS2Ab expression levels in vitro . Immunofluorescent techniques were used to examine the binding affinity between the expressed HBV preS2Ab and HBV-positive liver cells. ELISAs were also used to determine hepatitis B surface antigen (HBsAg) levels to assess the inhibitory effect of the preS2Ab against HBV infection in L02 cells. The inhibitory effect of preS2Ab against hepatic carcinogenesiswas studied with diethylnitrosamine (DEN)-induced hepatocellular carcinomas (HCCs) in HBV transgenic mice. RESULTS: The expression of HBV preS2Ab increased with increases in the multiplicity of infection (MOI) of Ad315-preS2Ab in L02 cells, with 350.87 ± 17.37 μg/L of preS2Ab when the MOI was 100 plaque forming units (pfu)/cell. The expressed preS2Abs could recognize liver cells from HBV transgenic mice. ELISA results showed that L02 cells expressing preS2Ab produced less HBsAg after treatment with the serum of HBV patients than parental L02 cells expressing no preS2Ab. HBV transgenic mice treated with Ad315-preS2Ab had fewer and smaller cancerous nodes after induction with DEN than mice treated with a blank Ad315 vector or untreated mice. Additionally, the administration of Ad315-preS2Ab could alleviate hepatic cirrhosis and decrease the serum levels of alanine transaminase and aspartate transaminase. CONCLUSION: Adenovirus-mediated HBV preS2Ab expression could inhibit HBV infection in L02 cells, and then inhibit DEN-induced hepatocellular carcinogenesis and protect hepatic function in HBV transgenic mice.

  20. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.

    Directory of Open Access Journals (Sweden)

    Yongfeng Fan

    Full Text Available The paralytic disease botulism is caused by botulinum neurotoxins (BoNT, multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC of BoNT serotype A (BoNT/A was targeted for generation of monoclonal antibodies (mAbs that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS. Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10-11 M to 3.53×10-8 M (mean KD 5.38×10-9 M and median KD 1.53×10-9 M, as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10-9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.

  1. Post-streptococcal auto-antibodies inhibit protein disulfide isomerase and are associated with insulin resistance.

    Directory of Open Access Journals (Sweden)

    Adi Aran

    Full Text Available Post-streptococcal autoimmunity affects millions worldwide, targeting multiple organs including the heart, brain, and kidneys. To explore the post-streptococcal autoimmunity spectrum, we used western blot analyses, to screen 310 sera from healthy subjects with (33% and without (67% markers of recent streptococcal infections [anti-Streptolysin O (ASLO or anti-DNAse B (ADB]. A 58 KDa protein, reacting strongly with post-streptococcal sera, was identified as Protein Disulfide Isomerase (PDI, an abundant protein with pleiotropic metabolic, immunologic, and thrombotic effects. Anti-PDI autoantibodies, purified from human sera, targeted similar epitopes in Streptolysin O (SLO, P51-61 and PDI (P328-338. The correlation between post-streptococcal status and anti-human PDI auto-immunity was further confirmed in a total of 2987 samples (13.6% in 530 ASLO positive versus 5.6% in 2457 ASLO negative samples, p<0.0001. Finally, anti-PDI auto-antibodies inhibited PDI-mediated insulin degradation in vitro (n = 90, p<0.001, and correlated with higher serum insulin (14.1 iu/ml vs. 12.2 iu/ml, n = 1215, p = 0.039 and insulin resistance (Homeostatic Model Assessment (HOMA 4.1 vs. 3.1, n = 1215, p = 0.004, in a population-based cohort. These results identify PDI as a major target of post-streptococcal autoimmunity, and establish a new link between infection, autoimmunity, and metabolic disturbances.

  2. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    International Nuclear Information System (INIS)

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with 51Cr, [3H]leucine, or, preferentially, with [3H]uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30 degree C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well

  3. Anti-DNase I antibodies in systemic lupus erythematosus: diagnostic value and share in the enzyme inhibition.

    Science.gov (United States)

    Trofimenko, A S; Gontar, I P; Zborovsky, A B; Paramonova, O V

    2016-04-01

    Diagnostic accuracy of anti-DNase I antibodies measurement in a differentiation between SLE and other autoimmune rheumatic diseases was evaluated. The share of anti-DNase I and actin in the DNase I activity decrease in SLE was established. Serum samples were obtained from 54 patients with verified SLE, 52 control patients with other autoimmune rheumatic diseases, and 44 healthy persons. Anti-DNase I concentrations were measured by ELISA. Free and actin inhibited DNase I activities were evaluated in the fresh serum samples. The appraisal of antibodies and actin effects on DNase I activity was made using multiple regression. Anti-DNase I antibodies were positive in 35 SLE and 8 control patients, without significant difference between the mean antibody concentrations. Sensitivity of this test was 64.81 %, and specificity-84.62 %. Mean free DNase I activity in SLE was somewhat lower than in the control group as a result of augmented frequency of extremely low enzyme activities. On the contrary, after the exclusion of the latter cases we have revealed elevated mean free DNase I activity in the other SLE patients comparing to the similar control subgroup. Unlike the controls, low serum DNase I activity in SLE arose not only from actin and antibody action, but also, in half of the cases, from unidentified factor, related to active SLE. The accuracy of the anti-DNase I antibodies measurement is approximate to the present reference standard of SLE diagnostics. We first demonstrated that neither antibodies nor actin caused DNase I activity decrease in SLE.

  4. The strong in vivo anti-tumor effect of the UIC2 monoclonal antibody is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Gábor Szalóki

    Full Text Available P-glycoprotein (Pgp extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR. The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA. The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX in KB-V1 (Pgp+ cells in vitro almost to the level of KB-3-1 (Pgp- cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs, it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC.

  5. The strong in vivo anti-tumor effect of the UIC2 monoclonal antibody is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity.

    Science.gov (United States)

    Szalóki, Gábor; Krasznai, Zoárd T; Tóth, Ágnes; Vízkeleti, Laura; Szöllősi, Attila G; Trencsényi, György; Lajtos, Imre; Juhász, István; Krasznai, Zoltán; Márián, Teréz; Balázs, Margit; Szabó, Gábor; Goda, Katalin

    2014-01-01

    P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC). PMID:25238617

  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. Epitope specificity and isotype of monoclonal anti-D antibodies dictate their ability to inhibit phagocytosis of opsonized platelets.

    Science.gov (United States)

    Kjaersgaard, Mimi; Aslam, Rukhsana; Kim, Michael; Speck, Edwin R; Freedman, John; Stewart, Donald I H; Wiersma, Erik J; Semple, John W

    2007-08-15

    Rh immune globulin (WinRho SDF; Cangene, Mississauga, ON, Canada) is an effective treatment for autoimmune thrombocytopenic purpura; however, maintaining a sustained supply for its use in autoimmune thrombocytopenic purpura and its primary indication, hemolytic disease of the newborn, makes the development of alternative reagents desirable. We compared Rh immune globulin and 6 human monoclonal anti-D antibodies (MoAnti-D) with differing isotypes and specificities for their ability to opsonize erythrocytes and inhibit platelet phagocytosis in an in vitro assay. Results demonstrated that opsonization of erythrocytes with Rh immune globulin significantly (P < .001) reduced phagocytosis of fluorescently labeled opsonized platelets in an Fc-dependent manner. Of the MoAnti-D that shared specificity but differed in isotype, only IgG3 antibodies could significantly (P < .001) inhibit platelet phagocytosis. In contrast, 2 MoAnti-D shared isotypes and differed in specificity; however, only one could significantly (P < .001) inhibit platelet phagocytosis. The results suggest that MoAnti-D epitope specificity and isotypes are critical requirements for optimal inhibition of opsonized platelet phagocytosis. PMID:17456719

  8. Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action.

    Science.gov (United States)

    Geoghegan, James C; Diedrich, Gundo; Lu, Xiaojun; Rosenthal, Kim; Sachsenmeier, Kris F; Wu, Herren; Dall'Acqua, William F; Damschroder, Melissa M

    2016-01-01

    CD73 (ecto-5'-nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzyme's function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states.

  9. Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action.

    Science.gov (United States)

    Geoghegan, James C; Diedrich, Gundo; Lu, Xiaojun; Rosenthal, Kim; Sachsenmeier, Kris F; Wu, Herren; Dall'Acqua, William F; Damschroder, Melissa M

    2016-01-01

    CD73 (ecto-5'-nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzyme's function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states. PMID:26854859

  10. Inhibition of Hepatitis C Virus-Like Particle Binding to Target Cells by Antiviral Antibodies in Acute and Chronic Hepatitis C

    Science.gov (United States)

    Steinmann, Daniel; Barth, Heidi; Gissler, Bettina; Schürmann, Peter; Adah, Mohammed I.; Gerlach, J. Tilman; Pape, Gerd R.; Depla, Erik; Jacobs, Dirk; Maertens, Geert; Patel, Arvind H.; Inchauspé, Geneviève; Liang, T. Jake; Blum, Hubert E.; Baumert, Thomas F.

    2004-01-01

    Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies. PMID:15308699

  11. Involvement of lymphocyte function-associated antigen-1 (LFA-1) in HIV infection: inhibition by monoclonal antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Mathiesen, Lars Reinhardt;

    1991-01-01

    Monoclonal antibodies (MAbs) against the alpha- and beta-chain of lymphocyte-associated antigen-1 (LFA-1) were examined for inhibition of HIV-1 infection in vitro. Infection of the T cell line MT4 and the monocytic cell line U937 by isolates HTLVIIIB and SSI-002, respectively was inhibited...... in a concentration dependent manner by MAb against the beta-chain but not against the alpha-chain. No cross-reactivity was found between MAb against LFA-1 and against the CD4 receptor (MAb Leu3a). MAbs against the beta-chain and the CD4 receptor were found to act synergistically in inhibiting HIV infection....... These data indicate that the beta-chain of LFA-1 in addition to the CD4 receptor may be involved in HIV infection in vitro....

  12. Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type.

    Science.gov (United States)

    Sandbulte, Matthew R; Gauger, Phillip C; Kitikoon, Pravina; Chen, Hongjun; Perez, Daniel R; Roth, James A; Vincent, Amy L

    2016-07-19

    The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and respiratory specimens of pigs vaccinated with intramuscular whole-inactivated virus (WIV), intranasal live-attenuated influenza virus (LAIV), and intranasal wild type (WT) IAV. NI titers were also analyzed in sera from an investigation of piglet vaccination in the presence of passive maternally-derived antibodies. Test antigens contained genetically divergent swine-lineage NA genes homologous or heterologous to the vaccines with mismatched hemagglutinin genes (HA). Naïve piglets responded to WIV and LAIV vaccines and WT infection with strong homologous serum NI titers. Cross-reactivity to heterologous NAs depended on the degree of genetic divergence between the NA genes. Bronchoalveolar lavage specimens of LAIV and WT-immunized groups also had significant NI titers against the homologous antigen whereas the WIV group did not. Piglets of vaccinated sows received high levels of passive NI antibody, but their NI responses to homologous LAIV vaccination were impeded. These data demonstrate the utility of the enzyme-linked lectin assay for efficient NI antibody titration of serum as well as respiratory tract secretions. Swine IAV vaccines that induce robust NI responses are likely to provide broader protection against the diverse and rapidly evolving IAV strains that circulate in pig populations. Mucosal antibodies to NA may be one of the protective immune mechanisms induced by LAIV vaccines.

  13. Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type.

    Science.gov (United States)

    Sandbulte, Matthew R; Gauger, Phillip C; Kitikoon, Pravina; Chen, Hongjun; Perez, Daniel R; Roth, James A; Vincent, Amy L

    2016-07-19

    The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and respiratory specimens of pigs vaccinated with intramuscular whole-inactivated virus (WIV), intranasal live-attenuated influenza virus (LAIV), and intranasal wild type (WT) IAV. NI titers were also analyzed in sera from an investigation of piglet vaccination in the presence of passive maternally-derived antibodies. Test antigens contained genetically divergent swine-lineage NA genes homologous or heterologous to the vaccines with mismatched hemagglutinin genes (HA). Naïve piglets responded to WIV and LAIV vaccines and WT infection with strong homologous serum NI titers. Cross-reactivity to heterologous NAs depended on the degree of genetic divergence between the NA genes. Bronchoalveolar lavage specimens of LAIV and WT-immunized groups also had significant NI titers against the homologous antigen whereas the WIV group did not. Piglets of vaccinated sows received high levels of passive NI antibody, but their NI responses to homologous LAIV vaccination were impeded. These data demonstrate the utility of the enzyme-linked lectin assay for efficient NI antibody titration of serum as well as respiratory tract secretions. Swine IAV vaccines that induce robust NI responses are likely to provide broader protection against the diverse and rapidly evolving IAV strains that circulate in pig populations. Mucosal antibodies to NA may be one of the protective immune mechanisms induced by LAIV vaccines. PMID:27325350

  14. Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit insulin action.

    OpenAIRE

    Roth, R A; Cassell, D J; Wong, K. Y.; Maddux, B A; Goldfine, I D

    1982-01-01

    Antibodies to the insulin receptor were prepared in BALB/c mice by immunization with IM-9 human lymphocytes, a cell type that has a large number of plasma membrane insulin receptors. The spleens of these mice were then removed, and their lymphocytes were fused to a mouse myeloma cell line, FO cells. After screening over 1,200 resulting hybrids, one stable hybrid was obtained that produced IgG1 antibodies directed towards the insulin receptor. This antibody blocked 125I-labeled insulin binding...

  15. Cell-cell transmission enables HIV-1 to evade inhibition by potent CD4bs directed antibodies.

    Directory of Open Access Journals (Sweden)

    Irene A Abela

    Full Text Available HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo.

  16. Cell-cell transmission enables HIV-1 to evade inhibition by potent CD4bs directed antibodies.

    Science.gov (United States)

    Abela, Irene A; Berlinger, Livia; Schanz, Merle; Reynell, Lucy; Günthard, Huldrych F; Rusert, Peter; Trkola, Alexandra

    2012-01-01

    HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo. PMID:22496655

  17. Development and evaluation of a monoclonal antibody-based inhibition ELISA for the quantification of chymosin in solution.

    Science.gov (United States)

    Rolet-Répécaud, O; Arnould, C; Dupont, D; Gavoye, S; Beuvier, E; Achilleos, C

    2015-05-20

    Chymosin is the major enzyme of natural rennet, traditionally used in cheese making for its high milk-clotting activity. For technical reasons, an accurate characterization of rennet should include its total clotting activity and also its enzymatic composition. Monoclonal antibodies specific to chymosin were obtained from mice immunized with purified bovine chymosin, and an inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of chymosin in solution. No cross-reactivity was observed with other milk-clotting enzymes commonly used in cheese making. The limit of detection and limit of quantification were 125 and 400 ng/mL, respectively. The values of precision within and among runs were 7.23 and 7.39%, respectively, and satisfying recovery, from 92 to 119%, was found for spiked samples. The inhibition ELISA was successfully applied to commercial rennets, and the results were consistent with those obtained using the standard chromatographic method (IDF 110: A, 1987).

  18. Plasmodium falciparum 19-kilodalton merozoite surface protein 1 (MSP1)-specific antibodies that interfere with parasite growth in vitro can inhibit MSP1 processing, merozoite invasion, and intracellular parasite development.

    Science.gov (United States)

    Moss, David K; Remarque, Edmond J; Faber, Bart W; Cavanagh, David R; Arnot, David E; Thomas, Alan W; Holder, Anthony A

    2012-03-01

    Merozoite surface protein 1 (MSP1) is a target for malaria vaccine development. Antibodies to the 19-kDa carboxy-terminal region referred to as MSP1(19) inhibit erythrocyte invasion and parasite growth, with some MSP1-specific antibodies shown to inhibit the proteolytic processing of MSP1 that occurs at invasion. We investigated a series of antibodies purified from rabbits immunized with MSP1(19) and AMA1 recombinant proteins for their ability to inhibit parasite growth, initially looking at MSP1 processing. Although significant inhibition of processing was mediated by several of the antibody samples, there was no clear relationship with overall growth inhibition by the same antibodies. However, no antibody samples inhibited processing but not invasion, suggesting that inhibition of MSP1 processing contributes to but is not the only mechanism of antibody-mediated inhibition of invasion and growth. Examining other mechanisms by which MSP1-specific antibodies inhibit parasite growth, we show that MSP1(19)-specific antibodies are taken up into invaded erythrocytes, where they persist for significant periods and result in delayed intracellular parasite development. This delay may result from antibody interference with coalescence of MSP1(19)-containing vesicles with the food vacuole. Antibodies raised against a modified recombinant MSP1(19) sequence were more efficient at delaying intracellular growth than those to the wild-type protein. We propose that antibodies specific for MSP1(19) can mediate inhibition of parasite growth by at least three mechanisms: inhibition of MSP1 processing, direct inhibition of invasion, and inhibition of parasite development following invasion. The balance between mechanisms may be modulated by modifying the immunogen used to induce the antibodies.

  19. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress

    NARCIS (Netherlands)

    Fox, Julie M.; Long, Feng; Edeling, Melissa A.; Lin, Hueylie; van Duijl-Richter, Mareike K. S.; Fong, Rachel H.; Kahle, Kristen M.; Smit, Jolanda M.; Jin, Jing; Simmons, Graham; Doranz, Benjamin J.; Crowe, James E.; Fremont, Daved H.; Rossmann, Michael G.; Diamond, Michael S.

    2015-01-01

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphav

  20. Antibody-dependent enhancement of dengue virus infection is inhibited by SA-17, a doxorubicin derivative

    NARCIS (Netherlands)

    Ayala Nunez, Vanesa; Jarupathirun, Patsaporn; Kaptein, Suzanne; Neyts, Johan; Smit, Jolanda

    2013-01-01

    Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus (DENV)-induced disease during a heterologous re-infection. Despite ADE's clinical impact, only a few antiviral compounds have been assessed for their anti-ADE activity. We reported earlier tha

  1. Inhibition of Staphylococcus epidermidis biofilm formation by rabbit polyclonal antibodies against the SesC protein.

    NARCIS (Netherlands)

    Shahrooei, M.; Hira, V.; Stijlemans, B.; Merckx, R.; Hermans, P.W.M.; Eldere, J. van

    2009-01-01

    Several well-studied proteins with defined roles in Staphylococcus epidermidis biofilm formation are LPXTG motif-containing proteins. Here, we investigate the possible use of the LPXTG motif-containing protein SesC (S. epidermidis surface protein C; accession no. NP_765787) as a target for antibodie

  2. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.

    Directory of Open Access Journals (Sweden)

    Atul Asati

    Full Text Available Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA

  3. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.

    Science.gov (United States)

    Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

    2016-01-01

    Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of

  4. Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.

    Science.gov (United States)

    Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

    2016-01-01

    Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of

  5. Ibuprofen and other widely used non-steroidal anti-inflammatory drugs inhibit antibody production in human cells.

    Science.gov (United States)

    Bancos, Simona; Bernard, Matthew P; Topham, David J; Phipps, Richard P

    2009-01-01

    The widely used non-steroidal anti-inflammatory drugs (NSAIDs) function mainly through inhibition of cyclooxygenases 1 and 2 (Cox-1 and Cox-2). Unlike Cox-1, Cox-2 is considered an inducible and pro-inflammatory enzyme. We previously reported that Cox-2 is upregulated in activated human B lymphocytes and using Cox-2 selective inhibitors that Cox-2 is required for optimal antibody synthesis. It is not known whether commonly used non-prescription and non-Cox-2 selective drugs also influence antibody synthesis. Herein, we tested a variety of Cox-1/Cox-2 non-selective NSAIDs, namely ibuprofen, tylenol, aspirin and naproxen and report that they blunt IgM and IgG synthesis in stimulated human peripheral blood mononuclear cells (PBMC). Ibuprofen had its most profound effects in inhibiting human PBMCs and purified B lymphocyte IgM and IgG synthesis when administered in the first few days after activation. As shown by viability assays, ibuprofen did not kill B cells. The implications of this research are that the use of widely available NSAIDs after infection or vaccination may lower host defense. This may be especially true for the elderly who respond poorly to vaccines and heavily use NSAIDs.

  6. 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.

  7. Growth inhibition of Staphylococcus aureus and escherichia coli strains by neutralizing IgY antibodies from ostrich egg yolk

    Directory of Open Access Journals (Sweden)

    Fernando Luiz Tobias

    2012-06-01

    Full Text Available Ostrich raising around the world have some key factors and farming profit depend largely on information and ability of farmers to rear these animals. Non fertilized eggs from ostriches are discharged in the reproduction season. Staphylococcus aureus and Escherichia coli are microorganisms involved in animal and human diseases. In order to optimize the use of sub products of ostrich raising, non fertilized eggs of four selected birds were utilized for development of polyclonal IgY antibodies. The birds were immunized (200ug/animal with purified recombinant staphylococcal enterotoxin C (recSEC and synthetic recRAP, both derived from S. aureus, and recBFPA and recEspB involved in E. coli pathogenicity, diluted in FCA injected in the braquial muscle. Two subsequent immunization steps with 21 days intervals were repeated in 0,85% saline in FIA. Blood and eggs samples were collected before and after immunization steps. Egg yolk immunoglobulins were purified by precipitation with 19% sodium sulfate and 20% ammonium sulphate methodologies. Purified IgY 50µL aliquots were incubated in 850µL BHI broth containing 50µL inoculums of five strains of S. aureus and five strains of E.coli during four hours at 37ºC. Growth inhibition was evaluated followed by photometry reading (DO550nm. Egg yolk IgY preparation from hiperimmunized birds contained antibodies that inhibited significantly (p<0,05 growth of strains tested. Potential use of ostrich IgY polyclonal antibodies as a diagnostic and therapeutic tool is proposed for diseased animals.

  8. Anti-epidermal growth factor receptor monoclonal antibody cetuximab inhibits EGFR/HER-2 heterodimerization and activation.

    Science.gov (United States)

    Patel, Dipa; Bassi, Rajiv; Hooper, Andrea; Prewett, Marie; Hicklin, Daniel J; Kang, Xiaoqiang

    2009-01-01

    Human carcinomas frequently express one or more members of the epidermal growth factor receptor family. Two family members, epidermal growth factor receptor (EGFR) and c-erbB2/neu (HER2), homodimerize or heterodimerize upon activation with ligand and trigger potent mechanisms of cellular proliferation, differentiation and migration. In this study, we examined the effect of the anti-EGFR monoclonal antibody Erbitux (cetuximab) on human tumor cells expressing both EGFR and HER2. Investigation of the effect of cetuximab on the activation of EGFR-EGFR, EGFR-HER2 and HER2-HER2 homodimers and heterodimers was conducted using the NCI-N87 human gastric carcinoma cell line. Treatment of NCI-N87 cells with cetuximab completely inhibited formation of EGFR-EGFR homodimers and EGFR-HER2 heterodimers. Activation of HER2-HER2 homodimers was not appreciably stimulated by exogenous ligand and was not inhibited by cetuximab treatment. Furthermore, cetuximab inhibited EGF-induced EGFR and HER2 phosphorylation in CAL27, NCI-H226 and NCI-N87 cells. The activation of downstream signaling molecules such as AKT, MAPK and STAT-3 were also inhibited by cetuximab in these cells. To examine the effect of cetuximab on the growth of tumors in vivo, athymic mice bearing established NCI-N87 or CAL27 xenografts were treated with cetuximab (1 mg, i.p., q3d). The growth of NCI-N87 and CAL27 tumors was significantly inhibited with cetuximab therapy compared to the control groups (p<0.0001 in both cases). In the CAL27 xenograft model, tumor growth inhibition by cetuximab treatment was similar to that by cetuximab and trastuzumab combination treatment. Immunohistological analysis of cetuximab-treated tumors showed a decrease in EGFR-HER2 signaling and reduced tumor cell proliferation. These results suggest that cetuximab may be useful in the treatment of carcinomas co-expressing EGFR and HER2. PMID:19082474

  9. Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

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    Merkel George

    2006-06-01

    Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

  10. Differential inhibition of lipopolysaccharide-induced phenomena by anti-tumor necrosis factor alpha antibody.

    OpenAIRE

    Vogel, S N; Havell, E A

    1990-01-01

    Tumor necrosis factor alpha (TNF alpha) has been implicated as a major mediator of lipopolysaccharide (LPS)-induced phenomena. Administration to mice of a polyclonal, monospecific antibody prepared against recombinant murine TNF alpha abolished detection of LPS-induced TNF alpha activity and significantly reduced levels of LPS-induced colony-stimulating factor but failed to reduce the production of LPS-induced interferon, corticosterone, or LPS-induced hypoglycemia.

  11. Immunisation with recombinant PfEMP1 domains elicits functional rosette-inhibiting and phagocytosis-inducing antibodies to Plasmodium falciparum.

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    Ashfaq Ghumra

    Full Text Available BACKGROUND: Rosetting is a Plasmodium falciparum virulence factor implicated in the pathogenesis of life-threatening malaria. Rosetting occurs when parasite-derived P. falciparum Erythrocyte Membrane Protein One (PfEMP1 on the surface of infected erythrocytes binds to human receptors on uninfected erythrocytes. PfEMP1 is a possible target for a vaccine to induce antibodies to inhibit rosetting and prevent severe malaria. METHODOLOGY/FINDINGS: We examined the vaccine potential of the six extracellular domains of a rosette-mediating PfEMP1 variant (ITvar9/R29var1 from the R29 parasite strain by immunizing rabbits with recombinant proteins expressed in E. coli. Antibodies raised to each domain were tested for surface fluorescence with live infected erythrocytes, rosette inhibition and phagocytosis-induction. Antibodies to all PfEMP1 domains recognized the surface of live infected erythrocytes down to low concentrations (0.02-1.56 µg/ml of total IgG. Antibodies to all PfEMP1 domains except for the second Duffy-Binding-Like region inhibited rosetting (50% inhibitory concentration 0.04-4 µg/ml and were able to opsonize and induce phagocytosis of infected erythrocytes at low concentrations (1.56-6.25 µg/ml. Antibodies to the N-terminal region (NTS-DBL1α were the most effective in all assays. All antibodies were specific for the R29 parasite strain, and showed no functional activity against five other rosetting strains. CONCLUSIONS/SIGNIFICANCE: These results are encouraging for vaccine development as they show that potent antibodies can be generated to recombinant PfEMP1 domains that will inhibit rosetting and induce phagocytosis of infected erythrocytes. However, further work is needed on rosetting mechanisms and cross-reactivity in field isolates to define a set of PfEMP1 variants that could induce functional antibodies against a broad range of P. falciparum rosetting parasites.

  12. A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission.

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    Isabel Fofana

    Full Text Available BACKGROUND AND AIMS: Hepatitis C virus (HCV infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. METHODS: Using genetic immunization, we produced four monoclonal antibodies (mAbs against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines. RESULTS: The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity. CONCLUSION: A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HCV infection.

  13. Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.

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    Juan Reguera

    Full Text Available The coronaviruses (CoVs are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10-20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN, a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs of two closely related CoV strains, transmissible gastroenteritis virus (TGEV and porcine respiratory CoV (PRCV, in complex with their receptor, porcine APN (pAPN, or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.

  14. Anti-caries DNA vaccine-induced secretory immunoglobulin A antibodies inhibit formation of Streptococcus mutans biofilms in vitro

    Institute of Scientific and Technical Information of China (English)

    Li HUANG; Qing-an XU; Chang LIU; Ming-wen FAN; Yu-hong LI

    2013-01-01

    Aim: To investigate the effects of anti-caries DNA vaccine-induced salivary secretory immunoglobulin A (S-IgA) antibodies on Streptococcus mutans (S.mutans) adherence and biofilms formation in vitro.Methods: Adult female Wistar rats were intranasally immunized with the anti-caries DNA vaccine pGJA-P/VAX.Their saliva samples were collected at different times after the immunization,and S-IgA antibody level in the saliva and its inhibition on S.mutans adherence were examined.The effects of S-IgA in the saliva with the strongest inhibitory effects were examined at 3 different stages,ie acquired pellicles,biofilm formation and production of mature biofilms.The number of viable bacteria and depth of the biofilm at 16 h in each stage were determined using counting colony forming units and using a confocal laser scanning microscopy (CLSM).The participation of S-IgA in acquired pellicles and its aggregation with S.mutans were also observed under CLSM.Results: The S-lgA titer in saliva reached its peak and exhibited the strongest inhibition on S.mutans adhesion at 10 weeks after the immunization.The colonies and depth of the biofilm in the saliva-pretreated group were 41.79% and 41.02%,respectively,less than the control group.The colonies and depth of the biofilm in the co-culture group were 27.4% and 22.81% less than the control group.The assembly of S.mutans and S-IgA was observed under CLSM after co-cultivation.In the mature-stage biofilm,no differences were observed between the different groups.Conclusion: These results demonstrate that the anti-caries DNA vaccine induces the production of specific S-IgA antibodies that may prevent dental caries by inhibiting the initial adherence of S.mutans onto tooth surfaces,thereby reducing the accumulation of S.mutans on the acquired pellicles.

  15. Neutralizing antibodies induced by recombinant virus-like particles of enterovirus 71 genotype C4 inhibit infection at pre- and post-attachment steps.

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    Zhiqiang Ku

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia-Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear. METHODS/FINDINGS: In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs. Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment. CONCLUSIONS: Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.

  16. Inhibition of Entamoeba histolytica proteolytic activity by human salivary IgA antibodies.

    Science.gov (United States)

    Guerrero-Manríquez, G G; Sánchez-Ibarra, F; Avila, E E

    1998-11-01

    Entamoeba histolytica is a protozoan parasite that causes amoebiasis in humans; as the infection occurs mainly in the intestinal epithelium, the secretory immune response of the host could have an influence on the outcome. Secretory IgA antibodies against E. histolytica have been detected in asymptomatic and symptomatic patients, but little is known about their protective role. E. histolytica cysteine proteases seem to be involved in the pathogenesis of amoebiasis; therefore, it is important to evaluate the human IgA response against these proteases and its effect on their enzymatic activity. When human saliva samples with and without antibodies against E. histolytica were tested by Western blot against one purified 70 kDa amoebic cysteine protease, 84% of anti-amoeba-positive samples recognized it. The secretory IgA purified from a pool of anti-protease-positive samples had a strong in vitro inhibitory effect on the E. histolytica proteolytic activity. These results suggest that this effect, if it occurs in vivo, could be an important protective factor against this parasite.

  17. Antibody-Mediated Inhibition of TNFR1 Attenuates Disease in a Mouse Model of Multiple Sclerosis

    OpenAIRE

    Williams, Sarah K.; Olaf Maier; Roman Fischer; Richard Fairless; Sonja Hochmeister; Aleksandar Stojic; Lara Pick; Doreen Haar; Sylvia Musiol; Maria K Storch; Klaus Pfizenmaier; Ricarda Diem

    2014-01-01

    Tumour necrosis factor (TNF) is a proinflammatory cytokine that is known to regulate inflammation in a number of autoimmune diseases, including multiple sclerosis (MS). Although targeting of TNF in models of MS has been successful, the pathological role of TNF in MS remains unclear due to clinical trials where the non-selective inhibition of TNF resulted in exacerbated disease. Subsequent experiments have indicated that this may have resulted from the divergent effects of the two TNF receptor...

  18. A therapeutic anti-CD4 monoclonal antibody inhibits T cell receptor signal transduction in mouse autoimmune cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    WANG Zhao-hui; LIAO Yu-hua; YUAN Jing; ZHANG Li; WANG Min; ZHANG Jing-hui; LIU Zhong-ping; DONG Ji-hua

    2007-01-01

    Background T cell immune abnormalities in patients with dilated cardiomyopathy (DCM) has been intensively studied over the past 10 years. Our previous study has suggested that immunization of mice with the peptides derived from human adenine nucleotide translocator (ANT) result in the production of autoantibodies against the ANT and histopathological changes similar to those in human DCM. The ANT peptides can induce autoimmune cardiomyopathy like DCM in Balb/c mice. In this study we aimed to focus on the molecular mechanism of T cells in the autoimmune cardiomyopathy mouse model by detecting the expression of the two T cell signaling molecules.Methods The ANT peptides were used to cause autoimmune cardiomyopathy in Balb/c mice. Anti-L3T4 or rat anti-mouse IgG was administered to the mice (n=6 in each group) simultaneously immunized with ANT. ELISA analysis was used to detect autoantibodies against the ANT peptides and the percentages of interferon-Y and interleukin-4 producing cells among splenic CD4+ lymphocytes was determined by using flow cytometry analysis. The expression of CD45 in spleen T cells was determined by immunohistochemistry and the mRNAs of T cell signaling molecules were detected by real-time PCR.Results Treatment of ANT immunized Balb/c mice with anti-CD4 mAb caused a reduction in the gene expression of P56lck and Zap-70 and a lower level of CD45 expression by spleen T cells. Aiso, a reverse of the Th1/Th2 ratio that results in the reduced production of antibodies against ANT was found in the anti-CD4 monoclonal antibodies (mAb)group. Whereas irrelevant antibody (rat anti-mouse IgG) did not suppress T cell signaling molecules nor inhibit CD45 expression, and control-antibody mice did not show any significant differences compared with the DCM group.Conclusion The results show that anti-CD4 mAb is a powerful inhibitor of the early initiating events of T cell receptor(TCR) signal transduction in mouse autoimmune dilated cardiomyopathy.

  19. A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo.

    Science.gov (United States)

    Lin, Hong; Zhang, Huiling; Wang, Jun; Lu, Meiping; Zheng, Feng; Wang, Changjun; Tang, Xiaojun; Xu, Ning; Chen, Renjie; Zhang, Dawei; Zhao, Ping; Zhu, Jin; Mao, Yuan; Feng, Zhenqing

    2014-03-01

    Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer.

  20. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

    Directory of Open Access Journals (Sweden)

    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  1. An inhibition enzyme immunoassay, using a human monoclonal antibody (K14) reactive with gp41 of HIV-1, for the serology of HIV-1 infections.

    NARCIS (Netherlands)

    V.J.P. Teeuwsen; J.J. Schalken; G. van der Groen (Guido); R. van den Akker (Ruud); J. Goudsmit (Jaap); A.D.M.E. Osterhaus (Ab)

    1991-01-01

    textabstractAn inhibition enzyme immunoassay (IEIA), using a human monoclonal antibody (K14) reactive with gp41 of HIV-1, was evaluated for its applicability to the serology of HIV-1 infections. Using panels of serum samples from seronegative and confirmed HIV-1-seropositive individuals, it was show

  2. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization

    DEFF Research Database (Denmark)

    Hansen, J E; Clausen, H; Nielsen, C;

    1990-01-01

    Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N...

  3. Comparison of an enzyme-linked immunosorbent assay, an immunofluorescence assay and a hemagglutination inhibition assay for detection of antibodies to K-papovavirus in mice.

    NARCIS (Netherlands)

    J. Groen (Jan); A.D.M.E. Osterhaus (Ab); H.W.J. Broeders; H.E.M. Spijkers (Ine)

    1989-01-01

    textabstractThe sensitivity of a newly developed enzyme-linked immunosorbent assay (ELISA) for detection of antibody to K virus was compared with the sensitivities of an immunofluorescence assay (IFA) and a hemagglutination inhibition assay (HIA). Specific pathogen-free BALB/c RIVM mice, 5 weeks old

  4. Amino acid metabolism inhibits antibody-driven kidney injury by inducing autophagy.

    Science.gov (United States)

    Chaudhary, Kapil; Shinde, Rahul; Liu, Haiyun; Gnana-Prakasam, Jaya P; Veeranan-Karmegam, Rajalakshmi; Huang, Lei; Ravishankar, Buvana; Bradley, Jillian; Kvirkvelia, Nino; McMenamin, Malgorzata; Xiao, Wei; Kleven, Daniel; Mellor, Andrew L; Madaio, Michael P; McGaha, Tracy L

    2015-06-15

    Inflammatory kidney disease is a major clinical problem that can result in end-stage renal failure. In this article, we show that Ab-mediated inflammatory kidney injury and renal disease in a mouse nephrotoxic serum nephritis model was inhibited by amino acid metabolism and a protective autophagic response. The metabolic signal was driven by IFN-γ-mediated induction of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity with subsequent activation of a stress response dependent on the eIF2α kinase general control nonderepressible 2 (GCN2). Activation of GCN2 suppressed proinflammatory cytokine production in glomeruli and reduced macrophage recruitment to the kidney during the incipient stage of Ab-induced glomerular inflammation. Further, inhibition of autophagy or genetic ablation of Ido1 or Gcn2 converted Ab-induced, self-limiting nephritis to fatal end-stage renal disease. Conversely, increasing kidney IDO1 activity or treating mice with a GCN2 agonist induced autophagy and protected mice from nephritic kidney damage. Finally, kidney tissue from patients with Ab-driven nephropathy showed increased IDO1 abundance and stress gene expression. Thus, these findings support the hypothesis that the IDO-GCN2 pathway in glomerular stromal cells is a critical negative feedback mechanism that limits inflammatory renal pathologic changes by inducing autophagy.

  5. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer.

    Science.gov (United States)

    Schanzer, Juergen M; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2-3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

  6. Effect of Low Molecular Weight Heparins (LMWHs on antiphospholipid Antibodies (aPL-mediated inhibition of endometrial angiogenesis.

    Directory of Open Access Journals (Sweden)

    Silvia D'Ippolito

    Full Text Available Antiphospholipid syndrome (APS is an autoimmune disorder characterized by vascular thrombosis and/or pregnancy morbidity in the presence of circulating antiphospholipid antibodies (aPL. Different pathogenic mechanisms for aPL-mediated pregnancy failure have been proposed. In particular a direct effect of aPL on both maternal and fetal side of the placental tissue has been reported, since their reactivity with β2-glycoprotein I (β2GPI makes them adhere to trophoblast and human endometrial endothelial cell (HEEC membranes. β2GPI can be recognized by aPL that, once bound, interfere with both trophoblast functions and with the HEEC differentiation.APS patients can be successfully treated with Low Molecular Weight Heparin (LMWH. Recent reports suggest that LMWH acts through mechanisms alternative to its well known anticoagulant effect, because of its ability to bind β2GPI. In our previous studies, we showed that LMWH is able to reduce the aPL binding to trophoblasts and restore cell invasiveness and differentiation. So far, however, no study has described its effects on endometrial angiogenesis.The aim of our research was to evaluate whether two LMWHs, tinzaparin and enoxaparin, have an effect on the aPL-inhibited endometrial angiogenesis. This prompted us to investigate: (i in vitro HEEC angiogenesis through a Matrigel assay; (ii VEGF secretion by ELISA; (iii matrix metalloproteinase-2 (MMP-2 activity by gelatin zymography; (iv Nuclear Factor-κB (NF-κB DNA binding activity by colorimetric assay; (v STAT-3 activation by a sandwich-ELISA kit. Furthermore, using an in vivo murine model we investigated the LMWHs effects on angiogenesis.We demonstrated that the addition of LMWHs prevents aPL-inhibited HEEC angiogenesis, both in vitro and in vivo, and is able to restore the aPL inhibited NF-κB and/or STAT-3 activity, the VEGF secretion and the MMPs activity.The demonstration of a beneficial role for LMWHs on the aPL-inhibited HEEC angiogenesis

  7. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  8. A recombinant humanized anti-cocaine monoclonal antibody inhibits the distribution of cocaine to the brain in rats.

    Science.gov (United States)

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

    2014-07-01

    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 steady state (Vdss) of 0.3 l/kg. Pretreatment with h2E2 produced a dramatic 8.8-fold increase in the area under the plasma cocaine concentration-time curve (AUC) and in brain a concomitant decrease of 68% of cocaine's AUC following an i.v. injection of an equimolar cocaine dose. Sequestration of cocaine in plasma by h2E2, shown via reduction of cocaine's Vdss, indicates potential clinical efficacy. Although the binding of cocaine to h2E2 in plasma should inhibit distribution and metabolism, the elimination of cocaine remained multicompartmental and was still rapidly eliminated from plasma despite the presence of h2E2. BE was the major cocaine metabolite, and brain BE concentrations were sixfold higher than in plasma, indicating that cocaine is normally metabolized in the brain. In the presence of h2E2, brain BE concentrations were decreased and plasma BE was increased, consistent with the observed h2E2-induced changes in cocaine disposition. The inhibition of cocaine distribution to the brain confirms the humanized mAb, h2E2, as a lead candidate for development as an immunotherapy for cocaine abuse. PMID:24733787

  9. Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to Caprine Arthritis-Encephalitis Virus: Diagnostic Tool for Successful Eradication

    OpenAIRE

    Herrmann, Lynn M.; Cheevers, William P.; McGuire, Travis C.; Adams, D. Scott; Hutton, Melinda M.; Gavin, William G.; Knowles, Donald P

    2003-01-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was evaluated for the detection of serum antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) in goats. This assay utilized 96-well microtiter plates containing CAEV-63 SU captured by monoclonal antibody (MAb) F7-299 and measured the competitive displacement of horseradish peroxidase-conjugated MAb GPB 74A binding by undiluted goat sera (F. Özyörük, W. P. Cheevers, G. A. Hullinger, T. C. McGu...

  10. Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Herrmann, Lynn M.; Cheevers, William P.; Marshall, Katherine L.; McGuire, Travis C.; Hutton, Melinda M.; Lewis, Gregory S.; Knowles, Donald P

    2003-01-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of bi...

  11. Structural and Functional Analysis of a C3b-specific Antibody That Selectively Inhibits the Alternative Pathway of Complement*S⃞

    OpenAIRE

    Katschke, Kenneth J.; Stawicki, Scott; Yin, JianPing; Steffek, Micah; Xi, Hongkang; Sturgeon, Lizette; Hass, Philip E.; Loyet, Kelly M.; DeForge, Laura; Wu, Yan; van Lookeren Campagne, Menno; Wiesmann, Christian

    2009-01-01

    Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in compl...

  12. Intracytoplasmic stable expression of IgG1 antibody targeting NS3 helicase inhibits replication of highly efficient hepatitis C Virus 2a clone

    OpenAIRE

    Clementi Massimo; Burioni Roberto; Liu Gerald; Prabhu Ramesh; Gunduz Feyza; Poat Bret; Hazari Sidhartha; Chandra Partha K; Garry Robert F; Dash Srikanta

    2010-01-01

    Abstract Background Hepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of...

  13. Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

    OpenAIRE

    Baltus, E; Hanocq-Quertier, J; Hanocq, F.; Brachet, J.

    1988-01-01

    The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the ...

  14. CCR3 monoclonal antibody inhibits airway eosinophilic inflammation and mucus overproduction in a mouse model of asthma

    Institute of Scientific and Technical Information of China (English)

    Hua-hao SHEN; Feng XU; Gen-sheng ZHANG; Shao-bin WANG; Wei-hua XU

    2006-01-01

    Aim: To explore the effect of a rat anti-mouse CC-chemokine receptor-3 (CCR3) monoclonal antibody (CCR3 mAb) on airway eosinophilia and mucus overproduction in asthmatic mice. Methods: An asthma model was sensitized and challenged by ovalbumin (OVA) in male C57BL/6 mice. Asthmatic mice were given dual administration (intraperitoneal injection and aerosol inhalation) of CCR3 mAb or nonspecific rat IgG (ns-IgG). The number of total and differential inflammatory cells in the bronchial alveolar lavage fluid (BALF) was counted. Eosinophils number, the goblet cell percentage (GCP) and airway mucus index (AMI) were measured in the lung tissues. Interleukin (IL)-5 levels in the BALF were examined. The expression of MUC5AC and the epidermal growth factor receptor (EGFR) mRNA in the lung tissues was detected by semi-quantitative RT-PCR. The results were compared among the groups. Results: CCR3 mAb significantly suppressed the increased eosinophils in the BALF and lung tissues in OVA-challenged mice compared with ns-IgG-treated mice. IL-5 levels in the BALF in CCR3 mAb and ns-IgG administration mice exhibited no obvious changes relative to OVA-challenged asthmatic mice. CCR3 mAb reduced the increased GCP and AMI after OVA challenge and decreased the enhanced expression of MUC5AC and EGFR mRNA in lung tissues in asthmatic animals. Conclusion: CCR3 mAb can significantly inhibit airway eosinophilia and mucus overproduction in asthmatic mice. Blockage of CCR3 may represent a new strategy to asthma therapy.

  15. Use of silver nanoparticles increased inhibition of cell-associated HIV-1 infection by neutralizing antibodies developed against HIV-1 envelope proteins

    Directory of Open Access Journals (Sweden)

    Garza Treviño Elsa N

    2011-09-01

    Full Text Available Abstract Background HIV/AIDS pandemic is a worldwide public health issue. There is a need for new approaches to develop new antiviral compounds or other therapeutic strategies to limit viral transmission. The envelope glycoproteins gp120 and gp41 of HIV are the main targets for both silver nanoparticles (AgNPs and neutralizing antibodies. There is an urgency to optimize the efficiency of the neutralizing antibodies (NABs. In this study, we demonstrated that there is an additive effect between the four NABs and AgNPs when combined against cell-associated HIV-1 infection in vitro Results Four NABs (Monoclonal antibody to HIV-1 gp41 126-7, HIV-1 gp120 Antiserum PB1 Sub 2, HIV-1 gp120 Antiserum PB1, HIV-1 gp120 Monoclonal Antibody F425 B4e8 with or without AgNPs of 30-50 nm in size were tested against cell free and cell-associated HIVIIIB virus. All NABs inhibited HIV-1 cell free infection at a dose response manner, but with AgNPs an antiviral additive effect was not achieved Although there was no inhibition of infection with cell-associated virus by the NABs itself, AgNPs alone were able to inhibit cell associated virus infection and more importantly, when mixed together with NABs they inhibited the HIV-1 cell associated infection in an additive manner. Discussion The most attractive strategies to deal with the HIV problem are the development of a prophylactic vaccine and the development of effective topical vaginal microbicide. For two decades a potent vaccine that inhibits transmission of infection of HIV has been searched. There are vaccines that elicit NABs but none of them has the efficacy to stop transmission of HIV-1 infection. We propose that with the addition of AgNPs, NABs will have an additive effect and become more potent to inhibit cell-associated HIV-1 transmission/infection. Conclusions The addition of AgNPs to NABs has significantly increased the neutralizing potency of NABs in prevention of cell-associated HIV-1 transmission

  16. IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

    Science.gov (United States)

    Nakatani, Yoshihiko; Nagaoka, Takumi; Hotta, Sayako; Utsunomiya, Iku; Yoshino, Hiide; Miyatake, Tadashi; Hoshi, Keiko; Taguchi, Kyoji

    2007-03-01

    We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

  17. Anomalous expression of Thy1 (CD90) in B-cell lymphoma cells and proliferation inhibition by anti-Thy1 antibody treatment

    Energy Technology Data Exchange (ETDEWEB)

    Ishiura, Yoshihito [Department of Biochemistry, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan); CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kotani, Norihiro, E-mail: kotani@kochi-u.ac.jp [CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kochi System Glycobiology Center, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan); Yamashita, Ryusuke [Department of Biochemistry, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan); CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Yamamoto, Harumi [Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Yoshida Shimo-Adachi, Sakyo, Kyoto 606-8501 (Japan); Kozutsumi, Yasunori [CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Yoshida Shimo-Adachi, Sakyo, Kyoto 606-8501 (Japan); Honke, Koichi [Department of Biochemistry, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan); CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kochi System Glycobiology Center, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan)

    2010-05-28

    The anti-CD20 monoclonal antibody (Ab) rituximab is accepted to be an effective therapeutic Ab for malignant B-cell lymphoma; however, discovery of other cell surface antigens is required for the option of antibody medicine. Considering that many tumor-associated antigens are glycans, we have searched glycoconjugates for the candidate antigens that therapeutic Abs target. To this end, we first focused on the difference in the glycogenes expression in terms of Epstein-Barr virus (EBV) infection of a Burkitt's lymphoma cell line, Akata. Using DNA array, flow cytometry and Western blotting, we found that Thy1 was highly expressed in EBV-positive Akata cells. Subsequently, Thy1 was found to be expressed in other B-cell lymphoma cell lines: BJAB, MutuI, and MutuIII, irrespective of EBV infection. Treatment of these cells with an anti-Thy1 monoclonal antibody inhibited proliferation more strongly than the therapeutic Ab rituximab. The B-cell lymphoma cell lines were classified based on the extent of the proliferation inhibition, which was not correlated with the expression level of Thy1. It is suggested that stable residence of receptor tyrosine kinases in lipid rafts sustains cell growth in B-cell lymphoma cells.

  18. Generation and Characterization of C305, a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA

    Institute of Scientific and Technical Information of China (English)

    Mei-Yun LIU; Wei HAN; Yan-Li DING; Tian-Hong ZHOU; Rui-Yang TIAN; Sheng-Li YANG; Hui LIU; Yi GONG

    2005-01-01

    B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS.After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

  19. Inhibition of the Production of Anti-OspA Borreliacidal Antibody with T Cells from Hamsters Vaccinated against Borrelia burgdorferi

    OpenAIRE

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

    1998-01-01

    The serious morbidity associated with Lyme borreliosis has focused considerable effort on the development of a comprehensive vaccine for protection against infection with Borrelia burgdorferi. Induction of borreliacidal antibody by vaccination or infection has been shown to correlate with protection of humans and animals against infection with the Lyme spirochete. In this report, we showed that high levels of borreliacidal antibody (titer of 1,280) were produced in vitro when T and B cells fr...

  20. Single Neutralizing Monoclonal Antibodies Targeting the VP1 GH Loop of Enterovirus 71 Inhibit both Virus Attachment and Internalization during Viral Entry

    Science.gov (United States)

    Ku, Zhiqiang; Ye, Xiaohua; Shi, Jinping; Wang, Xiaoli

    2015-01-01

    ABSTRACT Antibodies play a critical role in immunity against enterovirus 71 (EV71). However, how EV71-specific antibodies neutralize infections remains poorly understood. Here we report the working mechanism for a group of three monoclonal antibodies (MAbs) that potently neutralize EV71. We found that these three MAbs (termed D5, H7, and C4, respectively) recognize the same conserved neutralizing epitope within the VP1 GH loop of EV71. Single MAbs in this group, exemplified by D5, could inhibit EV71 infection in cell cultures at both the pre- and postattachment stages in a cell type-independent manner. Specifically, MAb treatment resulted in the blockade of multiple steps of EV71 entry, including virus attachment, internalization, and subsequent uncoating and RNA release. Furthermore, we show that the D5 and C4 antibodies can interfere with EV71 binding to its key receptors, including heparan sulfate, SCARB2, and PSGL-1, thus providing a possible explanation for the observed multi-inhibitory function of the MAbs. Collectively, our study unravels the mechanism of neutralization by a unique group of anti-EV71 MAbs targeting the conserved VP1 GH loop. The findings should enhance our understanding of MAb-mediated immunity against enterovirus infections and accelerate the development of MAb-based anti-EV71 therapeutic drugs. IMPORTANCE Enterovirus 71 (EV71) is a major causative agent of hand, foot, and mouth disease (HFMD), which has caused significant morbidities and mortalities in young children. Neither a vaccine nor an antiviral drug is available. Neutralizing antibodies are major protective components in EV71 immunity. Here, we unraveled an unusual mechanism of EV71 neutralization by a group of three neutralizing monoclonal antibodies (MAbs). All of these MAbs bound the same conserved epitope located at the VP1 GH loop of EV71. Interestingly, mechanistic studies showed that single antibodies in this MAb group could block EV71 attachment and internalization during

  1. Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: diagnostic tool for successful eradication.

    Science.gov (United States)

    Herrmann, Lynn M; Cheevers, William P; McGuire, Travis C; Adams, D Scott; Hutton, Melinda M; Gavin, William G; Knowles, Donald P

    2003-03-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was evaluated for the detection of serum antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) in goats. This assay utilized 96-well microtiter plates containing CAEV-63 SU captured by monoclonal antibody (MAb) F7-299 and measured the competitive displacement of horseradish peroxidase-conjugated MAb GPB 74A binding by undiluted goat sera (F. Ozyörük, W. P. Cheevers, G. A. Hullinger, T. C. McGuire, M. Hutton, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 8:44-51, 2001). Two hundred serum samples from goats in the United States were used to determine the sensitivity and specificity of cELISA based on the immunoprecipitation (IP) of [(35)S]methionine-labeled viral antigens as a standard of comparison. A positive cELISA was defined as >33.2% inhibition of MAb 74A binding based on 2 standard deviations above the mean percent inhibition of 140 IP-negative serum samples. At this cutoff value, there were 0 of 60 false-negative sera (100% sensitivity) and 5 of 140 false-positive sera (96.4% specificity). Additional studies utilized IP-monitored cELISA to establish a CAEV-free herd of 1,640 dairy goats. PMID:12626453

  2. Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion.

    Science.gov (United States)

    Sim, B K; Orlandi, P A; Haynes, J D; Klotz, F W; Carter, J M; Camus, D; Zegans, M E; Chulay, J D

    1990-11-01

    The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts. PMID:2229177

  3. Inhibition of HIV Env binding to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120

    Directory of Open Access Journals (Sweden)

    Wilson Ian A

    2006-07-01

    Full Text Available Abstract During natural HIV infection, an array of host receptors are thought to influence virus attachment and the kinetics of infection. In this study, to probe the interactions of HIV envelope (Env with various receptors, we assessed the inhibitory properties of various anti-Env monoclonal antibodies (mAbs in binding assays. To assist in detecting Env in attachment assays, we generated Fc fusions of full-length wild-type gp120 and several variable loop-deleted gp120s. Through investigation of the inhibition of Env binding to cell lines expressing CD4, CCR5, DC-SIGN, syndecans or combinations thereof, we found that the broadly neutralizing mAb, 2G12, directed to a unique carbohydrate epitope of gp120, inhibited Env-CCR5 binding, partially inhibited Env-DC-SIGN binding, but had no effect on Env-syndecan association. Furthermore, 2G12 inhibited Env attachment to primary monocyte-derived dendritic cells, that expressed CD4 and CCR5 primary HIV receptors, as well as DC-SIGN, and suggested that the dual activities of 2G12 could be valuable in vivo for inhibiting initial virus dissemination and propagation.

  4. Cyclosporine inhibits long-term survival in cardiac allografts treated with monoclonal antibody against CD45RB

    NARCIS (Netherlands)

    Parry, N; Lazarovits, AI; Wang, JJ; Garcia, B; Luke, P; Poppema, S; Zhong, R

    1999-01-01

    Background: We have previously reported that a monoclonal antibody to CD45RB is a novel immunosuppressive agent; however, the optimal regimen in cardiac allografts remains unknown. The present study was undertaken to determine the optimal protocol of this therapy and its interaction with cyclosporin

  5. IMMUNE INHIBITION OF VIRUS RELEASE FROM HUMAN AND NONHUMAN CELLS BY ANTIBODY TO VIRAL AND HOST-CELL DETERMINANTS

    NARCIS (Netherlands)

    SHARIFF, DM; DESPERBASQUES, M; BILLSTROM, M; GEERLIGS, HJ; WELLING, GW; WELLINGWESTER, S; BUCHAN, A; SKINNER, GRB

    1991-01-01

    Immune inhibition of release of the DNA virues, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and su

  6. Liposomes loaded with P. falciparum merozoite-derived proteins are highly immunogenic and produce invasion-inhibiting and anti-toxin antibodies.

    Science.gov (United States)

    Fotoran, Wesley L; Santangelo, Rachele M; Medeiros, Márcia M; Colhone, Marcelle; Ciancaglini, Pietro; Barboza, Renato; Marinho, Claudio Romero Farias; Stábeli, Rodrigo Guerino; Wunderlich, Gerhard

    2015-11-10

    The formulation of an effective vaccine against malaria is still a significant challenge and the induction of high anti-parasite antibody titers plus a sustained T cell response is mandatory for the success of such a vaccine. We have developed a nanoliposome-based structure which contains plasma membrane-associated proteins (PfMNP) of Plasmodium falciparum merozoites on its surface. Incorporation of parasite-derived proteins led to a significant increase in the size and dispersity of particles. Immunization of particles in BalbC and C57BL/6 mice led to high anti-MSP119 IgG titers (10(4)) after the first dose and reached a plateau (>10(6)) after the third dose. While very high titers were observed against the C-terminal domain of the vaccine candidate MSP1, only modest titers (≤10(3)) were detected against MSP2. The induced antibodies showed also a strong growth-inhibiting effect in reinvasion assays. In addition, PfMNP immunization generated antibodies which partially blocked the inflammatory response, probably by blocking TLR-induced activation of macrophages by malarial toxins such as GPI anchors. The results underline the potential of nanoliposome-based formulations as anti-malarial vaccines. PMID:26334481

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

    Science.gov (United States)

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

    2015-04-01

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

  8. Regulation of naturally occurring autoantibody-producing cells: detection of a suppressive substance which inhibits the secretion of antibodies directed against enzyme-treated isologous erythrocytes.

    Science.gov (United States)

    Moticka, E J

    1983-10-01

    Normal mice possess spleen cells capable of forming hemolytic plaques against bromelain-treated autologous red cells (Br MRBC). There is present in the serum of these same mice a substance which can inhibit the formation of these plaques. This substance is inhibitory to the secretion of these antibodies following incubation of spleen cells in 20% serum at 4 degrees C for 5 min. This substance is not inhibitory to the formation of anti-sheep erythrocyte plaques from mice either immunized or nonimmunized with sheep erythrocytes. Characterization of the substance indicates that it is neither soluble antigen nor specific antibody. However, inclusion of nanogram amounts of soluble antigen from bromelain-treated red cells in the assay mixture effectively neutralized the suppression. In addition, passage of serum through a mouse anti-Br MRBC antibody immunoadsorbent effectively removed the suppressive activity of the serum while suppression could be recovered in the acid eluate from such a column. This suggests that the mechanism of suppression brought about by incubation in serum is due to the action of a molecule possessing anti-idiotypic activity directed against the cell surface receptors of anti-Br MRBC B cells. Attempts to isolate the molecule based on the postulate that it is immunoglobulin in nature have been unsuccessful.

  9. In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

    Directory of Open Access Journals (Sweden)

    Daniel Volker

    2012-08-01

    Full Text Available Abstract Background IFNγ-producing CD4+CD25+Foxp3+ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins. Methods PBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ+ iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry. Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4+CD25+CD127-IFNγ+ PBL. Results High monoclonal antibody concentrations inhibited the induction of CD4+CD25+Foxp3+IFNγ+ PBL (anti-CD152, anti-CD279, anti-CD95: p +CD25+CD127-IFNγ+ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p +CD25+Foxp3+IFNγ+ PBL (rCD152 and rCD95: p +CD25+CD127-IFNγ+ PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-IFNγ- PBL (p +CD25+CD127-IFNγ- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p +CD25+CD127-IFNγ+ PBL (with the exception anti-CD28 monoclonal antibody: p +CD25+CD127-IFNγ- PBL but do not efficiently block suppressive iTreg function in co-cultures with CD4+CD25+CD127-IFNγ+ PBL. Conclusions CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ+ iTreg.

  10. Inhibition of the alternative complement activation pathway in traumatic brain injury by a monoclonal anti-factor B antibody: a randomized placebo-controlled study in mice

    Directory of Open Access Journals (Sweden)

    Holers V Michael

    2007-05-01

    Full Text Available Abstract Background The posttraumatic response to traumatic brain injury (TBI is characterized, in part, by activation of the innate immune response, including the complement system. We have recently shown that mice devoid of a functional alternative pathway of complement activation (factor B-/- mice are protected from complement-mediated neuroinflammation and neuropathology after TBI. In the present study, we extrapolated this knowledge from studies in genetically engineered mice to a pharmacological approach using a monoclonal anti-factor B antibody. This neutralizing antibody represents a specific and potent inhibitor of the alternative complement pathway in mice. Methods A focal trauma was applied to the left hemisphere of C57BL/6 mice (n = 89 using a standardized electric weight-drop model. Animals were randomly assigned to two treatment groups: (1 Systemic injection of 1 mg monoclonal anti-factor B antibody (mAb 1379 in 400 μl phosphate-buffered saline (PBS at 1 hour and 24 hours after trauma; (2 Systemic injection of vehicle only (400 μl PBS, as placebo control, at identical time-points after trauma. Sham-operated and untreated mice served as additional negative controls. Evaluation of neurological scores and analysis of brain tissue specimens and serum samples was performed at defined time-points for up to 1 week. Complement activation in serum was assessed by zymosan assay and by murine C5a ELISA. Brain samples were analyzed by immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL histochemistry, and real-time RT-PCR. Results The mAb 1379 leads to a significant inhibition of alternative pathway complement activity and to significantly attenuated C5a levels in serum, as compared to head-injured placebo-treated control mice. TBI induced histomorphological signs of neuroinflammation and neuronal apoptosis in the injured brain hemisphere of placebo-treated control mice for up to 7 days. In contrast, the

  11. CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

    Science.gov (United States)

    Amash, Alaa; Wang, Lin; Wang, Yawen; Bhakta, Varsha; Fairn, Gregory D; Hou, Ming; Peng, Jun; Sheffield, William P; Lazarus, Alan H

    2016-04-15

    Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions. PMID:26944929

  12. Nuclear Factor κB is Required for Tumor Growth Inhibition Mediated by Enavatuzumab (PDL192), a Humanized Monoclonal Antibody to TweakR.

    Science.gov (United States)

    Purcell, James W; Kim, Han K; Tanlimco, Sonia G; Doan, Minhtam; Fox, Melvin; Lambert, Peter; Chao, Debra T; Sho, Mien; Wilson, Keith E; Starling, Gary C; Culp, Patricia A

    2014-01-01

    TweakR is a TNF receptor family member, whose natural ligand is the multifunctional cytokine TWEAK. The growth inhibitory activity observed following TweakR stimulation in certain cancer cell lines and the overexpression of TweakR in many solid tumor types led to the development of enavatuzumab (PDL192), a humanized IgG1 monoclonal antibody to TweakR. The purpose of this study was to determine the mechanism of action of enavatuzumab's tumor growth inhibition and to provide insight into the biology behind TweakR as a cancer therapeutic target. A panel of 105 cancer lines was treated with enavatuzumab in vitro; and 29 cell lines of varying solid tumor backgrounds had >25% growth inhibition in response to the antibody. Treatment of sensitive cell lines with enavatuzumab resulted in the in vitro and in vivo (xenograft) activation of both classical (p50, p65) and non-classical (p52, RelB) NFκB pathways. Using NFκB DNA binding functional ELISAs and microarray analysis, we observed increased activation of NFκB subunits and NFκB-regulated genes in sensitive cells over that observed in resistant cell lines. Inhibiting NFκB subunits (p50, p65, RelB, p52) and upstream kinases (IKK1, IKK2) with siRNA and chemical inhibitors consistently blocked enavatuzumab's activity. Furthermore, enavatuzumab treatment resulted in NFκB-dependent reduction in cell division as seen by the activation of the cell cycle inhibitor p21 both in vitro and in vivo. The finding that NFκB drives the growth inhibitory activity of enavatuzumab suggests that targeting TweakR with enavatuzumab may represent a novel cancer treatment strategy.

  13. Nuclear Factor κB is required for tumor growth inhibition mediated by enavatuzumab (PDL192, a humanized monoclonal antibody to TweakR

    Directory of Open Access Journals (Sweden)

    James W. Purcell

    2014-01-01

    Full Text Available TweakR is a TNF receptor family member, whose natural ligand is the multifunctional cytokine TWEAK. The growth inhibitory activity observed following TweakR stimulation in certain cancer cell lines and the overexpression of TweakR in many solid tumor types led to the development of enavatuzumab (PDL192, a humanized IgG1 monoclonal antibody to TweakR. The purpose of this study was to determine the mechanism of action of enavatuzumab’s tumor growth inhibition and to provide insight into the biology behind TweakR as a cancer therapeutic target. A panel of 105 cancer lines was treated with enavatuzumab in vitro; and 29 cell lines of varying solid tumor backgrounds had >25% growth inhibition in response to the antibody. Treatment of sensitive cell lines with enavatuzumab resulted in the in vitro and in vivo (xenograft activation of both classical (p50, p65 and non-classical (p52, RelB NFκB pathways. Using NFκB DNA binding functional ELISAs and microarray analysis, we observed increased activation of NFκB subunits and NFκB regulated genes in sensitive cells over that observed in resistant cell lines. Inhibiting NFκB subunits (p50, p65, RelB, p52 and upstream kinases (IKK1, IKK2 with siRNA and chemical inhibitors consistently blocked enavatuzumab’s activity. Furthermore, enavatuzumab treatment resulted in NFκB-dependent reduction in cell-division as seen by the activation of the cell cycle inhibitor p21 both in vitro and in vivo. The finding that NFκB drives the growth inhibitory activity of enavatuzumab suggests that targeting TweakR with enavatuzumab may represent a novel cancer treatment strategy.

  14. Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay

    OpenAIRE

    J.J.N. Ngeranwa; S.P. Shompole; E.H. Venter; Wambugu, A.; J.E. Crafford; Penzhorn, B.L.

    2008-01-01

    The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra) and domestic animal (cattle, sheep and goat) populations was studied in wildlife / livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA), using a recombinant antigen (MSP-5) from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was ...

  15. Detection of serum antibodies to ovine progressive pneumonia virus in sheep by using a caprine arthritis-encephalitis virus competitive-inhibition enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Herrmann, Lynn M; Cheevers, William P; Marshall, Katherine L; McGuire, Travis C; Hutton, Melinda M; Lewis, Gregory S; Knowles, Donald P

    2003-09-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity. PMID:12965917

  16. A unique anti-CD115 monoclonal antibody which inhibits osteolysis and skews human monocyte differentiation from M2-polarized macrophages toward dendritic cells.

    Science.gov (United States)

    Haegel, Hélène; Thioudellet, Christine; Hallet, Rémy; Geist, Michel; Menguy, Thierry; Le Pogam, Fabrice; Marchand, Jean-Baptiste; Toh, Myew-Ling; Duong, Vanessa; Calcei, Alexandre; Settelen, Nathalie; Preville, Xavier; Hennequi, Marie; Grellier, Benoit; Ancian, Philippe; Rissanen, Jukka; Clayette, Pascal; Guillen, Christine; Rooke, Ronald; Bonnefoy, Jean-Yves

    2013-01-01

    Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163(+)CD64(+) M2-polarized suppressor macrophages, skewing their differentiation toward CD14(-)CD1a(+) dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.

  17. 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.

    Science.gov (United States)

    Ozyörük, F; Cheevers, W P; Hullinger, G A; McGuire, T C; Hutton, M; Knowles, D 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 chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection. PMID:11139194

  18. Infection of macrophages and dendritic cells with primary R5-tropic human immunodeficiency virus type 1 inhibited by natural polyreactive anti-CCR5 antibodies purified from cervicovaginal secretions.

    Science.gov (United States)

    Eslahpazir, Jobin; Jenabian, Mohammad-Ali; Bouhlal, Hicham; Hocini, Hakim; Carbonneil, Cédric; Grésenguet, Gérard; Mbopi Kéou, François-Xavier; LeGoff, Jérôme; Saïdi, Héla; Requena, Mary; Nasreddine, Nadine; de Dieu Longo, Jean; Kaveri, Srinivas V; Bélec, Laurent

    2008-05-01

    Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection. PMID:18353923

  19. 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...

  20. Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step

    DEFF Research Database (Denmark)

    Sabo, Michelle C; Luca, Vincent C; Prentoe, Jannick;

    2011-01-01

    (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection...

  1. Measurement of antibodies to avian influenza virus A(H7N7) in humans by hemagglutination inhibition test.

    NARCIS (Netherlands)

    Meijer, Adam; Bosman, Arnold; Kamp, Esther E H M van de; Wilbrink, Berry; Du Ry van Beest Holle, Mirna; Koopmans, Marion P G

    2006-01-01

    During the epizootic of highly pathogenic avian influenza A(H7N7) in 2003 in The Netherlands, RT-PCR and culture confirmed infection was detected in 89 persons who were ill. A modified hemagglutination inhibition (HI) test using horse erythrocytes and 2 hemagglutinating units of virus was applied to

  2. A natural bacterial-derived product, the metalloprotease arazyme, inhibits metastatic murine melanoma by inducing MMP-8 cross-reactive antibodies.

    Directory of Open Access Journals (Sweden)

    Felipe V Pereira

    Full Text Available The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

  3. A Natural Bacterial-Derived Product, the Metalloprotease Arazyme, Inhibits Metastatic Murine Melanoma by Inducing MMP-8 Cross-Reactive Antibodies

    Science.gov (United States)

    Pereira, Felipe V.; Ferreira-Guimarães, Carla A.; Paschoalin, Thaysa; Scutti, Jorge A. B.; Melo, Filipe M.; Silva, Luis S.; Melo, Amanda C. L.; Silva, Priscila; Tiago, Manoela; Matsuo, Alisson L.; Juliano, Luiz; Juliano, Maria A.; Carmona, Adriana K.; Travassos, Luiz R.; Rodrigues, Elaine G.

    2014-01-01

    The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent. PMID:24788523

  4. A novel mechanism for antibody-based anthrax toxin neutralization: inhibition of prepore-to-pore conversion.

    Science.gov (United States)

    Mechaly, Adva; Levy, Haim; Epstein, Eyal; Rosenfeld, Ronit; Marcus, Hadar; Ben-Arie, Einat; Shafferman, Avigdor; Ordentlich, Arie; Mazor, Ohad

    2012-09-21

    Protective antigen (PA), a key component of anthrax toxin, mediates the entry of lethal factor (LF) or edema factor (EF) through a membranal pore into target cells. We have previously reported the isolation and chimerization of cAb29, an anti-PA monoclonal antibody that effectively neutralizes anthrax toxin in an unknown mechanism. The aim of this study was to elucidate the neutralizing mechanism of this antibody in vitro and to test its ability to confer post-exposure protection against anthrax in vivo. By systematic evaluation of the steps taking place during the PA-based intoxication process, we found that cAb29 did not interfere with the initial steps of intoxication, namely its ability to bind to the anthrax receptor, the consecutive proteolytic cleavage to PA(63), oligomerization, prepore formation, or LF binding. However, the binding of cAb29 to the prepore prevented its pH-triggered transition to the transmembranal pore, thus preventing the last step of intoxication, i.e. the translocation of LF/EF into the cell. Epitope mapping, using a phage display peptide library, revealed that cAb29 binds the 2α(1) loop in domain 2 of PA, a loop that undergoes major conformational changes during pore formation. In vivo, we found that 100% of anthrax-infected rabbits survived when treated with cAb29 12 h after exposure. In conclusion, these experiments demonstrate that cAb29 exerts its potent neutralizing activity in a unique manner by blocking the prepore-to-pore conversion process. PMID:22869370

  5. Generation and characterization of a tetraspanin CD151/integrin α6β1-binding domain competitively binding monoclonal antibody for inhibition of tumor progression in HCC

    Science.gov (United States)

    Cai, Jia-Bin; Huang, Xiao-Yong; Wu, Chao; Zhang, Lu; Kang, Qiang; Liu, Li-Xin; Xie, Nan; Shen, Zao-Zhuo; Hu, Mei-Yu; Cao, Ya; Qiu, Shuang-Jian; Sun, Hui-Chuan; Zhou, Jian; Fan, Jia; Shi, Guo-Ming

    2016-01-01

    Our previous studies revealed that tetraspanin CD151 plays multiple roles in the progression of hepatocellular carcinoma (HCC) by forming a functional complex with integrin α6β1. Herein, we generated a monoclonal antibody (mAb) that dissociates the CD151/integrin α6β1 complex, and we evaluated its bioactivity in HCCs. A murine mAb, tetraspanin CD151 (IgG1, called CD151 mAb 9B), was successfully generated against the CD151-integrin α6β1 binding site of CD151 extracellular domains. Co-immunoprecipitation using CD151 mAb 9B followed by Western blotting detected a 28 kDa protein. Both immunofluorescent and immunohistochemical staining showed a good reactivity of CD151 mAb 9B in the plasma membrane and cytoplasm of HCC cells, as well as in liver cells. In vitro assays demonstrated that CD151 mAb 9B could inhibit neoangiogenesis and both the mobility and the invasiveness of HCC cells. An in vivo assay showed that CD151 mAb 9B inhibited tumor growth potential and HCC cells metastasis. We successfully produced a CD151 mAb 9B targeting the CD151/integrin α6β1-binding domain, which not only can displayed good reactivity to the CD151 antigen but also prevented tumor progression in HCC. PMID:26756217

  6. Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib.

    Science.gov (United States)

    Muz, Barbara; Azab, Feda; de la Puente, Pilar; Rollins, Scott; Alvarez, Richard; Kawar, Ziad; Azab, Abdel Kareem

    2015-01-01

    Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

  7. Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib

    Directory of Open Access Journals (Sweden)

    Barbara Muz

    2015-01-01

    Full Text Available Multiple myeloma (MM is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1 play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

  8. The use of homologous virus in the haemagglutination-inhibition assay after vaccination with Newcastle disease virus strain La Sota or clone30 leads to an over estimation of protective serum antibody titres

    NARCIS (Netherlands)

    Maas, R.A.; Oei, H.L.; Kemper, S.; Koch, G.; Visser, L.

    1998-01-01

    We evaluated the influence of the use of the Newcastle disease virus (NDV)-strains Ulster and La Sota in the haemagglutination inhibition (HI) assay for the measurement of antibody titres after NDV vaccination. The use of the homologous La Sota antigen in the HI assay after Clone30 and La Sota vacci

  9. High efficacy of anti DBL4e-VAR2CSA antibodies in inhibition of CSA-binding Plasmodium falciparum-infected erythrocytes from pregnant women

    DEFF Research Database (Denmark)

    Magistrado, Pamela A; Minja, Daniel; Doritchamou, Justin;

    2011-01-01

    Malaria during pregnancy is a major cause of intra-uterine growth-retardation and infant death in sub-Saharan Africa. Ideally, this could be prevented by a vaccine delivered before the first pregnancy. Antibodies against domain DBL4¿ from VAR2CSA has been shown to inhibit adhesion of laboratory...

  10. Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    J.J.N. Ngeranwa

    2008-09-01

    Full Text Available The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra and domestic animal (cattle, sheep and goat populations was studied in wildlife / livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA, using a recombinant antigen (MSP-5 from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA, as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers.

  11. Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Ngeranwa, J J N; Shompole, S P; Venter, E H; Wambugu, A; Crafford, J E; Penzhorn, B L

    2008-09-01

    The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra) and domestic animal (cattle, sheep and goat) populations was studied in wildlife/livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA), using a recombinant antigen (MSP-5) from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA), as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers. PMID:19040134

  12. Antibody-Mediated Inhibition of the FGFR1c Isoform Induces a Catabolic Lean State in Siberian Hamsters.

    Science.gov (United States)

    Samms, Ricardo J; Lewis, Jo E; Lory, Alex; Fowler, Maxine J; Cooper, Scott; Warner, Amy; Emmerson, Paul; Adams, Andrew C; Luckett, Jeni C; Perkins, Alan C; Wilson, Dana; Barrett, Perry; Tsintzas, Kostas; Ebling, Francis J P

    2015-11-16

    Hypothalamic tanycytes are considered to function as sensors of peripheral metabolism. To facilitate this role, they express a wide range of receptors, including fibroblast growth factor receptor 1 (FGFR1). Using a monoclonal antibody (IMC-H7) that selectively antagonizes the FGFR1c isoform, we investigated possible actions of FGFR1c in a natural animal model of adiposity, the Siberian hamster. Infusion of IMC-H7 into the third ventricle suppressed appetite and increased energy expenditure. Likewise, peripheral treatment with IMC-H7 decreased appetite and body weight and increased energy expenditure and fat oxidation. A greater reduction in body weight and caloric intake was observed in response to IMC-H7 during the long-day fat state as compared to the short-day lean state. This enhanced response to IMC-H7 was also observed in calorically restricted hamsters maintained in long days, suggesting that it is the central photoperiodic state rather than the peripheral adiposity that determines the response to FGFR1c antagonism. Hypothalamic thyroid hormone availability is controlled by deiodinase enzymes (DIO2 and DIO3) expressed in tanycytes and is the key regulator of seasonal cycles of energy balance. Therefore, we determined the effect of IMC-H7 on hypothalamic expression of these deiodinase enzymes. The reductions in food intake and body weight were always associated with decreased expression of DIO2 in the hypothalamic ependymal cell layer containing tanycytes. These data provide further support for the notion the tanycytes are an important component of the mechanism by which the hypothalamus integrates central and peripheral signals to regulate energy intake and expenditure.

  13. Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5'-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3'-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP)(73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn't. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.

  14. Thyroid Antibodies

    Science.gov (United States)

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

  15. Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model

    Directory of Open Access Journals (Sweden)

    Megas Françoise

    2010-03-01

    Full Text Available Abstract Background Antibodies directed against haemagglutinin, measured by the haemagglutination inhibition (HI assay are essential to protective immunity against influenza infection. An HI titre of 1:40 is generally accepted to correspond to a 50% reduction in the risk of contracting influenza in a susceptible population, but limited attempts have been made to further quantify the association between HI titre and protective efficacy. Methods We present a model, using a meta-analytical approach, that estimates the level of clinical protection against influenza at any HI titre level. Source data were derived from a systematic literature review that identified 15 studies, representing a total of 5899 adult subjects and 1304 influenza cases with interval-censored information on HI titre. The parameters of the relationship between HI titre and clinical protection were estimated using Bayesian inference with a consideration of random effects and censorship in the available information. Results A significant and positive relationship between HI titre and clinical protection against influenza was observed in all tested models. This relationship was found to be similar irrespective of the type of viral strain (A or B and the vaccination status of the individuals. Conclusion Although limitations in the data used should not be overlooked, the relationship derived in this analysis provides a means to predict the efficacy of inactivated influenza vaccines when only immunogenicity data are available. This relationship can also be useful for comparing the efficacy of different influenza vaccines based on their immunological profile.

  16. Evaluation of enzyme-linked immunosorbent assays and a haemagglutination inhibition tests for the detection of antibodies to Newcastle disease virus in village chickens using a Bayesian approach.

    Science.gov (United States)

    Chaka, H; Thompson, P N; Goutard, F; Grosbois, V

    2015-04-01

    Newcastle disease (ND) is an endemic disease in village chickens in Ethiopia with substantial economic importance. The sensitivity (Se) and specificity (Sp) of a blocking enzyme-linked immunosorbent assay (bELISA, Svanova Biotech), indirect ELISA (iELISA, Laboratoire Service International) and haemagglutination inhibition (HI) test for ND virus (NDV) antibody detection were evaluated in a Bayesian framework in the absence of a gold standard test, on sera collected from unvaccinated chickens kept under the village production system in household flocks and at markets in two woredas (i.e. districts) of the Eastern Shewa zone, Ethiopia. The outcomes of the iELISA test differed dramatically from those of the two other tests with 92% of the samples testing positive as compared with less than 15% for bELISA and HI. iELISA results were also inconsistent with previous estimations of Newcastle serological prevalence. The information provided by the iELISA test was thus considered as highly unreliable, probably due to an extremely low specificity, and thus not considered in the Bayesian models aiming at estimating serological prevalence and test performance parameters. Bayesian modelling of HI and bELISA test results suggested that bELISA had both the highest Se (86.6%; 95% posterior credible interval (PCI): 61.8%; 98.5%), and the highest Sp (98.3%; 95% PCI: 97.2%; 99.5%), while HI had a Se of 80.2% (95% PCI: 59.1%; 94.3%), and a Sp of 96.1% (95% PCI: 95.1%; 97.4%). Model selection and the range of the posterior distribution of the correlation between bELISA and HI test outcomes for truly seropositive animals (median at 0.461; PCI: -0.055; 0.894) suggested a tendency for bELISA and HI to detect the same truly positive animals and to fail to detect the same truly positive animals. The use of bELISA in screening and surveillance for NDV antibodies is indicated given its high Se and Sp, in addition to its ease of automation to handle large numbers of samples compared to HI. The

  17. Inhibition of phagocytosis of complement C3- or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol).

    OpenAIRE

    Arnaout, M. A.; Todd, R F; Dana, N; Melamed, J; Schlossman, S F; Colten, H R

    1983-01-01

    Events that lead to phagocytosis of complement (C3)- or IgG-coated particles after their interaction with specific cell surface receptors are poorly understood. Two mouse monoclonal antibodies (an IgM and an IgG2a) to a human granulocyte-monocyte surface membrane differentiation antigen (Mol) inhibited ingestion by granulocytes both of oil Red O particles opsonized with normal human serum or with IgG and of sheep erythrocytes sensitized with IgG. In addition, they specifically inhibited roset...

  18. The NTS-DBL2X region of VAR2CSA Induces cross-reactive antibodies that inhibit adhesion of several Plasmodium falciparum isolates to chondroitin sulfate A

    DEFF Research Database (Denmark)

    Bigey, Pascal; Gnidehou, Sédami; Doritchamou, Justin;

    2011-01-01

    -adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. Results. Antibodies targeting the N-terminal sequence (NTS......) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping...

  19. A novel trifunctional IgG-like bispecific antibody to inhibit HIV-1 infection and enhance lysis of HIV by targeting activation of complement

    Directory of Open Access Journals (Sweden)

    Tomlinson Stephen

    2010-06-01

    Full Text Available Abstract Background The complement system is not only a key component of innate immunity but also provides a first line of defense against invading pathogens, especially for viral pathogens. Human immunodeficiency virus (HIV, however, possesses several mechanisms to evade complement-mediated lysis (CoML and exploit the complement system to enhance viral infectivity. Responsible for this intrinsic resistance against complement-mediated virolysis are complement regulatory membrane proteins derived from the host cell that inherently downregulates complement activation at several stages of the cascade. In addition, HIV is protected from complement-mediated lysis by binding soluble factor H (fH through the viral envelope proteins, gp120 and gp41. Whereas inhibition of complement activity is the desired outcome in the vast majority of therapeutic approaches, there is a broader potential for complement-mediated inhibition of HIV by complement local stimulation. Presentation of the hypothesis Our previous studies have proven that the complement-mediated antibody-dependent enhancement of HIV infection is mediated by the association of complement receptor type 2 bound to the C3 fragment and deposited on the surface of HIV virions. Thus, we hypothesize that another new activator of complement, consisting of two dsFv (against gp120 and against C3d respectively linked to a complement-activating human IgG1 Fc domain ((anti-gp120 × anti-C3d-Fc, can not only target and amplify complement activation on HIV virions for enhancing the efficiency of HIV lysis, but also reduce the infectivity of HIV through blocking the gp120 and C3d on the surface of HIV. Testing the hypothesis Our hypothesis was tested using cell-free HIV-1 virions cultivated in vitro and assessment of virus opsonization was performed by incubating appropriate dilutions of virus with medium containing normal human serum and purified (anti-gp120 × anti-C3d-Fc proteins. As a control group, viruses

  20. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    Directory of Open Access Journals (Sweden)

    Nidiane D R Prado

    Full Text Available Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II, two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs and immunoglobulin frameworks (FRs of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718 were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607 neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

  1. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments

    Science.gov (United States)

    Prado, Nidiane D. R.; Pereira, Soraya S.; da Silva, Michele P.; Morais, Michelle S. S.; Kayano, Anderson M.; Moreira-Dill, Leandro S.; Luiz, Marcos B.; Zanchi, Fernando B.; Fuly, André L.; E. F. Huacca, Maribel; Fernandes, Cleberson F.; Calderon, Leonardo A.; Zuliani, Juliana P.; Soares, Andreimar M.; Stabeli, Rodrigo G.; F. C. Fernandes, Carla

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  2. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    Science.gov (United States)

    Prado, Nidiane D R; Pereira, Soraya S; da Silva, Michele P; Morais, Michelle S S; Kayano, Anderson M; Moreira-Dill, Leandro S; Luiz, Marcos B; Zanchi, Fernando B; Fuly, André L; Huacca, Maribel E F; Fernandes, Cleberson F; Calderon, Leonardo A; Zuliani, Juliana P; Pereira da Silva, Luiz H; Soares, Andreimar M; Stabeli, Rodrigo G; Fernandes, Carla F C

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  3. Lysine at position 222 of the goat prion protein inhibits the binding of monoclonal antibody F99/97.6.1.

    Science.gov (United States)

    Mazza, Maria; Guglielmetti, Chiara; Pagano, Marianna; Sciuto, Simona; Ingravalle, Francesco; Martucci, Francesca; Caramelli, Maria; Acutis, Pier Luigi

    2012-09-01

    Prion protein (PrP) is encoded by the PRNP gene, which is highly polymorphic in goats, with polymorphisms encoding amino acid substitutions at the protein level. In the current study, the reactivity of monoclonal antibody (mAb) F99/97.6.1 in binding PrP from goats polymorphic at PRNP codon 222 was investigated. Nervous tissue from 30 scrapie-negative goats with 3 different genotypes (222Q/Q, 222Q/K, and 222K/K) was analyzed by Western blot using mAbs P4 and F99/97.6.1. Although PrP was detected in all 30 samples by mAb P4, detection of PrP by mAb F99/97.6.1 was limited to 222Q/Q (12/12). No PrP was detected by mAb F99/97.6.1 in the 222K/K samples (n = 6), and the signal intensity of mAb F99/97.6.1 for PrP was lower for the 222Q/K samples (12/12 samples). To further investigate these results, additional Western blot analyses were performed, and the PrP signals detected by mAbs F99/97.6.1 and SAF84 were then quantified. The mean F99/SAF84 ratio (± standard deviation) calculated for the 222Q/Q group was 0.73 ± 1.26, and the mean for the 222Q/K group was 0.27 ± 1.31. Statistical analysis of these values evidenced statistically significant differences between the 222Q/Q and 222Q/K samples. The results of the study thus revealed an inhibition by lysine at position 222 on the binding of mAb F99/97.6.1 to goat PrP. This has implications for the use of mAb F99/97.6.1 for diagnostic purposes. Because the 222K allele could be a target for genetic selection in goats, the differential reactivity of mAb F99/97.6.1 could be exploited with a genotyping test setup. PMID:22914824

  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. Antibody of predetermined specificity to a carboxy-terminal region of H-ras gene products inhibits their guanine nucleotide-binding function.

    OpenAIRE

    Srivastava, S. K.; Lacal, J C; Reynolds, S.H.; Aaronson, S A

    1985-01-01

    The high prevalence of ras oncogenes in human tumors has given increasing impetus to efforts aimed at elucidating the structure and function of their p21 products. To identify functionally important domains of the p21 protein, antibodies were generated against synthetic peptides corresponding to various regions of the protein. Antibodies directed against a synthetic peptide fragment corresponding to amino acid residues 161 to 176 in the carboxy-terminal region of the H-ras-encoded p21 molecul...

  6. Functional effects of anticardiolipin antibodies.

    Science.gov (United States)

    Harris, E N; Pierangeli, S S

    1996-10-01

    The 'lupus anticoagulant' phenomenon is the best documented functional effect of antiphospholipid (aPL) antibodies, occurring either by inhibition of the prothrombinase and/or Factor X activation reactions. Understanding the mechanism by which aPL antibodies inhibit phospholipid dependent coagulation reactions may yield important clues about their 'thrombogenic effects' in vivo. We conducted a series of studies to determine the specificity, diversity, and mechanism by which aPL antibodies inhibit phospholipid dependent reactions. Results showed that purified immunoglobulins with lupus anticoagulant and anti-cardiolipin activities were absorbed by negatively charged phospholipids and both activities were recovered from the phospholipid-antibody precipitate. Purified aPL antibodies inhibited the prothrombinase reaction in a plasma free system in which beta 2-glycoprotein 1 (beta 2-GP1) was absent. Affinity purified aPL antibodies had 25-50 times the inhibitory activity of immunoglobulin preparations. The phospholipid binding proteins, beta 2-GPI and placental anticoagulant protein I (PAP I), independently inhibited the prothrombinase reaction, and when these proteins were combined with aPL, inhibition of the prothrombinase reaction was additive. Antibodies of syphilis had no inhibitory effect, partially accounted for by lack of specificity for phosphotidylserine (PS). Although aPL antibodies inhibited the protein C activation reaction, there was no correlation of these activities with inhibition of the prothrombinase reaction. Together, these results show that aPL exert their effects by interaction with negatively charged phospholipids, in particular phosphotidylserine, but lack of correlation between inhibition of the prothrombinase and protein C activation reactions, suggests that the nature of the coagulation protein is also important. PMID:8902763

  7. Single-chain antibody-based gene therapy: Inhibition of tumor growth by in situ production of phage-derived antibodies blocking functionally active sites of cell-associated matrices

    DEFF Research Database (Denmark)

    Sanz, Laura; Kristensen, Peter; Blanco, Belén;

    2002-01-01

    Experimental evidence suggests that blocking the interactions between endothelial cells and extracellular matrix (ECM) components may provide a potent and general strategy to inhibit tumor neovascularization. Based on these considerations, we have focused our efforts on laminin, component of the ...

  8. Differential Inhibition of the TGF-β Signaling Pathway in HCC Cells Using the Small Molecule Inhibitor LY2157299 and the D10 Monoclonal Antibody against TGF-β Receptor Type II.

    Directory of Open Access Journals (Sweden)

    Francesco Dituri

    Full Text Available We investigated blocking the TGF-β signaling pathway in HCC using two small molecule inhibitors (LY2157299, LY2109761 and a neutralizing humanized antibody (D10 against TGF-βRII. LY2157299 and LY2109761 inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. LY2157299 inhibited HCC migration and cell growth independently of the expression levels of TGF-βRII. In contrast to LY2157299, D10 showed a reduction in pSMAD2 only after a short exposure. This study supports the use of LY2157299 in clinical trials, and presents new insights into TGF-β receptor cycling in cancer cells.

  9. Comparison of neutralizing and hemagglutination-inhibiting antibody responses to influenza A virus vaccination of human immunodeficiency virus-infected individuals

    NARCIS (Netherlands)

    Benne, CA; Harmsen, M; Tavares, L; Kraaijeveld, CA; De Jong, JC

    1998-01-01

    A neutralization enzyme immunoassay (N-EIA) was used to determine the neutralizing serum antibody titers to influenza A/Taiwan/1/86 (H1N1) and Beijing/353/89 (H3N2) viruses after vaccination of 51 human immunodeficiency virus (HIV) type 1-infected individuals and 10 healthy noninfected controls agai

  10. The potential role of functional inhibition of T regulatory cells by anti-TGFβ antibody in photodynamic therapy of renal cancer

    Science.gov (United States)

    Mroz, Pawel; Hamblin, Michael R.

    2011-03-01

    Photodynamic therapy (PDT) has been shown to be an effective locally ablative anti-cancer treatment that has the additional advantage of inducing tumor-directed immune response. We hypothesized that PDT could be combined with anti-transforming growth factor (TGF) beta antibody that does not significantly affect the population of cytotoxic T lymphocytes (CTL) but at the same time, has the potential to decrease the immunosuppressive effects of regulatory T-cells (Treg) mediated by TGF beta. This hypothesis was tested with aTGF-beta antibody combined with BPD-mediated PDT in a BALB/c renal cell carcinoma model. Evidence of positive benefits of the combination therapy over individual treatments alone was obtained.

  11. Human Immunodeficiency Virus Type 1 Escape from Cyclotriazadisulfonamide-Induced CD4-Targeted Entry Inhibition Is Associated with Increased Neutralizing Antibody Susceptibility▿

    Science.gov (United States)

    Vermeire, Kurt; Van Laethem, Kristel; Janssens, Wouter; Bell, Thomas W.; Schols, Dominique

    2009-01-01

    Continuous specific downmodulation of CD4 receptor expression in T lymphocytes by the small molecule cyclotriazadisulfonamide (CADA) selected for the CADA-resistant human immunodeficiency virus type 1 (HIV-1) NL4.3 virus containing unique mutations in the C4 and V5 regions of gp120, likely stabilizing the CD4-binding conformation. The amino acid changes in Env were associated with decreased susceptibility to anti-CD4 monoclonal antibody treatment of the cells and with higher susceptibility of the virus to soluble CD4. In addition, the acquired ability of a CADA-resistant virus to infect cells with low CD4 expression was associated with an increased susceptibility of the virus to neutralizing antibodies from sera of several HIV-1-infected patients. PMID:19570853

  12. VIRAL ANTIBODIES IN PRESCHOOL CHILDREN

    Directory of Open Access Journals (Sweden)

    S. Saidi

    1974-08-01

    Full Text Available One hundred sera from children 1 - 6 years of age, representative of a large serum collection, were tested for the prevalence of antibodies against different viruses. Hemagglutination-inhibition (HI antibodies were found in 68% for measles; 61 % for rubella; 75'% for influenza A2/Hong Kong/68, 16% for influenza B/Md./59, 0% for group A arboviruses, 10% for group B arboviruses, 3% for phlebotomus fever group and 4% for Congo-Crimean hemorrhagic fever (C-CHF group of arboviruses Poliomyelitis-neutralizing antibodies for type 1, 2 and 3 were 90%; 85% and 84%~ respectively. Antibody to EH virus was detected in 84% of the sera by immuno-fluorescence. None of the sera were positive for hepatitis-B antigen or antibody by immuno-precipitation test. The prevalence of some viral antibodies found in this survey are compared with results obtained from surveys in other parts of the country.

  13. Structural basis of LaDR5, a novel agonistic anti-death receptor 5 (DR5 monoclonal antibody, to inhibit DR5/TRAIL complex formation

    Directory of Open Access Journals (Sweden)

    Qiao Chunxia

    2012-07-01

    Full Text Available Abstract Background As a member of the TNF superfamily, TRAIL could induce human tumor cell apoptosis through its cognate death receptors DR4 or DR5, which can induce formation of the death inducing signaling complex (DISC and activation of the membrane proximal caspases (caspase-8 or caspase-10 and mitochondrial pathway. Some monoclonal antibodies against DR4 or DR5 have been reported to have anti-tumor activity. Results In this study, we reported a novel mouse anti-human DR5 monoclonal antibody, named as LaDR5, which could compete with TRAIL to bind DR5 and induce the apoptosis of Jurkat cells in the absence of second cross-linking in vitro. Using computer-guided molecular modeling method, the 3-D structure of LaDR5 Fv fragment was constructed. According to the crystal structure of DR5, the 3-D complex structure of DR5 and LaDR5 was modeled using molecular docking method. Based on distance geometry method and intermolecular hydrogen bonding analysis, the key functional domain in DR5 was predicted and the DR5 mutants were designed. And then, three mutants of DR5 was expressed in prokaryotic system and purified by affinity chromatograph to determine the epitope of DR5 identified by LaDR5, which was consistent with the theoretical results of computer-aided analysis. Conclusions Our results demonstrated the specific epitope located in DR5 that plays a crucial role in antibody binding and even antineoplastic bioactivity. Meanwhile, revealed structural features of DR5 may be important to design or screen novel drugs agonist DR5.

  14. Blockade of the interleukin-2 receptor by anti-Tac antibody inhibits the generation of antigen-nonspecific suppressor T cells in vitro.

    OpenAIRE

    Oh-Ishi, T; Goldman, C K; Misiti, J; Waldmann, T. A.

    1988-01-01

    The role of interleukin 2 (IL-2) in the activation of suppressor T cells was investigated by using the monoclonal antibody anti-Tac, which blocks the binding of IL-2 to the 55-kDa peptide of the high-affinity IL-2 receptor. Anti-Tac was added to an antigen-nonspecific suppressor system in which Con A-induced suppressor T cells were generated during a preculture period, and their effects on immunoglobulin production were assessed in second, indicator cultures containing pokeweed mitogen and pe...

  15. Antibody to Heat Shock Protein 70 (HSP70) Inhibits Human T-Cell Lymphoptropic Virus Type I (HTLV-I) Production by Transformed Rabbit T-Cell Lines

    OpenAIRE

    Hanan Fallouh; Wahib Mahana

    2012-01-01

    Adult T cell leukemia is a fatal malignant transformation caused by the human T-cell lymphoptropic virus type I (HTLV-I). HTLV-I is only associated with the development of this disease in a small percentage of infected individuals. Using two rabbit transformed T-cell lines; RH/K30 (asymptomatic) and RH/K34 (leukemogenic), we have investigated the expression of heat shock proteins (HSP) 90 and 70 and the role of anti-HSPs antibodies on virus production. HSPs surface expression was higher on RH...

  16. Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4.

    Directory of Open Access Journals (Sweden)

    Samuele E Burastero

    Full Text Available To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.

  17. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization

    DEFF Research Database (Denmark)

    Hansen, J E; Clausen, H; Nielsen, C;

    1990-01-01

    ), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 125I-labeled gp120. The MAbs therefore define...

  18. Bovine Colostrum Contains Immunoglobulin G Antibodies against Intimin, EspA, and EspB and Inhibits Hemolytic Activity Mediated by the Type Three Secretion System of Attaching and Effacing Escherichia coli▿

    Science.gov (United States)

    Vilte, Daniel A.; Larzábal, Mariano; Cataldi, Ángel A.; Mercado, Elsa C.

    2008-01-01

    Enterohemorrhagic Escherichia coli (EHEC) is the main cause of hemolytic-uremic syndrome, an endemic disease in Argentina which had an incidence in 2005 of 13.9 cases per 100,000 children younger than 5 years old. Cattle appear to be a major reservoir of EHEC, and a serological response to EHEC antigens has been demonstrated in natural and experimental infections. In the current study, antibodies against proteins implicated in EHEC's ability to form attaching and effacing lesions, some of which are exported to the host cell via a type three secretion system (TTSS), were identified in bovine colostrum by Western blot analysis. Twenty-seven (77.0%) of the 35 samples examined contained immunoglobulin G (IgG) antibodies against the three proteins assayed in this study: EspA, EspB, and the carboxy-terminal 280 amino acids of γ-intimin, an intimin subtype associated mainly with O157:H7 and O145:H- serotypes. Every colostrum sample was able to inhibit, in a range between 45.9 and 96.7%, the TTSS-mediated hemolytic activity of attaching and effacing E. coli. The inhibitory effect was partially mediated by IgG and lactoferrin. In conclusion, we found that early colostrum from cows contains antibodies, lactoferrin, and other unidentified substances that impair TTSS function in attaching and effacing E. coli strains. Bovine colostrum might act by reducing EHEC colonization in newborn calves and could be used as a prophylactic measure to protect non-breast-fed children against EHEC infection in an area of endemicity. PMID:18562563

  19. Monoclonal Antibody 16D10 to the C-Terminal Domain of the Feto-Acinar Pancreatic Protein Binds to Membrane of Human Pancreatic Tumoral SOJ-6 Cells and Inhibits the Growth of Tumor Xenografts

    Directory of Open Access Journals (Sweden)

    Laurence Panicot-Dubois

    2004-11-01

    Full Text Available Feto-acinar pancreatic protein (FAPP characterized by mAbJ28 reactivity is a specific component associated with ontogenesis and behaves as an oncodevelopment-associated antigen. We attempted to determine whether pancreatic tumoral SOJ-6 cells are expressed at their surface FAPP antigens and to examine if specific antibodies directed against these FAPP epitopes could decrease the growth of pancreatic tumors in a mice model. For this purpose, we used specific antibodies against either the whole FAPP, the O-glycosylated C-terminal domain, or the N-terminal domain of the protein. Our results indicate that SOJ-6 cells expressed at their surface a 32-kDa peptide corresponding to the C-terminal domain of the FAPP. Furthermore, we show, by using endoproteinase Lys-C or geldanamycin, a drug able to impair the FAPP secretion, that this 32-kDa peptide expressed on the SOJ-6 cell surface comes from the degradation of the FAPP. Finally, an in vivo prospective study using a preventative tumor model in nude mice indicates that targeting this peptide by the use of mAb16D10 inhibits the growth of SOJ-6 xenografts. The specificity of mAb16D10 for pancreatic tumors and the possibility to obtain recombinant structures of mucin-like peptides recognized by mAb16D10 and mAbJ28 are promising tools in immunologic approaches to cure pancreatic cancers.

  20. Inhibition of IL-10 production by maternal antibodies against Group B Streptococcus GAPDH confers immunity to offspring by favoring neutrophil recruitment.

    Directory of Open Access Journals (Sweden)

    Pedro Madureira

    2011-11-01

    Full Text Available Group B Streptococcus (GBS is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10 by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab'(2 fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/- mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a

  1. Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available The receptor tyrosine kinase (RTK ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL. There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8 induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC and antibody dependent cellular cytotoxicity (ADCC. The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375 including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

  2. Simplified fluorescent inhibition microtest for the titration of rabies neutralizing antibodies Microteste de inibição de fluorescência simplificado para titulação de anticorpos antirábicos

    Directory of Open Access Journals (Sweden)

    S.R. Favoretto

    1993-04-01

    Full Text Available A simplified fluorescence inhibition microtest (SFIMT was standardized for the evaluation of antirabies serum neutralizing antibodies based on the rapid fluorescent focus inhibition test (RFFIT and the fluorescence inhibition microtest (FIMT. The simplified test showed reproductibility similar to that of the FIMT with advantages as easier executation and quicker reading. A simple pre-treatment of Brazilian microplates produced for immune enzymatic assays (PROSIL gave equivalent results and substantial coast reduction, in relation to imported plates (DIFCO. The simplified test can be easily implemented in less sophisticated laboratories, as alternative to the mouse serum neutralization test, still the most largely employed in Brazil, or even to others as RFFIT and FIMT.No presente trabalho foi padronizada uma microtécnica simplificada para a avaliação de anticorpos neutralizantes anti-rábicos com base no RFFIT e no FIMT. Este teste mostrou reprodutibilidade comparável a dos testes originais, sendo de execução mais simples e de leitura mais rápida. Uma microplaca de fabricação nacional para testes imuno-enzimáticos (PROSIL Ind. e Com. pode ser utilizada com resultados equivalentes aos de microplacas Difco ou similar, com substancial redução de custos, mediante tratamento simples e rápido. O teste estudado pode ser implantado facilmente em laboratórios menos sofisticados substituindo com grandes vantagens a prova de soro-neutralização em camundongos ainda a mais empregada rotineiramente no Brasil.

  3. Anti-EphA2 Antibodies Decrease EphA2 Protein Levels in Murine CT26 Colorectal and Human MDA-231 Breast Tumors But Do Not Inhibit Tumor Growth

    Directory of Open Access Journals (Sweden)

    David Kiewlich

    2006-01-01

    Full Text Available The EphA2 receptor tyrosine kinase has been shown to be over-expressed in cancer and a monoclonal antibody (mAb that activates and down-modulates EphA2 was reported to inhibit the growth of human breast and lung tumor xenografts in nude mice. Reduction of EphA2 levels by treatment with anti-EphA2 siRNA also inhibited tumor growth, suggesting that the anti-tumor effects of these agents are mediated by decreasing the levels of EphA2. As these studies employed human tumor xenograft models in nude mice with reagents whose crossreactivity with murine EphA2 is unknown, we generated a mAb (Ab20 that preferentially binds, activates, and induces the degradation of murine EphA2. Treatment of established murine CT26 colorectal tumors with Ab20 reduced EphA2 protein levels to ~12% of control tumor levels, yet had no effect on tumor growth. CT26 tumor cell colonization of the lung was also not affected by Ab20 administration despite having barely detectable levels of EphA2. We also generated and tested a potent agonistic mAb against human EphA2 (1G9-H7. No inhibition of human MDA-231 breast tumor xenograft growth was observed despite evidence for >85% reduction of EphA2 protein levels in the tumors. These results suggest that molecular characteristics of the tumors in addition to EphA2 over-expression may be important for predicting responsiveness to EphA2-directed therapies.

  4. Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains.

    Science.gov (United States)

    Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo; Komiya, Eriko; Dang, Nam H; Iwao, Noriaki; Ohnuma, Kei; Morimoto, Chikao

    2016-05-20

    CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulating the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis.

  5. 抗 Eppin 抗体抑制人精子活性运动及相关机制%Inhibition of sperm activated motility by the anti-Eppin antibody and the potential mechanisms

    Institute of Scientific and Technical Information of China (English)

    龙燕; 李庆; 顾爱华

    2013-01-01

    目的:阐明抗附睾蛋白酶抑制剂( Eppin )抗体抑制人精子动力及分析相关机制。方法:将正常男性精子经过percoll分选后分为7组:空白对照组,抗体对照组与40μg · ml -1抗兔多克隆抗体孵育,其余5组分别与不同浓度抗Eppin抗体(浓度分别为2.5、5、10、20和40μg · ml -1)共同孵育4 h后进行精子动力测定,线粒体膜电位(Δψ)、ATP浓度及电镜镜检检测。结果:随着抗Eppin抗体浓度的增加,精子活力下降并呈浓度依赖趋势;当抗Eppin抗体浓度超过5μg · ml-1,精子Δψ及产生ATP浓度显著下降( P<0.05);部分精子线粒体正常环形结构发生异常改变,亚线粒体膜变薄甚至消失。结论:抗Eppin抗体与人精子共孵育后精子线粒体功能及超微结构异常,导致精子活性运动显著下降。%Objective:To clarify the inhibition of sperm activated motility by the anti-Eppin antibody and the potential mechanisms .Methods: Semen samples were obtained by masturbation from normal donors .The motile sperm were collected through discontinuous percoll gradient centrifugation and divided into 7 groups .The sperm in one group were incubated with the 40 μg· ml -1 anti-rabbit antibody and other five groups were incubated with the 2.5, 5, 10, 20 or 40 μg · ml-1 anti-Eppin antibody .Results: After the sperm treated with the anti-Eppin antibody , the results of computer-assisted semen analysis , mitochondrial membrane potential (Δψ) together with ATP production assays measurement and the images of electron microscopy show the reduction of the activated motility,Δψand ATP production , and the aberrations of the helical pattern of mitochondria with the partial loss of sub-mitochondrial reticulum .Conclusion: After the human sperm incubated with the anti-Eppin antibody , the aberrations in ultra structure and the functions of the mitochondria of the sperm result in the reduction of the sperm

  6. The anti-TNF-α antibody infliximab inhibits the expression of fat-transporter-protein FAT/CD36 in a selective hepatic-radiation mouse model.

    Science.gov (United States)

    Martius, Gesa; Cameron, Silke; Rave-Fränk, Margret; Hess, Clemens F; Wolff, Hendrik A; Malik, Ihtzaz A

    2015-01-01

    Previously, we reported a radiation-induced inflammation triggering fat-accumulation through fatty-acid-translocase/cluster of differentiation protein 36 (FAT/CD36) in rat liver. Furthermore, inhibition of radiation-induced FAT/CD36-expression by anti-tumor necrosis factor-α (anti-TNF-α) (infliximab) was shown in vitro. The current study investigates fat-accumulation in a mouse-model of single-dose liver-irradiation (25-Gray) and the effect of anti-TNF-α-therapy on FAT/CD36 gene-expression. Mice livers were selectively irradiated in vivo in presence or absence of infliximab. Serum- and hepatic-triglycerides, mRNA, and protein were analyzed by colorimetric assays, RT-PCR, Immunofluorescence and Western-Blot, respectively. Sudan-staining was used demonstrating fat-accumulation in tissue. In mice livers, early (1-3 h) induction of TNF-α-expression, a pro-inflammatory cytokine, was observed. It was followed by elevated hepatic-triglyceride level (6-12 h), compared to sham-irradiated controls. In contrast, serum-triglyceride level was decreased at these time points. Similar to triglyceride level in mice livers, Sudan staining of liver cryosections showed a quick (6-12 h) increase of fat-droplets after irradiation. Furthermore, expression of fat-transporter-protein FAT/CD36 was increased at protein level caused by radiation or TNF-α. TNF-α-blockage by anti-TNF-α showed an early inhibition of radiation-induced FAT/CD36 expression in mice livers. Immunohistochemistry showed basolateral and cytoplasmic expression of FAT/CD36 in hepatocytes. Moreover, co-localization of FAT/CD36 was detected with α-smooth muscle actin (α-SMA+) cells and F4/80+ macrophages. In summary, hepatic-radiation triggers fat-accumulation in mice livers, involving acute-phase-processes. Accordingly, anti-TNF-α-therapy prevented early radiation-induced expression of FAT/CD36 in vivo. PMID:25739082

  7. Insulin-like growth factor-I receptor (IGF-IR) targeting with monoclonal antibody cixutumumab (IMC-A12) inhibits IGF-I action in endometrial cancer cells.

    Science.gov (United States)

    Attias-Geva, Zohar; Bentov, Itay; Ludwig, Dale L; Fishman, Ami; Bruchim, Ilan; Werner, Haim

    2011-07-01

    Specific insulin-like growth factor-I receptor (IGF-IR) targeting emerged in recent years as a promising therapeutic strategy in cancer. Endometrial cancer is the most common gynaecological cancer in the Western world. The aim of this study was to evaluate the potential of cixutumumab (IMC-A12, ImClone Systems), a fully human monoclonal antibody against the IGF-IR, to inhibit IGF-I-mediated biological actions and cell signalling events in four endometrial carcinoma-derived cell lines (ECC-1, Ishikawa, USPC-1 and USPC-2). Our results demonstrate that cixutumumab was able to block the IGF-I-induced autophosphorylation of the IGF-IR. In addition, the PI3K and MAPK downstream signalling pathways were also inactivated by cixutumumab in part of the cell lines. Prolonged (24h and 48h) exposures to cixutumumab reduced IGF-IR expression. Furthermore, confocal microscopy of GFP-tagged receptors shows that cixutumumab treatment led to IGF-IR redistribution from the cell membrane to the cytoplasm. Antiapoptotic effects were evaluated by cleavage of caspase 3 and PARP, and mitogenicity and transformation by proliferation and cell cycle assays. Results obtained showed that cixutumumab abrogated the IGF-I-stimulated increase in proliferation rate, and increased caspase-3 and PARP cleavage, two markers of apoptosis. Of importance, cixutumumab had no effect neither on insulin receptor (IR) expression nor on IGF-I activation of IR. In summary, in a cellular model of endometrial cancer cixutumumab was able to inhibit the IGF-I-induced activation of intracellular cascades, apoptosis and proliferation.

  8. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M.

    2013-04-16

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  9. Anti-influenza M2e antibody

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M. (Santa Fe, NM)

    2011-12-20

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  10. A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines.

    Directory of Open Access Journals (Sweden)

    Lanying Du

    Full Text Available An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS, is caused by a novel coronavirus (MERS-CoV. It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588 in the truncated receptor-binding domain (RBD: residues 367-606 of MERS-CoV spike (S protein fused with human IgG Fc fragment (S377-588-Fc is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4, the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients' lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.

  11. Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVβ3 ectodomain-17E6 Fab complex.

    Science.gov (United States)

    Mahalingam, Bhuvaneshwari; Van Agthoven, Johannes F; Xiong, Jian-Ping; Alonso, José Luis; Adair, Brian D; Rui, Xianliang; Anand, Saurabh; Mehrbod, Mehrdad; Mofrad, Mohammad R K; Burger, Christa; Goodman, Simon L; Arnaout, M Amin

    2014-05-16

    The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.

  12. Mechanism of human antibody-mediated neutralization of Marburg virus.

    Science.gov (United States)

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. PMID:25723164

  13. 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

  14. 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

  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. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  17. Immunocytochemical and Immunohistochemical Staining with Peptide Antibodies.

    Science.gov (United States)

    Friis, Tina; Pedersen, Klaus Boberg; Hougaard, David; Houen, Gunnar

    2015-01-01

    Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/adsorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions, and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

  18. Polyclonal anti-idiotypic antibodies mimicking the small cell lung carcinoma antigen cluster-5A interact with a panel of antibodies and induce specific immune response in animals.

    OpenAIRE

    Zwicky, C.; Stahel, R A; Jaksche, H.; Waibel, R.; Lehmann, H. P.; Loibner, H

    1991-01-01

    Polyclonal anti-idiotypic antibodies (ab2) were generated by immunising goats with the murine IgG2a monoclonal antibody SWA20 which recognises the SCLC antigen cluster-5A, a tumour-associated sialoglycoprotein. Ab2 was shown to bind specifically to antibody SWA20, but not to isotype matched control antibodies. Pre-incubation with ab2 completely inhibited target cell binding of antibody SWA20 and of four other antibodies to cluster-5A antigen, while no effect was seen with antibodies to cluste...

  19. 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

  20. Analysis of hemagglutination inhibition antibody level in patients with influenza A H1N1%甲型H1N1流感患者血清流感病毒血凝抑制抗体滴度变化分析

    Institute of Scientific and Technical Information of China (English)

    沈宏辉; 毛远丽; 侯俊; 赵敏; 徐军; 白冰珂; 赵静; 胡燕; 貌盼勇; 李伯安

    2012-01-01

    目的 了解甲型H1N1流感患者发病后体内流感病毒特异性血凝抑制抗体滴度变化.方法 采集28例甲型H1N1流感患者发病后不同时间的血清,采用血凝抑制试验测定其抗体滴度并分析.结果 患者距发病1、5、15、22、37、49天和58天的血凝抑制抗体滴度分别为5.36、9.39、39.02、57.99、137.92、55.19和57.99,患者的最高抗体滴度几何均数为148.55,其中96.4%( 27/28)的患者在病程中产生保护性抗体和发生抗体血清转换.结论 甲型H1N1流感患者能有效产生保护性水平的抗体,有较好的免疫反应.%Objective To understand the hemagglutination inhibition antibody level in patients with influenza A H1N1.Methods Sera from 28 patients with influenza A H1N1 at different time points after illness onset were collected and measured by hemagglutination inhibition assay.Results The serum hemagglutination inhibition antibody tilers at 1,5,15,22,37,49 and 58 days after illness onset were 5.36、9.39、39.02、57.99、137.92、55.19 and 57.99 respectively.The top geometric mean tiler of hemagglutination inhibition antibody was 148.55.The antibody seroconversion rate and seroprotection rate were occurred in 96.4% (27/28)of patients.Conclusion The patients with influenza A H1N1 have effective immune response.

  1. The determination of rubella hemaogglutination inhibition antibodies protective concentration with RV-IgG standard%利用RV-IgG标准品对风疹疫苗免疫后血清血凝抑制抗体保护浓度的测定

    Institute of Scientific and Technical Information of China (English)

    赵冠棋; 程喆歆; 陈曦; 王燕; 徐维祯; 王华庆; 谷鸿喜

    2012-01-01

    目的 利用风疹病毒的IgG标准品(RV-IgG)进行定量ELISA试验和血凝抑制试验,以测定风疹疫苗免疫后血清血凝抑制(HI)抗体的保护浓度,并验证试剂盒的准确性.方法 RV-IgG标准品和10对风疹疫苗免疫前后血清的定量ELISA试验;风疹病毒血凝素的粗提以及血凝试验;RV-IgG标准品的血凝抑制试验测定其血凝完全被抑制时RV-IgG浓度.结果 将RV-IgG标准品稀释为8个不同浓度梯度做定量ELISA,所测得的浓度值与已知浓度相比无统计学意义(P >0.05);10对风疹疫苗免疫前后人血清抗体浓度进行比较,免疫后血清抗体浓度显著升高(t =2.151,P<0.05);风疹病毒血凝素的凝集效价为1∶128,RV-IgG标准品血凝抑制效价为1∶32,对应的HI抗体浓度为25 IU/ml.结论 实验结果证实血清中风疹病毒HI抗体保护浓度为25 IU/ml;风疹疫苗初次免疫后血清抗体保护性阳转率可达到80%.%Objective To determine the rubella hemaogglutination inhibition (HI) antibodies concentration in persons with rubella vaccine immune serum by RV-IgG standard-based quantitative ELISA and HI test,and verify the accuracy of the kit.Methods RV-IgG standard which was diluted to eight different concentrations,and 10 pairs of serum samples before and after immunized were used to detected the rubella virus serum antibody by quantitative ELISA test,respectively.The hemagglutination tests were performed with rubella virus haemagglutinin which were crudely extracted.The hemagglutination inhibition tests were respectively proceeded with RV-IgG standard and serum samples.After the comparison of the results,rubella antibody protective concentration was confirmed.Results There was no significant difference ( t =2.151,P > 0.05 ) between the measured concentration and the known concentration of RV-IgG standard diluted by the quantitative ELISA test.Comparing 10 pairs of the serum antibody level before and after vaccination,the concentration of

  2. A survey of the prevalence of penicillin-specific IgG, IgM and IgE antibodies detected by ELISA and defined by hapten inhibition, in patients with suspected penicillin allergy and in healthy volunteers.

    OpenAIRE

    Christie, G.; Coleman, J W; Newby, S; McDiarmaid-Gordon, A; Hampson, J P; Breckenridge, A M; Park, B.K.

    1988-01-01

    1. IgG, IgM and IgE anti-benzylpenicilloyl (BPO) antibody activities were determined by enzyme-linked immunosorbent assay (ELISA) in sera from 100 patients who claimed to be allergic to penicillin, and from 50 healthy volunteers. Continuous frequency distributions for all three classes of anti-BPO antibody, defined as differential binding (delta OD) to BPO-human serum albumin (HSA) and HSA, were obtained for both groups. 2. For IgM and IgE classes the anti-BPO activities were slightly but sta...

  3. Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

    International Nuclear Information System (INIS)

    Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL was comparable to that of 125I-oPRL by unlabeled oPRL, while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82

  4. Studies on Purification of Methamidophos Monoclonal Antibodies and Comoarative Immunoactivity of Purified Antibodies

    Institute of Scientific and Technical Information of China (English)

    SU-QING ZHAO; YUAN-MING SUN; CHUN-YAN ZHANG; XIAO-YU HUANG; HOU-RUI ZHANG; ZHEN-YU ZHU

    2003-01-01

    Objective To purify Methamidophos (Met) monoclonal antibodies with two methods andcompare immune activity of purified antibodies. Method Caprylic acid ammonium sulphateprecipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method wereused to purify Met monoclonal antibodies, UV spectrum scanning was used to determine proteincontent and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linkedimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mLand 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASPmethod. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was181.26 μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03 μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29 ug/mL and 3.42 μg/mL,respectively. Conclusion Antibodies purified by SPA method are better than those by CAASPmethod, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelledtesting paper for analyzing Met residue in vegetable and drink water.

  5. Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody%抗人精子膜蛋白抗体1对小鼠精子顶体反应和精子-透明带结合的抑制作用

    Institute of Scientific and Technical Information of China (English)

    G.Y.Cheng; J.L.Shi; M.Wang; Y.Q.Hu; C.M.Liu; Y.F.Wang; C.Xu

    2007-01-01

    Aim:To investigate the possible functions of human sperm membrane protein (hSMP-1) in the process of fertilization.Methods: A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 (anti-rhSMP-1) antibodies. Sperm acrosome reaction and spermzona pellucida (ZP) binding assay were carried out in 10-week-old BALB/c mice. Results: Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 (P < 0.05). Conclusion: The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

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

    NARCIS (Netherlands)

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

    1984-01-01

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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Hirai M

    2002-06-01

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

  9. Cross-domain inhibition of TACE ectodomain

    DEFF Research Database (Denmark)

    Tape, Christopher J; Willems, Sofie H; Dombernowsky, Sarah L;

    2011-01-01

    target for the treatment of cancer and arthritis. Prior attempts to antagonize cell-surface TACE activity have focused on small-molecule inhibition of the metalloprotease active site. Given the highly conserved nature of metalloprotease active sites, this paradigm has failed to produce a truly specific...... individual antibody variable domains to desired epitopes. The resulting "cross-domain" human antibody is a previously undescribed selective TACE antagonist and provides a unique alternative to small-molecule metalloprotease inhibition....

  10. 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

  11. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  12. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  13. Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine production: inhibition by a non-anaphylactogenic anti-IgE antibody

    OpenAIRE

    1996-01-01

    Elevated levels of immunoglobulin (Ig) E are associated with bronchial asthma, a disease characterized by eosinophilic inflammation of the airways. Activation of antigen-specific T helper (Th) 2 cells in the lung with the subsequent release of interleukin (IL) 4 and IL-5 is believed to play an important role in the pathogenesis of this disease. In this study, we have used a non-anaphylactogenic anti-mouse-IgE antibody to investigate the relationship between IgE, airway eosinophil infiltration...

  14. Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

    DEFF Research Database (Denmark)

    Owens, T; Fazekas de St Groth, B

    1987-01-01

    The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4...... the formation of TCR complexes and so prevent activation. However, by increasing the epitope density of the activating ligand, the avidity of the T cell/ligand interaction can be increased sufficiently to prevent this disruption.(ABSTRACT TRUNCATED AT 400 WORDS)...

  15. Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7

    DEFF Research Database (Denmark)

    Clausen, Mads Hartvig; Willats, William George Tycho; Knox, J. Paul

    2003-01-01

    A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens...

  16. Anti-human CD73 monoclonal antibody inhibits metastasis formation in human breast cancer by inducing clustering and internalization of CD73 expressed on the surface of cancer cells

    DEFF Research Database (Denmark)

    Terp, Mikkel G; Olesen, Kristina A; Christensen, Eva Arnspang;

    2013-01-01

    -linking of CD73, because both whole IgG anti-CD73 AD2 mAb and Fab' fragments thereof exhibited this effect. Ex vivo treatment of different breast cancer cell lines with anti-CD73 AD2 mAb before i.v. injection into mice inhibited extravasation/colonization of circulating tumor cells and significantly reduced...

  17. Rational Design of CXCR4 Specific Antibodies with Elongated CDRs

    Science.gov (United States)

    2015-01-01

    The bovine antibody (BLV1H12) which has an ultralong heavy chain complementarity determining region 3 (CDRH3) provides a novel scaffold for antibody engineering. By substituting the extended CDRH3 of BLV1H12 with modified CXCR4 binding peptides that adopt a β-hairpin conformation, we generated antibodies specifically targeting the ligand binding pocket of CXCR4 receptor. These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range. In addition, they inhibit SDF-1-dependent signal transduction and cell migration in a transwell assay. Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a Kd of 0.9 nM. This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future. PMID:25041362

  18. 禽蛋卵黄抗体检测中影响血凝抑制试验结果的因素分析%Analysis of Factors Influencing the Hemagglutination Inhibition Test in the Detection Process of Egg Yolk Antibody

    Institute of Scientific and Technical Information of China (English)

    庄金秋; 梅建国; 王艳

    2014-01-01

    血凝抑制试验是检测有血凝性的家禽疾病抗体效价最常用的一种方法,具有简便、快速、灵敏、准确等特点,已被广泛应用于各兽医检测机构和基层养禽场,但是由于试验操作过程中的不规范操作和细节注意不够,导致不正确检测结果出现的现象时有发生,笔者结合工作实践现将禽蛋卵黄抗体检测过程中影响血凝抑制试验结果的影响因素进行分析归纳总结,以期能对生产应用提供参考。%Hemagglutination inhibition test was widely-used laboratory methods for determination of antibody levels of poultry disease, and has the advantages of simple protocle, rapid, sensitive and accurate, used genera1ly in anima1 husbandry practice, are effected by many factors. So the testing process is not standardized operation and attention to detail is not enough, cause incorrect results have occurred from time to time. This paper mainIy discussed the infIuencing factors by combining the working practice of the factors affecting the hemagglutination inhibition test results of egg yolk antibody detection in the analysis are summarized as follows,in order to provide reference for clinical application.

  19. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud;

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen-...... for combating HER2+ breast cancer. © 2013 by Tabriz University of Medical Sciences.......Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen......-binding fragments in cancer targeting and therapy. VHHs express low immunogenicity, are highly robust and easy to manufacture and have the ability to recognize hidden or uncommon epitopes. We highlight the utility of VHH in design of new molecular, multifunctional particulate and immune cell-based systems...

  20. Engineering antibody therapeutics.

    Science.gov (United States)

    Chiu, Mark L; Gilliland, Gary L

    2016-06-01

    The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. PMID:27525816

  1. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    Full Text Available BACKGROUND: Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

  2. RBC Antibody Screen

    Science.gov (United States)

    ... the baby is Rh-positive and the mother's antibody status is negative for anti-D, the mother is given additional RhIG. This test also may be used to help diagnose autoimmune-related hemolytic anemia ... when a person produces antibodies against his or her own RBC antigens. This ...

  3. Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. I. Complement-mediated bactericidal activity of antibodies to a serum susceptible strain of E. coli of the serotype O 111.

    Science.gov (United States)

    Reiter, B; Brock, J H

    1975-01-01

    Bovine colostral whey diulted in unheated bovine milk or in Kolmer saline containing 5 per cent pre-colostral calf serum was bactericidal for Escherichia coli NCTC 9703 (serotype O 111). Whey diluted in saline without pre-colostral calf serum was inactive. Bactericidal activity was abolished by heating at 56 degrees for 30 minutes or by pre-incubating with N-acetyl-L-tyrosine ethylester, indicating that complement present in milk or pre-colostral calf serum was involved in the bactericidal activity. Undiluted colostral whey was inactive even in the presence of up to 20 per cent pre-colostral calf serum, probably due to a prozone effect. Milk heated at 80 degrees for 10 minutes was also shown to have a limited anti-complementary effect. Post-colostral milk from some cows had bactericidal activity, whereas similar milks from other cows were either inactive or active only after addition of pre-colostral calf serum, indicating that antibodies to E. coli or complement or both may or may not be present in the post-colostral milk of different cows. The implications of these findings in relation to the bacterial flora of the intestinal tract of neonates are discussed. PMID:1090521

  4. Selection of Recombinant Human Antibodies.

    Science.gov (United States)

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  5. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

  6. 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...

  7. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    Science.gov (United States)

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects.

  8. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    Science.gov (United States)

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects. PMID:24293819

  9. Detection of antitrophoblast antibodies in the sera of patients with anticardiolipin antibodies and fetal loss.

    Science.gov (United States)

    McCrae, K R; DeMichele, A M; Pandhi, P; Balsai, M J; Samuels, P; Graham, C; Lala, P K; Cines, D B

    1993-11-01

    Women with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with ACLA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P trophoblast cells. In most cases, sera from which ACLA were adsorbed by cardiolipin-containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an approximately 62-kD protein from the trophoblast cell surface, stimulated the release of arachidonic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with ACLA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise. PMID:7693045

  10. Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays.

    Science.gov (United States)

    Neal, Frances; Arnold, Joanne; Rossant, Christine J; Podichetty, Sadhana; Lowne, David; Dobson, Claire; Wilkinson, Trevor; Colley, Caroline; Howes, Rob; Vaughan, Tristan J

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets. PMID:26450103

  11. HIV Antibody Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? HIV Antibody and HIV Antigen (p24) Share this page: Was this page helpful? Also known as: HIV Screening Tests; AIDS Test; AIDS Screen; HIV Serology; ...

  12. Antinuclear antibody panel

    Science.gov (United States)

    ... blood may be due to: Chronic liver disease Collagen vascular disease Drug-induced lupus erythematosus Myositis (inflammatory muscle disease) ... Saunders; 2011:chap 51. Read More Antibody Arthritis Collagen vascular disease Drug-induced lupus erythematosus Liver disease Scleroderma Systemic ...

  13. Anti-cartilage antibody.

    Science.gov (United States)

    Greenbury, C L; Skingle, J

    1979-08-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change.

  14. Antibody tumor penetration

    Science.gov (United States)

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue. PMID:18541331

  15. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  16. DNA repair and DNA antibodies during experimental mycoplasma arthritis

    International Nuclear Information System (INIS)

    To clarify the pathogenesis of a mycoplasma induced arthritis in rats, investigations were carried out on the influence of mycoplasma infection on DNA repair and the occurrence of DNA antibodies. During acute and subacute stage of the experimentally induced arthritis an inhibition of DNA repair could be observed. Besides the results indicated a correlation between reduced or inhibited DNA repair and the appearance of DNA antibodies could be found. The DNA-repair behaviour after the mycoplasma infection was compared with the influence of γ-irradiation

  17. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi;

    2014-01-01

    for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

  18. Engineering antibodies for cancer therapy.

    Science.gov (United States)

    Boder, Eric T; Jiang, Wei

    2011-01-01

    The advent of modern antibody engineering has led to numerous successes in the application of these proteins for cancer therapy in the 13 years since the first Food and Drug Administration approval, which has stimulated active interest in developing more and better drugs based on these molecules. A wide range of tools for discovering and engineering antibodies has been brought to bear on this challenge in the past two decades. Here, we summarize mechanisms of monoclonal antibody therapeutic activity, challenges to effective antibody-based treatment, existing technologies for antibody engineering, and current concepts for engineering new antibody formats and antibody alternatives as next generation biopharmaceuticals for cancer treatment.

  19. Changes in the Properties of the Thyrotropin Receptor Antibody in Patients with Graves’ Disease after Radioiodine Treatment

    OpenAIRE

    Cho, Bo Youn; Shong, Young Kee; Chung, June-Key; Lee, Myung Chul; Lee, Hong Kyu; Koh, Chang-Soon; Min, Hun Ki

    1990-01-01

    We investigated the effect of a single dose of 131I upon thyrotropin receptor antibodies (TRAb) in 21 patients with Graves’ disease. The thyrotropin receptor antibodies were assessed by parallel measurements of thyrotropin binding inhibitor immunoglobulin (TBII), thyroid stimulating antibody (TSAb) and thyroid stimulation blocking antibody (TSBAb) in serum by radoreceptor assay, stimulation of adenlate cyclase and inhibition of TSH-stimulated adenylate cyclase activation in FRTL-5 cells, resp...

  20. Fab-mediated binding of drug-dependent antibodies to platelets in quinidine- and quinine-induced thrombocytopenia.

    OpenAIRE

    Christie, D J; Mullen, P C; Aster, R H

    1985-01-01

    Platelets coated with quinine- or quinidine-induced antibodies form rosettes around protein A-Sepharose beads and normal platelets form rosettes about protein A-Sepharose beads coated with these antibodies. These reactions occurred only in the presence of sensitizing drug. Platelets also formed rosettes about protein A-Sepharose beads coated with an anti-PIA1 antibody, but drug was not required. Formation of rosettes between antibody-coated platelets and protein A-Sepharose was inhibited by F...

  1. Enhancing blockade of Plasmodium falciparum erythrocyte invasion: assessing combinations of antibodies against PfRH5 and other merozoite antigens.

    Directory of Open Access Journals (Sweden)

    Andrew R Williams

    Full Text Available No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50 values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.

  2. Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I.

    Science.gov (United States)

    Shibayama, Mineko; Serrano-Luna, José de Jesús; Rojas-Hernández, Saúl; Campos-Rodríguez, Rafael; Tsutsumi, Victor

    2003-03-01

    In this work, we analyzed the in vitro interaction of human secretory immunoglobulin A (sIgA) antibodies with Naegleria fowleri trophozoites and the capacity of these antibodies to inhibit amoeba adherence to collagen type I. We also studied N. fowleri antigens that are recognized by sIgA, using immunoblot assays. Immunocytochemical analysis of the interaction showed a redistribution of antigens on the surface of trophozoites by sIgA antibodies. Ultrastructural analysis of antibody-amoeba interaction showed that besides the patching and cap formation, parasites were capable of eliminating the antigen-antibody complex produced on the surface. sIgA antibodies were capable of inhibiting the in vitro adhesion of trophozoites to collagen type I. We suggest that nonsymptomatic infections by N. fowleri may stimulate a local specific immunity that prevents trophozoite adhesion and invasion of nasal mucosa.

  3. Inhibition of breast cancer cell proliferation and migration by monoclonal antibody m590 specific for insulin-like growth factor receptor type Ⅰ%抗胰岛素样生长因子Ⅰ型受体单克隆抗体m590抑制乳腺癌细胞增生和迁移

    Institute of Scientific and Technical Information of China (English)

    符晓阳; 陈超; 潘华雄; 黄韬; 章美云; 郑鸿

    2012-01-01

    Objective To investigate the mechanism(s) that a human-mouse chimeric monoclonal anti-insulin-like growth factor receptor type Ⅰ (IGF-IR) antibody,m590,inhibits breast cancer cells proliferation and migration.Methods Binding of m590 to IGF-IR in 30 breast cancer samples was detected by using immunohistochemistry.Western blotting,immunocytofluorescence and other tests were used for analyzing the effects of m590 on breast cancer cells ( MCF-7 ) proliferation,survival,adhesion and migration as well as signaling pathways.Results m590 specifically bound to IGF-IR was overexpressed in invasive ductal carcinoma of the breast (87% of cases).Treatment of MCF-7 cells with m590 significantly suppressed IGF-I-induced phosphorylation of ERK and Akt,which led to reduced cell proliferation by 58%and increased apoptosis.m590 also inhibited IGF-Ⅰ-induced polymerization of F-actin and relocation of vinculin in cell edge,resulting in dramatically decreased cell migration by 56% and cell adhesion by 55%.Furthermore,herceptin in combination with m590 synergistically inhibited IGF-IR-induced phosphorylation of ERK and AKT,and decreased MCF-7 cells proliferation by 76% as compared with IGF-I treatment alone.Conclusion m590 is an effective anti-IGF-IR antibody and may have the potential in clinical use for diagnosis and treatment of breast cancer.%目的 探讨抗胰岛素样生长因子Ⅰ型受体(IGF-IR)抗体,m590,抑制乳腺癌细胞增生、迁移及其机制.方法 免疫组织化学染色30例乳腺浸润性导管癌标本;免疫印迹法、免疫荧光细胞染色及其他试验检测m590 对MCF-7细胞的作用及其机制.结果 87%的乳腺浸润性导管癌高表达IGF-IR;m590抑制胰岛素样生长因子Ⅰ(IGF-I)诱导的细胞外调节蛋白激酶(ERK)和蛋白激酶B(Akt)磷酸化以及细胞骨架蛋白(F-actin)和细胞骨架黏合班蛋白(Vinculin)在细胞内的分布,使细胞增生、迁移和黏附分别降低至58%、56%和55

  4. Maternal antibodies: clinical significance, mechanism of interference with immune responses, and possible vaccination strategies

    Directory of Open Access Journals (Sweden)

    Stefan eNiewiesk

    2014-09-01

    Full Text Available Neonates have an immature immune system which cannot adequately protect against infectious diseases. Early in life, immune protection is accomplished by maternal antibodies transferred from mother to offspring. However, decaying maternal antibodies inhibit vaccination as is examplified by the inhibition of seroconversion after measles vaccination. This phenomenon has been described in both human and veterinary medicine and is independent of the type of vaccine being used. This review will discuss the use of animal models for vaccine research. I will review clinical solutions for inhibition of vaccination by maternal antibodies, and the testing and development of potentially effective vaccines. These are based on new mechanistic insight about the inhibitory mechanism of maternal antibodies. Maternal antibodies inhibit the generation of antibodies whereas the T cell response is usually unaffected. B cell inhibition is mediated through a cross-link between B-cell receptor (BCR with the Fcg receptor IIB (FcgRIIB by a vaccine-antibody complex. In animal experiments, this inhibition can be partially overcome by injection of a vaccine-specific monoclonal IgM antibody. IgM stimulates the B-cell directly through cross-linking the BCR via complement protein C3d and antigen to the complement receptor 2 (CR2 signaling complex. In addition, it was shown that interferon alpha binds to the CD21 chain of CR2 as well as the interferon receptor and that this dual receptor usage drives B cell responses in the presence of maternal antibodies. In lieu of immunizing the infant the concept of maternal immunization as a strategy to protect neonates has been proposed. This approach would still not solve the question of how to immunize in the presence of maternal antibodies but would defer the time of infection to an age where infection might not have such a detrimental outcome as in neonates. I will review successful examples and potential challenges of implementing

  5. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate......-2. Based on the presented data we suggest that affinity maturation of the model antibody proceeds through multiple incremental steps of subtle improvements. We moreover conclude that the best affinity improved candidates are likely to be obtained through optimization of both the antigen...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  6. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    1979-03-15

    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  7. Pulmonary biology of anti-interleukin 5 antibodies

    Directory of Open Access Journals (Sweden)

    Egan RW

    1997-01-01

    Full Text Available Interleukin 5 (IL-5 is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulate in the lungs during asthma. We have studied anti IL-5 antibodies on allergic responses in mice, guinea pigs and monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single dose of 0.3 mg/kg, i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, a humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 did not cause immunosuppression in guinea pigs. Studies with this antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

  8. Competency development in antibody production in cancer cell biology

    Energy Technology Data Exchange (ETDEWEB)

    Park, M.S.

    1998-12-01

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

  9. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  10. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  11. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

    NARCIS (Netherlands)

    Dolk, E.; Vaart, M. van der; Lutje Hulsik, D.; Vriend, G.; Haard, H. de; Spinelli, S.; Cambillau, C.; Frenken, L.; Verrips, T.

    2005-01-01

    As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain

  12. Analysis of Antibodies Directed against Merozoite Surface Protein 1 of the Human Malaria Parasite Plasmodium falciparum

    Science.gov (United States)

    Woehlbier, Ute; Epp, Christian; Kauth, Christian W.; Lutz, Rolf; Long, Carole A.; Coulibaly, Boubacar; Kouyaté, Bocar; Arevalo-Herrera, Myriam; Herrera, Sócrates; Bujard, Hermann

    2006-01-01

    The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria. PMID:16428781

  13. Analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Woehlbier, Ute; Epp, Christian; Kauth, Christian W; Lutz, Rolf; Long, Carole A; Coulibaly, Boubacar; Kouyaté, Bocar; Arevalo-Herrera, Myriam; Herrera, Sócrates; Bujard, Hermann

    2006-02-01

    The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.

  14. Human oxidation-specific antibodies reduce foam cell formation and atherosclerosis progression

    DEFF Research Database (Denmark)

    Tsimikas, Sotirios; Miyanohara, Atsushi; Hartvigsen, Karsten;

    2011-01-01

    We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibod...... antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis....

  15. Treatment of Antibody-Mediated Rejection in Kidney Transplantation

    Directory of Open Access Journals (Sweden)

    Magdalena Durlik

    2013-07-01

    Full Text Available Antibody-mediated rejection (AMR is a relatively rare but severe complication in kidney transplantation associated with increased risk of graft loss. Diagnosis of acute and chronic AMR is based on typical histological hallmarks, deposition of C4d in peritubular capillaries and presence of donor-specific antibodies (DSA. Many novel and attractive treatment options have become available in recent years: antibody removal and production inhibition (plasmapheresis, IVIg, B cell depletion (rituximab, plasma cell depletion and apoptosis (bortezomib, and complement activation inhibition (eculizumab. Standard therapy is based on PP and IVIg. Preliminary results with new agents are encouraging but require randomised clinical trials and long-term follow-up.

  16. Detection of B-lymphocytes secreting antibodies to Dermatophagoides antigens

    DEFF Research Database (Denmark)

    Sparholt, S H; Barington, T; Schou, C

    1991-01-01

    An enzyme-linked immunospot assay (ELI-spot assay) has been established to count individual cells secreting antibodies to Dermatophagoides spp. allergens. Initial optimization of the assay was performed using Der p I-specific murine hybridoma cell lines. Inhibition with soluble purified allergen ...

  17. Antibody to porcine parvovirus in warthog (Phacochoerus aethiopicus).

    Science.gov (United States)

    Thomson, G R; Peenze, I

    1980-03-01

    Haemagglutination inhibiting antibody to porcine parvovirus was shown to be widespread in all but one of the warthog populations sampled from South Africa and Zimbabwe Rhodesia. In some instances titres as high as greater than or equal to 1/20 000 were detected. PMID:7454234

  18. A new tool for plant cell biology: in vivo antibody uptake in plant protoplasts.

    Science.gov (United States)

    Brière, C; Barthou, H; Petitprez, M

    2004-07-01

    We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.

  19. 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...

  20. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  1. Hemagglutinin inhibition assay with swine sera

    Science.gov (United States)

    Hemagglutination is based on the ability of certain viruses to agglutinate red blood cells (RBC) of certain animal species by formation of cross-linking lattices between RBC. Antibodies that have the ability to inhibit the hemagglutination property of influenza A viruses are generally thought to pro...

  2. Lupus anticoagulants and antiphospholipid antibodies

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000547.htm Lupus anticoagulants and antiphospholipid antibodies To use the sharing features on this page, please enable JavaScript. Lupus anticoagulants are antibodies against substances in the lining ...

  3. Recombinant antibodies and tumor targeting

    OpenAIRE

    Sheikholvaezin, Ali

    2006-01-01

    Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates. In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice. One hybridoma, producing the monoclonal antibody ...

  4. CXCR4单克隆抗体抑制乳腺癌MCF-7细胞裸鼠移植瘤的实验研究%CXCR4 monoclonal antibody inhibits the growth of MCF-7 breast cancer xenograft in nude mice

    Institute of Scientific and Technical Information of China (English)

    杜成; 谢晓冬

    2012-01-01

    Objective:To investigate the effect of CXCR4 monoclonal antibody( CXCR4 mAb )on the growth of human MCF - 7 breast cancer xenograft subcutaneously implanted in nude mice and to explore the antitumor mechanism of CXCR4 mAb. Methods: 8 - week - old Balb/c femal nude mice were adopted to establish a subcutaneously tumor- bearing model. Treating animals with CXCR4 mAb. The inhibitory effects of CXCR4 mAb on tumor growth were assessed after treatment. Immunohistochemical staining was performed to examine the expressions of PCNA,Caspase- 3 and VEGF. Results: CXCR4 mAb significantly inhibited the growth of breast cancer xenograft in nude mice. Inhibition ratio of tumor volum reached 71. 4% . After treatment, CXCR4 mAb markedly downregulated the expressions of PCNA and VEGF while upregulated the expression of Caspase - 3. Conclusion: CXCR4 mAb might inhibit breast cancer growth in vivo via the induction of cancer cell apoptosis and inhibition of cancer cell proliferation and tumor angiogenesis.%目的 研究CXC趋化因子受体4(CXC chemokine receptor 4,CXCR4)单克隆抗体(CXCR4 mAb)对人乳腺癌MCF-7细胞裸鼠皮下移植瘤生长的影响,并初步探讨CXCR4 mAb抗肿瘤的作用机制.方法 采用8周龄Balb/c雌性裸鼠,建立乳腺癌MCF-7细胞裸鼠皮下移植瘤模型.运用CXCR4 mAb进行干预,从整体水平观察CXCR4 mAb对肿瘤生长的影响,采用免疫组织化学法检测肿瘤组织中增殖细胞核抗原(PCNA)、半胱氨酸天冬氨酸酶3(Caspase-3)和血管内皮细胞生长因子(VEGF)的表达情况.结果 CXCR4 mAb可明显抑制移植瘤的生长,瘤体抑制率达到71.4%;CXCR4 mAb治疗后的肿瘤组织中PCNA和VEGF表达明显下降,而Caspase-3表达上升.结论 CXCR4 mAb可能是通过抑制肿瘤细胞增殖、促进肿瘤细胞凋亡及抑制肿瘤血管形成而发挥抗肿瘤生长的作用.

  5. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  6. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    Science.gov (United States)

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed. PMID:25264572

  7. 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...

  8. Fcγ receptor-mediated inflammation inhibits axon regeneration.

    Directory of Open Access Journals (Sweden)

    Gang Zhang

    Full Text Available Anti-glycan/ganglioside antibodies are the most common immune effectors found in patients with Guillain-Barré Syndrome, which is a peripheral autoimmune neuropathy. We previously reported that disease-relevant anti-glycan autoantibodies inhibited axon regeneration, which echo the clinical association of these antibodies and poor recovery in Guillain-Barré Syndrome. However, the specific molecular and cellular elements involved in this antibody-mediated inhibition of axon regeneration are not previously defined. This study examined the role of Fcγ receptors and macrophages in the antibody-mediated inhibition of axon regeneration. A well characterized antibody passive transfer sciatic nerve crush and transplant models were used to study the anti-ganglioside antibody-mediated inhibition of axon regeneration in wild type and various mutant and transgenic mice with altered expression of specific Fcγ receptors and macrophage/microglia populations. Outcome measures included behavior, electrophysiology, morphometry, immunocytochemistry, quantitative real-time PCR, and western blotting. We demonstrate that the presence of autoantibodies, directed against neuronal/axonal cell surface gangliosides, in the injured mammalian peripheral nerves switch the proregenerative inflammatory environment to growth inhibitory milieu by engaging specific activating Fcγ receptors on recruited monocyte-derived macrophages to cause severe inhibition of axon regeneration. Our data demonstrate that the antibody orchestrated Fcγ receptor-mediated switch in inflammation is one mechanism underlying inhibition of axon regeneration. These findings have clinical implications for nerve repair and recovery in antibody-mediated immune neuropathies. Our results add to the complexity of axon regeneration in injured peripheral and central nervous systems as adverse effects of B cells and autoantibodies on neural injury and repair are increasingly recognized.

  9. 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.

  10. 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

  11. Engineering antibodies by yeast display.

    Science.gov (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  12. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)

    吴竞生

    2008-01-01

    @@ Antiphospholipid antibodies (APA) APA is a big category for all kinds of negative charge phospholipid or lecithin - a protein complex autoantibodies or the same antibody, through its recognition of antigen (target protein) different, and phospholipids or lecithin - protein complex combination of various rely on the interference Phospholipid clotting and anti-coagulation factor, and promote endothelial cells, platelets, complement activation and play a role. APA including lupus anticoagulant(LA) and anticardiolipin antibody (ACA), In addition, there are anti-β2 glycoprotein-I (β2-GPI) antibody, anti-prothrombin (a- PT) antibody, anti-lysophosphatidic acid antibody and anti-phosphatidylserine antibody, and so on. APA as the main target of phospholipid-binding protein, including β2-GPI, prothrombin, annexin, protein C (PC) and protein S (PS), plasminogen, and so on.

  13. The use of combinations of monoclonal antibodies in clinical oncology.

    Science.gov (United States)

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

    2015-12-01

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

  14. Demonstration onf anticollagen antibodies in rheumatoid arthritis synovial fluids by 14C-radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Menzel, J.; Steffen, C.; Kolarz, G.; Kojer, M.; Smolen, J.

    1978-03-01

    Twenty-seven synovial fluids from rheumatoid arthritis (RA) patients and 17 synovial fluids from controls were investigated in a new radioimmunoassay for anticollagen antibodies. In vitro labeled human 14C-collagen of type I in native or denatured state was used as antigen. Passive hemagglutination was used in comparison. Parameters for defining positive results in radioimmunoassay were evaluated on the basis of control synovial fluids. Synovial fluids from 20 RA patients (74%) showed antibodies to denatured collagen; synovial fluids from 8 RA patients (30%) also demonstrated antibodies to native collagen. Control fluids of posttraumatic effusions were negative; among the other controls synovial fluid from 1 psoriatic arthritis patient reacted positively. Inhibition experiments showed that antibodies to denatured collagen cross-reacted with native collagen. Inhibition with human denatured type III collagen displayed strong cross-reactivity of anti-type I collagen antibodies with type III collagen.

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-01-01

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

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

    Science.gov (United States)

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

    1988-01-01

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

  18. Comparisons of the effect of naturally acquired maternal pertussis antibodies and antenatal vaccination induced maternal tetanus antibodies on infant's antibody secreting lymphocyte responses and circulating plasma antibody levels.

    Science.gov (United States)

    Ahmad, Shaikh Meshbahuddin; Alam, Jahangir; Afsar, Nure Alam; Huda, Nazmul; Kabir, Yearul; Qadri, Firdausi; Raqib, Rubhana; Stephensen, Charles B

    2016-04-01

    The goal of this study was to explore the effects of trans-placental tetanus toxoid (TT) and pertussis (PT) antibodies on an infant's response to vaccination in the context of antenatal immunization with tetanus but not with pertussis. 38 mothers received a single dose of TT vaccine during pregnancy. Infants received tetanus and pertussis vaccines at 6, 10 and 14 wk of age. TT and PT anti-IgG secretion by infant lymphocytes was measured at 15 wk. Plasma antibodies were measured at 6 wk (pre-vaccination), 15 wk and 1 y of age. Prior to vaccination, TT and PT antibody were detected in 94.6% and 15.2% of infants. At 15 wk anti-TT-IgG and anti-PT-IgG in plasma was increased by 7-9 fold over pre-vaccination levels, while at 1 y plasma anti-TT-IgG was decreased by approximately 5-fold from the peak and had returned to near the pre-vaccination level. At 1 y plasma anti-PT-IgG was decreased by 2-fold 1 yfrom the 15 wk level. However, 89.5% and 82.3% of infants at 1 y had protective levels of anti-TT and anti-PT IgG, respectively. Pre-vaccination plasma IgG levels were associated with lower vaccine-specific IgG secretion by infant lymphocytes at 15 wk (p < 0.10). This apparent inhibition was seen for anti-TT-IgG at both 15 wk (p < 0.05) and t 1 y (p < 0.10) of age. In summary, we report an apparent inhibitory effect of passively derived maternal antibody on an infants' own antibody response to the same vaccine. However, since the cut-off values for protective titers are low, infants had protective antibody levels throughout infancy. PMID:27176823

  19. Prevalence of mycoplasma antibodies in lesser prairie-chicken sera.

    Science.gov (United States)

    Hagen, Christian A; Crupper, Scott S; Applegate, Roger D; Robel, Robert J

    2002-01-01

    Serologic testing by the serum plate agglutination (SPA) procedure was performed to detect the presence of cross-reacting antibodies to Mycoplasma meleagridis, Mycoplasma synoviae, and Mycoplasma gallisepticum in lesser prairie-chickens (Tympanuchus pallidicinctus) trapped over a 2-yr period in Finney and Kearny counties of southwestern Kansas. Sera examined from birds (n = 50) obtained in March-April 2000 tested positive for M meleagridis, M. synoviae, and M. gallisepticum at levels of 6%, 10%, and 10%, respectively, for the population examined. Mycoplasma meleagridis antibodies were detected in 3 samples (2.7%), M. synoviae antibodies in 2 samples (1.7%), and M. gallisepticum antibodies in 3 samples (2.7%) from birds (n = 112) collected in March-April 2001. Data obtained by SPA can result in false positives and should be verified by additional procedures such as the hemagglutination-inhibition test. Low amounts of sera prohibited this additional testing. Thus, the positive SPA results should be considered presumptive for the presence of Mycoplasma antibodies. Although Mycoplasma antibodies have been detected in wild turkeys (Meleagris gallopavo) from Kingman and Butler counties in Kansas, this report is the first of possible mycoplasmosis in Finney and Kearny counties, Kansas. All birds testing positive by this procedure should be considered as potential carriers of Mycoplasma and should not be used in relocation efforts.

  20. Strong antitumor activities of IgG3 antibodies to a human melanoma-associated ganglioside

    Energy Technology Data Exchange (ETDEWEB)

    Hellstroem, I.; Brankovan, V.; Hellstroem, K.E.

    1985-03-01

    Three mouse monoclonal IgG3 antibodies, 2B2, IF4, and MG-21, recognize a G/sub D3/ ganglioside antigen that is expressed at the cell surface of most human melanomas. All three antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro when tested with human lymphocytes or effector cells in a 2-hr or 4-hr /sup 51/Cr-release test, and one antibody, MG-21, also gives strong complement-dependent cytotoxicity with human serum. Antibody 2B2, which gives ADDC also in the presence of mouse lymphocytes, inhibited the outgrowth of a human melanoma in nude mice, but antibody IF4, which showed no ADCC with mouse lymphocyte effectors, did not.

  1. The antibody Hijikata Tatsumi

    Directory of Open Access Journals (Sweden)

    Éden Peretta

    2012-11-01

    Full Text Available Considered one of the most influential modern dance representatives in Japan, Tatsumi Hijikata’s work was a milestone in the Japanese post-war experimental artistic scene. Heretic son of his time, he staged a fertile mix of artistic and cultural influences, overlapping subversive elements of European arts and philosophy with radical references from pre-modern Japanese culture. In this way he built the foundations of its unstable antibody, its political-artistic project of dissolution of a organism, both physical and social.

  2. Metrics for antibody therapeutics development

    OpenAIRE

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approv...

  3. Empowered Antibody Therapies - IBC conference.

    Science.gov (United States)

    Herold, Jens

    2010-10-01

    The Empowered Antibody Therapies conference, held in Burlingame, CA, USA, included topics covering new therapeutic developments in the field of multispecific antibodies. This conference report highlights selected presentations on DVD-Igs from Abbott Laboratories, ImmTACs from Immunocore, 'Dock-and-Lock' technology from Immunomedics, the bispecific BiTE antibody blinatumomab from Micromet, and Triomabs from TRION Pharma and Fresenius Biotech. PMID:20878591

  4. 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

  5. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Science.gov (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function. PMID:26205082

  6. Antineutrophil cytoplasmic antibodies

    Directory of Open Access Journals (Sweden)

    G.D. Sebastiani

    2011-06-01

    Full Text Available Antineutrophil cytoplasmic antibodies (ANCA are predominantly IgG autoantibodies directed against constituents of primary granules of neutrophils and monocytes’ lysosomes. Although several antigenic targets have been identified, those ANCA directed to proteinase 3 or myeloperoxidase are clinically relevant, whereas the importance of other ANCA remains unknown. Both are strongly associated with small vessel vasculitides, the ANCA-associated vasculitides, which include Wegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome, and the localised forms of these diseases (eg, pauci-immune necrotising and crescentic glomerulonephritis. ANCA is a useful serological test to assist in diagnosis of small-vessel vasculitides. 85-95% of patients with Wegener’s granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. There is increasing evidence that myeloperoxidase- ANCA are directly involved in the pathogenesis of necrotizing vasculitis. This is less clear for proteinase 3-ANCA, markers for Wegener’s granulomatosis. With respect to proteinase 3-ANCA, complementary proteinase 3, a peptide translated from the antisense DNA strand of proteinase 3 and homologous to several microbial peptides, may be involved in induction of proteinase 3-antineutrophil cytoplasmic autoantibodies.

  7. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity.

    OpenAIRE

    Forsayeth, J R; Caro, J F; Sinha, M K; Maddux, B A; Goldfine, I D

    1987-01-01

    Three mouse monoclonal antibodies were produced that reacted with the alpha subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate ...

  8. Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α

    Directory of Open Access Journals (Sweden)

    Ceran Ceyhan

    2012-10-01

    Full Text Available Abstract Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α. Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells

  9. A peptide mimic blocks the cross-reaction of anti-DNA antibodies with glomerular antigens.

    Science.gov (United States)

    Xia, Y; Eryilmaz, E; Der, E; Pawar, R D; Guo, X; Cowburn, D; Putterman, C

    2016-03-01

    Anti-DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross-reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti-DNA antibodies to self-antigens is isotype-dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti-DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9-11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti-DNA IgG isotypes to glomerular antigens. The PL9-11 panel of IgG anti-DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12-mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme-linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or 'ALW') which binds to all PL9-11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9-11 anti-DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti-DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti-DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti-DNA antibody binding to renal tissue.

  10. Different binding of stimulatory-type and blocking-type TSH receptor antibody with guinea-pig testis membrane.

    Science.gov (United States)

    Inui, T; Ochi, Y; Hachiya, T; Chen, W; Nakajima, Y; Kajita, Y; Ogura, H

    1991-11-01

    A receptor assay using [125I]bTSH-binding to guinea-pig testis membrane was developed. Unlabelled hCG and FSH inhibited [125I]bTSH binding. In patients with Graves' disease and in untreated hyperthyroid patients, almost all long-acting thyroid stimulators and thyroid-stimulating antibodies, respectively did not inhibit [125I]bTSH binding, which on the other hand was inhibited by thyroid stimulation blocking antibodies in patients with primary hypothyroidism. When the inhibitory effect on the binding of [125I]hCG and 125I-synthetic alpha-subunit peptide (alpha 26-46) of hCG to testis membrane was examined, bTSH resulted in a significant inhibition. However, all three kinds of TSH receptor antibodies had no inhibitory effect. This study demonstrated 1. interaction of alpha-subunit of TSH and hCG with the testicular receptor; 2. binding of thyroid stimulation-blocking antibody and lack of binding of thyroid-stimulating antibody to the testicular TSH receptor in spite of binding of these TSH receptor antibodies to the thyroidal TSH receptor, and 3. lack of binding of thyroid-stimulating antibody and thyroid stimulation-blocking antibody to the testicular gonadotropin receptor. PMID:1684686

  11. Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

    International Nuclear Information System (INIS)

    Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. The authors have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG/sub 2b/, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter

  12. Development and Optimization of High-Throughput Methods To Measure Plasmodium falciparum-Specific Growth Inhibitory Antibodies

    OpenAIRE

    Persson, Kristina E. M.; Lee, Chee T.; Marsh, Kevin; Beeson, James G.

    2006-01-01

    Antibodies that inhibit replication of Plasmodium falciparum in erythrocytes are thought to be important both in acquired immunity to malaria and as mediators of immunity generated by candidate blood-stage vaccines. However, several constraints have limited the study of these functional antibodies in population studies and vaccine trials. We report the development and optimization of high-throughput growth inhibition assays with improved sensitivity that use minimal volumes of test serum. The...

  13. Carbohydrate Biopolymers Enhance Antibody Responses to Mucosally Delivered Vaccine Antigens

    Science.gov (United States)

    Bacon, A.; Makin, J.; Sizer, P. J.; Jabbal-Gill, I.; Hinchcliffe, M.; Illum, L.; Chatfield, S.; Roberts, M.

    2000-01-01

    We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 μg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1). PMID:10992483

  14. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening.

    Science.gov (United States)

    Li, Yan; Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D; Hiraga, Kaori

    2015-08-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  15. Targeting of Antibodies using Aptamers

    OpenAIRE

    Missailidis, Sotiris

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  16. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptide...

  17. Pathogenic role of antiphospholipid antibodies

    NARCIS (Netherlands)

    Salmon, J. E.; de Groot, P. G.

    2008-01-01

    The antiphospholipid antibody syndrome (APS) is characterized by recurrent arterial and venous thrombosis and/or pregnancy in association with antiphospholipid (aPL) antibodies. The pathogenic mechanisms in APS that lead to in vivo injury are incompletely understood. Recent evidence suggests that AP

  18. Metrics for antibody therapeutics development.

    Science.gov (United States)

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed. PMID:20930555

  19. Metrics for antibody therapeutics development.

    Science.gov (United States)

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

  20. Anti-plasminogen antibodies compromise fibrinolysis and associate with renal histology in ANCA-associated vasculitis.

    Science.gov (United States)

    Berden, Annelies E; Nolan, Sarah L; Morris, Hannah L; Bertina, Rogier M; Erasmus, Dianhdra D; Hagen, E Christiaan; Hayes, Donal P; van Tilburg, Nico H; Bruijn, Jan A; Savage, Caroline O S; Bajema, Ingeborg M; Hewins, Peter

    2010-12-01

    Antibodies recognizing plasminogen, a key component of the fibrinolytic system, associate with venous thrombotic events in PR3-ANCA vasculitis. Here, we investigated the prevalence and function of anti-plasminogen antibodies in independent UK and Dutch cohorts of patients with ANCA-associated vasculitis (AAV). We screened Ig isolated from patients (AAV-IgG) and healthy controls by ELISA. Eighteen of 74 (24%) UK and 10/38 (26%) Dutch patients with AAV had anti-plasminogen antibodies compared with 0/50 and 1/61 (2%) of controls. We detected anti-plasminogen antibodies in both PR3-ANCA- and MPO-ANCA-positive patients. In addition, we identified anti-tissue plasminogen activator (tPA) antibodies in 13/74 (18%) patients, and these antibodies were more common among patients with anti-plasminogen antibodies (P = 0.011). Eighteen of 74 AAV-IgG (but no control IgG) retarded fibrinolysis in vitro, and this associated with anti-plasminogen and/or anti-tPA antibody positivity. Only 4/18 AAV-IgG retarded fibrinolysis without harboring these antibodies; dual-positive samples retarded fibrinolysis to the greatest extent. Patients with anti-plasminogen antibodies had significantly higher percentages of glomeruli with fibrinoid necrosis (P < 0.05) and cellular crescents (P < 0.001) and had more severely reduced renal function than patients without these antibodies. In conclusion, anti-plasminogen and anti-tPA antibodies occur in AAV and associate with functional inhibition of fibrinolysis in vitro. Seropositivity for anti-plasminogen antibodies correlates with hallmark renal histologic lesions and reduced renal function. Conceivably, therapies that enhance fibrinolysis might benefit a subset of AAV patients.

  1. Effects of tau domain-specific antibodies and intravenous immunoglobulin on tau aggregation and aggregate degradation.

    Science.gov (United States)

    Esteves-Villanueva, Jose O; Trzeciakiewicz, Hanna; Loeffler, David A; Martić, Sanela

    2015-01-20

    Tau pathology, including neurofibrillary tangles, develops in Alzheimer's disease (AD). The aggregation and hyperphosphorylation of tau are potential therapeutic targets for AD. Administration of anti-tau antibodies reduces tau pathology in transgenic "tauopathy" mice; however, the optimal tau epitopes and conformations to target are unclear. Also unknown is whether intravenous immunoglobulin (IVIG) products, currently being evaluated in AD trials, exert effects on pathological tau. This study examined the effects of anti-tau antibodies targeting different tau epitopes and the IVIG Gammagard on tau aggregation and preformed tau aggregates. Tau aggregation was assessed by transmission electron microscopy and fluorescence spectroscopy, and the binding affinity of the anti-tau antibodies for tau was evaluated by enzyme-linked immunosorbent assays. Antibodies used were anti-tau 1-150 ("D-8"), anti-tau 259-266 ("Paired-262"), anti-tau 341-360 ("A-10"), and anti-tau 404-441 ("Tau-46"), which bind to tau's N-terminus, microtubule binding domain (MBD) repeat sequences R1 and R4, and the C-terminus, respectively. The antibodies Paired-262 and A-10, but not D-8 and Tau-46, reduced tau fibrillization and degraded preformed tau aggregates, whereas the IVIG reduced tau aggregation but did not alter preformed aggregates. The binding affinities of the antibodies for the epitope for which they were specific did not appear to be related to their effects on tau aggregation. These results confirm that antibody binding to tau's MBD repeat sequences may inhibit tau aggregation and indicate that such antibodies may also degrade preformed tau aggregates. In the presence of anti-tau antibodies, the resulting tau morphologies were antigen-dependent. The results also suggested the possibility of different pathways regulating antibody-mediated inhibition of tau aggregation and antibody-mediated degradation of preformed tau aggregates. PMID:25545358

  2. Molecular mechanisms of antibody-mediated neutralisation of flavivirus infection.

    Science.gov (United States)

    Pierson, Theodore C; Diamond, Michael S

    2008-01-01

    Flaviviruses are a group of positive-stranded RNA viruses that cause a spectrum of severe illnesses globally in more than 50 million individuals each year. While effective vaccines exist for three members of this group (yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses), safe and effective vaccines for several other flaviviruses of clinical importance, including West Nile and dengue viruses, remain in development. An effective humoral immune response is critical for protection against flaviviruses and an essential goal of vaccine development. The effectiveness of virus-specific antibodies in vivo reflects their capacity to inhibit virus entry and spread through several mechanisms, including the direct neutralisation of virus infection. Recent advances in our understanding of the structural biology of flaviviruses, coupled with the use of small-animal models of flavivirus infection, have promoted significant advances in our appreciation of the factors that govern antibody recognition and inhibition of flaviviruses in vitro and in vivo. In this review, we discuss the properties that define the potency of neutralising antibodies and the molecular mechanisms by which they inhibit virus infection. How recent advances in this area have the potential to improve the development of safe and effective vaccines and immunotherapeutics is also addressed. PMID:18471342

  3. Preparation of Anti-Idiotypic Antibody against Avian Influenza Virus Subtype H9

    Institute of Scientific and Technical Information of China (English)

    BaoquanLi; JunPeng; ZhongxiangNiu; XunheYin; FaxiaoLiu

    2005-01-01

    To generate monoclonal anti-idiotypic antibodies (mAb2) against avian influenza virus subtype H9 (H9 AIV), BALB/c mice were immunized with purified chicken anti-H9-AIV IgG and the splenocytes of immunized mice were fused with myeloma cells NS-1. Hybridoma cells were screened by indirect enzyme-linked immunosorbent assays with both chicken and rabbit anti-H9-AIV IgG as coating antigens. One hybridoma cell clone secreting monoclonal antibody against idiotypes shared by both chicken and rabbit anti-H9-AIV IgG was established. Experiments demonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AIV IgG and to induce chickens to generate hemagglutination inhibition antibodies, indicating this anti-species-sharing-idiotypic antibody bore the internal image of hemagglutinin on avian influenza virus. Cellular & Molecular Immunology. 2005;2(2):155-157.

  4. Preparation of Anti-Idiotypic Antibody against Avian Influenza Virus Subtype H9

    Institute of Scientific and Technical Information of China (English)

    Baoquan Li; Jun Peng; Zhongxiang Niu; Xunhe Yin; Faxiao Liu

    2005-01-01

    To generate monoclonal anti-idiotypic antibodies (mAb2) against avian influenza virus subtype H9 (H9 AIV),BALB/c mice were immunized with purified chicken anti-H9-AIV IgG and the splenocytes of immunized mice were fused with myeloma cells NS-1. Hybridoma cells were screened by indirect enzyme-linked immunosorbent assays with both chicken and rabbit anti-H9-AIV IgG as coating antigens. One hybridoma cell clone secreting monoclonal antibody against idiotypes shared by both chicken and rabbit anti-H9-AIV IgG was established. Experiments demonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AIV IgG and to induce chickens to generate hemagglutination inhibition antibodies, indicating this anti-species-sharing-idiotypic antibody bore the internal image of hemagglutinin on avian influenza virus. Cellular & Molecular Immunology. 2005;2(2):155-157.

  5. 题目:抗CD69单克隆抗体对哮喘小鼠的气道炎症抑制作用研究%Anti-CD69 monoclonal antibody treatment inhibits airway inflammation in a mouse model of asthma

    Institute of Scientific and Technical Information of China (English)

    Hui-ying WANG; Yu DAI; Jiao-li WANG; Xu-yan YANG; Xin-guo JIANG

    2015-01-01

    目的:气道慢性炎症和高反应性是哮喘最主要的病理生理表现,CD69是一种与免疫细胞活化、增殖及凋亡密切相关的共刺激分子,本研究旨在观察抗CD69单克隆抗体在哮喘小鼠模型中抑制气道炎症和高反应性的作用及机制探讨。创新点:(1)与大多数关于哮喘的药物研究多为造模前提前用药以阻断炎症的进展相比,本研究将抗CD69单抗作用于气道已经形成的炎症,与临床上哮喘的治疗更接近;(2)抗CD69单抗的独特作用在于它特异性地作用于气道活化的炎症细胞,与已有的哮喘药物以糖皮质激素相比,可能的全身反应少。方法:鸡卵白蛋白致敏激发制备哮喘模型(图1),Buxco系统测试哮喘小鼠的气道高反应性(图6)。结论:抗CD69单克隆抗体可抑制哮喘小鼠已经形成的气道炎症,其作用效果与地塞米松预处理相当。本研究为临床哮喘的急性发作治疗和管理提供了新的可能靶点。%Objective: Airway inflammation and airway hyper-responsiveness (AHR) are principle pathological mani-festations of asthma. Cluster of differentiation 69 (CD69) is a well-known co-stimulatory factor associated with the activation, proliferation as well as apoptosis of immune cels. This study aims to examine the effect of anti-CD69 monoclonal antibody (mAb) on the pathophysiology of a mouse model of asthma. Methods: A murine model of oval-bumin (OVA)-induced alergic airway inflammation was used in this study. Briefly, mice were injected with 20μg chicken OVA intraperitonealy on Days 0 and 14, folowed by aerosol provocation with 1% (0.01 g/ml) OVA on Days 24, 25, and 26. Anti-CD69 mAb or isotype IgG was injected intraperitoneally after OVA chalenge; dexamethasone (DXM) was administrated either before or after OVA chalenge. AHR, mucus production, and eosinophil infiltration in the peribronchial area were examined. The levels of granulocyte

  6. Anti-CD69 monoclonal antibody treatment inhibits airway inflammation in a mouse model of asthma%题目:抗CD69单克隆抗体对哮喘小鼠的气道炎症抑制作用研究

    Institute of Scientific and Technical Information of China (English)

    Hui-ying WANG; Yu DAI; Jiao-li WANG; Xu-yan YANG; Xin-guo JIANG

    2015-01-01

    Objective: Airway inflammation and airway hyper-responsiveness (AHR) are principle pathological mani-festations of asthma. Cluster of differentiation 69 (CD69) is a well-known co-stimulatory factor associated with the activation, proliferation as well as apoptosis of immune cels. This study aims to examine the effect of anti-CD69 monoclonal antibody (mAb) on the pathophysiology of a mouse model of asthma. Methods: A murine model of oval-bumin (OVA)-induced alergic airway inflammation was used in this study. Briefly, mice were injected with 20μg chicken OVA intraperitonealy on Days 0 and 14, folowed by aerosol provocation with 1% (0.01 g/ml) OVA on Days 24, 25, and 26. Anti-CD69 mAb or isotype IgG was injected intraperitoneally after OVA chalenge; dexamethasone (DXM) was administrated either before or after OVA chalenge. AHR, mucus production, and eosinophil infiltration in the peribronchial area were examined. The levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) in bronchoalveolar lavage fluid (BALF) were also assayed as indices of airway inflammation on Day 28 folowing OVA injection. Results: Pretreatment with DXM together with anti-CD69 mAb treatment after OVA provoca-tion completely inhibited AHR, eosinophil infiltration and mucus overproduction, and significantly reduced BALF IL-5. However, treatment with DXM alone after OVA chalenge only partially inhibited AHR, eosinophil infiltration and mucus overproduction, and did not diminish BALF IL-5. Treatment with either DXM or anti-CD69 mAb did not alter the con-centration of BALF GM-CSF. Conclusions: Anti-CD69 mAb treatment inhibits established airway inflammation as effectively as DXM pretreatment. This study provides a potential alternative therapeutic opportunity for the clinical management of asthma and its exacerbation.%目的:气道慢性炎症和高反应性是哮喘最主要的病理生理表现,CD69是一种与免疫细胞活化、增殖及凋亡密切相

  7. DARPins: a true alternative to antibodies.

    Science.gov (United States)

    Stumpp, Michael T; Amstutz, Patrick

    2007-03-01

    Designed ankyrin repeat proteins (DARPins) are a promising class of non-immunoglobulin proteins that can offer advantages over antibodies for target binding in drug discovery and drug development. DARPins have been successfully used, for example, for the inhibition of kinases, proteases and drug-exporting membrane proteins. DARPins specifically targeting the cancer marker HER2 have also been generated and were shown to function in both in vitro diagnostics and in vivo tumor targeting. DARPins are ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads because of their favorable molecular properties, including small size and high stability. The low-cost production in bacteria and the rapid generation of many target-specific DARPins make the DARPin approach useful for drug discovery. Additionally, DARPins can be easily generated in multispecific formats, offering the potential to target an effector DARPin to a specific organ or to target multiple receptors with one molecule composed of several DARPins.

  8. DARPins: a true alternative to antibodies.

    Science.gov (United States)

    Stumpp, Michael T; Amstutz, Patrick

    2007-03-01

    Designed ankyrin repeat proteins (DARPins) are a promising class of non-immunoglobulin proteins that can offer advantages over antibodies for target binding in drug discovery and drug development. DARPins have been successfully used, for example, for the inhibition of kinases, proteases and drug-exporting membrane proteins. DARPins specifically targeting the cancer marker HER2 have also been generated and were shown to function in both in vitro diagnostics and in vivo tumor targeting. DARPins are ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads because of their favorable molecular properties, including small size and high stability. The low-cost production in bacteria and the rapid generation of many target-specific DARPins make the DARPin approach useful for drug discovery. Additionally, DARPins can be easily generated in multispecific formats, offering the potential to target an effector DARPin to a specific organ or to target multiple receptors with one molecule composed of several DARPins. PMID:17436550

  9. Specific inhibition of Escherichia coli ferrienterochelin uptake by a normal human serum immunoglobulin.

    OpenAIRE

    Moore, D G; Earhart, C F

    1981-01-01

    Normal human serum contains an enterochelin-specific antibody which presumably acts with transferrin to hinder iron assimilation by enterochelin-producing pathogens. This antibody can be isolated from serum by sodium sulfate fractionation or affinity chromatography by employing an enterochelin-derived ligand (2,3-dihydroxy-N-benzoyl-L-serine) attached to aminohexyl Sepharose 4B. In assays of iron uptake by whole cells, the antibody inhibited enterochelin-directed uptake but not that mediated ...

  10. Maternal Antibody Protected Chicks from Growth Retardation and Immunosuppression Induced by Early Reticuloendotheliosis Virus Infection

    Institute of Scientific and Technical Information of China (English)

    SUN Shu-hong; GUI Zhi-zhong; QU Li-xin

    2007-01-01

    To determine if the maternal antibody from breeders vaccinated with cell culture-adapted reticuloendotheliosis virus (REV) could protect chicks from early REV infection, one-day-old chicks with or without anti-REV maternal antibodies were inoculated with REV, and then their growth rates and antibody tilers to Newcastle disease virus (NDV) and avian influenza virus (AIV), after vaccination with inactivated vaccines, were compared. This study indicated that REV infection could cause growth retardation and severely inhibit immune reactions to inactivated vaccines against NDV and Avian influenza virus (AIV, H9 and H5) in one-day-old broilers without maternal antibodies specific to REV. Maternal antibody from breeders vaccinated with an attenuated REV vaccine effectively protected REV-challenged birds from growth retardation and immunosuppression on antibody reactions to NDV and AIV vaccines. Four weeks after vaccination, the HI liters to NDV, AIV-H9, and AIV-H5 in maternal antibody positive and negative groups were 3.36±2.04 versus 1.58±1.69 (P<0.01), 6.27±3.87 versus 0.71±1.60(P<0.01), and 6.72 versus 0.54±1.44(p<0.01). Maternal antibodies from breeders vaccinated with REV vaccine could successfully protect chicks from REV infection and effectively prevent REV-induced growth retardation and immunosuppression in antibody responses to NDV and AIV.

  11. IgE antibodies against snake venoms.

    Science.gov (United States)

    Alonso, A; Scavini, L M; Marino, G A; Rodríguez, S M

    1995-01-01

    A similar event was detected in the clinical records of a small group of atopic patients living in the northern provinces of Argentina, i.e., they were bitten by a snake of the Bothrops species (or yarará) during their rural activities (woodcutters, cattle-drivers and farmers). Those who were bitten twice suffered an acute episode of hives and angioedema within 15 minutes after the snake bite. The presence of specific antibodies against Bothrops alternata (Ba) extract was detected by means of RAST for IgE and Ouchterlony and Boyden for IgG. The Ouchterlony also demonstrated crossreactivity among the venoms of the Bothrops species and the positivity of the six fractions obtained by DEAE-cellulose column fractionation against the horse anti-Ba serum. The Ba antigen induced a definite inhibition of the RAST. We presume that hives and angioedema in atopic patients immediately after a second snake bite could be attributed to the presence of a specific IgE antibody against the venom, and must not be misinterpreted with the toxic effects that appear later. PMID:7551202

  12. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

    DEFF Research Database (Denmark)

    Da Roit, F.; Engelberts, P. J.; Taylor, R. P.;

    2015-01-01

    the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre......-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells...

  13. Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

    Directory of Open Access Journals (Sweden)

    Ferenc A Scheeren

    Full Text Available BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5. Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

  14. An update on antibody-based immunotherapies for Clostridium difficile infection

    Science.gov (United States)

    Hussack, Greg; Tanha, Jamshid

    2016-01-01

    Clostridium difficile continues to be one of the most prevalent hospital-acquired bacterial infections in the developed world, despite the recent introduction of a novel and effective antibiotic agent (fidaxomicin). Alternative approaches under investigation to combat the anaerobic Gram-positive bacteria include fecal transplantation therapy, vaccines, and antibody-based immunotherapies. In this review, we catalog the recent advances in antibody-based approaches under development and in the clinic for the treatment of C. difficile infection. By and large, inhibitory antibodies that recognize the primary C. difficile virulence factors, toxin A and toxin B, are the most popular passive immunotherapies under investigation. We provide a detailed summary of the toxin epitopes recognized by various antitoxin antibodies and discuss general trends on toxin inhibition efficacy. In addition, antibodies to other C. difficile targets, such as surface-layer proteins, binary toxin, motility factors, and adherence and colonization factors, are introduced in this review. PMID:27536153

  15. Effect of anti-carbohydrate antibodies on HIV infection in a monocytic cell line (U937)

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Clausen, H;

    1991-01-01

    Monoclonal antibodies (mAbs) against carbohydrate epitopes of gp120 have recently been found to inhibit HIV infection of lymphocytes in vitro thereby opening new possibilities for vaccine considerations. Antibody-dependent enhancement of infection has however come increasingly into focus....... This study therefore investigated the neutralization of HIV in a monocytic cell line (U937) using mAbs against these carbohydrate gp120-epitopes. While antibodies against one of the epitopes (AI) neutralized infection of U937 cells despite binding to the Fc-receptor, one mAb against the sialosyl-Tn epitope...... enhanced infection. This enhancement was independent of complement and could be blocked by mAb Leu3a against the CD4-receptor. The study indicated that enhancement of infection in monocytic cells can occur by the same anti-carbohydrate antibodies that neutralize infection in lymphocytes, and that antibody...

  16. 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

  17. Th2 cytokines inhibit lymphangiogenesis.

    Directory of Open Access Journals (Sweden)

    Ira L Savetsky

    Full Text Available Lymphangiogenesis is the process by which new lymphatic vessels grow in response to pathologic stimuli such as wound healing, inflammation, and tumor metastasis. It is well-recognized that growth factors and cytokines regulate lymphangiogenesis by promoting or inhibiting lymphatic endothelial cell (LEC proliferation, migration and differentiation. Our group has shown that the expression of T-helper 2 (Th2 cytokines is markedly increased in lymphedema, and that these cytokines inhibit lymphatic function by increasing fibrosis and promoting changes in the extracellular matrix. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphatic function is clear, the direct effects of Th2 cytokines on isolated LECs remains poorly understood. Using in vitro and in vivo studies, we show that physiologic doses of interleukin-4 (IL-4 and interleukin-13 (IL-13 have profound anti-lymphangiogenic effects and potently impair LEC survival, proliferation, migration, and tubule formation. Inhibition of these cytokines with targeted monoclonal antibodies in the cornea suture model specifically increases inflammatory lymphangiogenesis without concomitant changes in angiogenesis. These findings suggest that manipulation of anti-lymphangiogenic pathways may represent a novel and potent means of improving lymphangiogenesis.

  18. The antineutrophil antibody in uveitis.

    OpenAIRE

    Young, D W

    1991-01-01

    Ninety eight patients with uveitis of various types were tested for the presence of the antineutrophil antibody or ANCA by an indirect immunofluorescence method. This antibody is found in patients with diseases associated with small vessel vasculitis, including Wegener's granulomatosis and microscopic polyarteritis. Eleven true positive cases were found. A positive test was not associated with the anatomical site of the uveitis but was related to the time course of the disease. In particular ...

  19. Development of Anti-Isoproturon Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    LI Fang-shi; SUN Feng; LIU Xian-jin; CUI Heng-hua

    2007-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropyl)-3-(4-isopropylphenyl)-1-methylurca (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin(OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen,with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry.The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6×105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.0 × 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.07 mg mL-1.The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and

  20. Antibodies to watch in 2014.

    Science.gov (United States)

    Reichert, Janice M

    2014-01-01

    Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed. PMID:24284914

  1. 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)

  2. 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

  3. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo

    2014-10-09

    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http://www.biocomputing.it/tabhu CONTACT: anna.tramontano@uniroma1.it, pierpaolo.olimpieri@uniroma1.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  4. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  5. 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.

  6. The development and specificity of antiidiotypic antibodies in renal transplant recipients receiving single-donor blood transfusions.

    Science.gov (United States)

    Phelan, D L; Rodey, G E; Anderson, C B

    1989-07-01

    Multiple pretransplant sera obtained from alloimmunized renal transplant recipients were tested for the presence of antiidiotypic-like antibodies (AB2) that inhibit donor-specific HLA antibodies in the microlymphocytotoxicity assay. Fourteen patients received repetitive single-donor blood transfusions (SDT). In this patient group, sera were collected prior to each blood transfusion and prior to transplantation. Three additional patients were studied in whom prior donor-specific HLA antibodies had been lost over a period of 6 months preceding transplantation. Donor-specific AB2-like antibodies were found in the sera of 13/14 SDT patients who did not develop HLA antibodies, and in the 3 patients who had lost donor-specific HLA antibodies. All patients had received prior random blood transfusions in the year preceding the study. Five (38%) of the SDT patients had detectable donor-specific AB2 prior to the initiation of single-donor blood transfusion, presumably related to previous blood transfusions. In the remaining six SDT patients in whom complete serum sets were available, AB2 always appeared after the first blood transfusion. The specificity of HLA antibodies inhibited by AB2 was studied, and antibodies against HLA-A, -B, -C, -DR, and DQw were all identified. Thus, there was no predilection for patients to develop AB2 against locus-specific HLA gene products. This study also confirms the apparent polymorphism of putative crossreactive idiotypes. Approximately 25% of donor-specific HLA antibodies were not inhibited by relevant AB2. This study confirms and extends previous observations that alloimmunization is associated in many patients with the development of antiidiotypic-like antibodies that are capable of inhibiting the binding and cytotoxicity of HLA alloantibodies. PMID:2473550

  7. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

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

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are...

  8. Production of recombinant antibodies using bacteriophages

    OpenAIRE

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

    2014-01-01

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

  9. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  10. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Forsayeth, J.R.; Caro, J.F.; Sinha, M.K.; Maddux, B.A.; Goldfine, I.D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the ..cap alpha.. subunit of the human insulin receptor. All three both immunoprecipitated /sup 125/I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited /sup 125/I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  11. Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity

    International Nuclear Information System (INIS)

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity

  12. 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

  13. Acquisition of growth-inhibitory antibodies against blood-stage Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Fiona J McCallum

    Full Text Available BACKGROUND: Antibodies that inhibit the growth of blood-stage Plasmodium falciparum may play an important role in acquired and vaccine-induced immunity in humans. However, the acquisition and activity of these antibodies is not well understood. METHODS: We tested dialysed serum and purified immunoglobulins from Kenyan children and adults for inhibition of P. falciparum blood-stage growth in vitro using different parasite lines. Serum antibodies were measured by ELISA to blood-stage parasite antigens, extracted from P. falciparum schizonts, and to recombinant merozoite surface protein 1 (42 kDa C-terminal fragment, MSP1-42. RESULTS: Antibodies to blood-stage antigens present in schizont protein extract and to recombinant MSP1-42 significantly increased with age and were highly correlated. In contrast, growth-inhibitory activity was not strongly associated with age and tended to decline marginally with increasing age and exposure, with young children demonstrating the highest inhibitory activity. Comparison of growth-inhibitory activity among samples collected from the same population at different time points suggested that malaria transmission intensity influenced the level of growth-inhibitory antibodies. Antibodies to recombinant MSP1-42 were not associated with growth inhibition and high immunoglobulin G levels were poorly predictive of inhibitory activity. The level of inhibitory activity against different isolates varied. CONCLUSIONS: Children can acquire growth-inhibitory antibodies at a young age, but once they are acquired they do not appear to be boosted by on-going exposure. Inhibitory antibodies may play a role in protection from early childhood malaria.

  14. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    Science.gov (United States)

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  15. Selection of apoptotic cell specific human antibodies from adult bone marrow.

    Directory of Open Access Journals (Sweden)

    Caroline Grönwall

    Full Text Available Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.

  16. Quantifying homologous and heterologous antibody titre rises after influenza virus infection.

    Science.gov (United States)

    Freeman, G; Perera, R A P M; Ngan, E; Fang, V J; Cauchemez, S; Ip, D K M; Peiris, J S M; Cowling, B J

    2016-08-01

    Most influenza virus infections are associated with mild disease. One approach to estimate the occurrence of influenza virus infections in individuals is via repeated measurement of humoral antibody titres. We used baseline and convalescent antibody titres measured by haemagglutination inhibition (HI) and viral neutralization (VN) assays against influenza A(H1N1), A(H3N2) and B viruses to investigate the characteristics of antibody rises following virologically confirmed influenza virus infections in participants in a community-based study. Multivariate models were fitted in a Bayesian framework to characterize the distribution of changes in antibody titres following influenza A virus infections. In 122 participants with PCR-confirmed influenza A virus infection, homologous antibody titres rose by geometric means of 1·2- to 10·2-fold after infection with A(H1N1), A(H3N2) and A(H1N1)pdm09. Significant cross-reactions were observed between A(H1N1)pdm09 and seasonal A(H1N1). Antibody titre rises for some subtypes and assays varied by age, receipt of oseltamivir treatment, and recent receipt of influenza vaccination. In conclusion, we provided a quantitative description of the mean and variation in rises in influenza virus antibody titres following influenza virus infection. The multivariate patterns in boosting of antibody titres following influenza virus infection could be taken into account to improve estimates of cumulative incidence of infection in seroepidemiological studies. PMID:27018720

  17. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins

    Science.gov (United States)

    Sormanni, Pietro; Aprile, Francesco A.; Vendruscolo, Michele

    2015-01-01

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions. PMID:26216991

  18. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins.

    Science.gov (United States)

    Sormanni, Pietro; Aprile, Francesco A; Vendruscolo, Michele

    2015-08-11

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions.

  19. Isolation of Human Antibodies Against Hepatitis E From Phage Display Library by Metal Affinity Chromatography

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC). Methods Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis. Results His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically. Conclusion Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.

  20. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    Science.gov (United States)

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  1. Antibodies: an alternative for antibiotics?

    Science.gov (United States)

    Berghman, L R; Abi-Ghanem, D; Waghela, S D; Ricke, S C

    2005-04-01

    In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases. PMID:15844826

  2. Antibodies to watch in 2016.

    Science.gov (United States)

    Reichert, Janice M

    2016-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015. PMID:26651519

  3. Mechanisms of resistance to HER family targeting antibodies

    International Nuclear Information System (INIS)

    The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Receptor activation via ligand binding leads to downstream signaling that influence cell proliferation, angiogenesis, invasion and metastasis. Aberrant expression or activity of EGFR and HER2 have been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and breast cancer. With this, intense efforts have been made to inhibit the activity of the EGFR and HER2 by designing antibodies against the ligand binding domains (cetuximab, panitumumab and trastuzumab) or small molecules against the tyrosine kinase domains (erlotinib, gefitinib, and lapatinib). Both approaches have shown considerable clinical promise. However, increasing evidence suggests that the majority of patients do not respond to these therapies, and those who show initial response ultimately become refractory to treatment. While mechanisms of resistance to tyrosine kinase inhibitors have been extensively studied, resistance to monoclonal antibodies is less well understood, both in the laboratory and in the clinical setting. In this review, we discuss resistance to antibody-based therapies against the EGFR and HER2, similarities between these resistance profiles, and strategies to overcome resistance to HER family targeting monoclonal antibody therapy.

  4. PCSK9 Antibodies for the Treatment of Hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Ioanna Gouni-Berthold

    2014-12-01

    Full Text Available The serine protease proprotein convertase subtilisin/kexin type 9 (PCSK9 binds to the low-density lipoprotein (LDL receptor (LDLR and directs it to lysosomes for intracellular degradation. This results in decreased numbers of LDLR available on the hepatic cell surface to bind LDL particles and remove them from the circulation and therefore to a subsequent increase in circulating LDL-cholesterol (LDL-C plasma levels. Since 2003, when the role of PCSK9 in LDL-C metabolism was discovered, there have been major efforts to develop efficient and safe methods to inhibit it. Amongst those, monoclonal antibodies against PCSK9 are the furthest in development, with multiple phase 3 trials already published and with cardiovascular endpoint trials currently underway. Two fully human monoclonal antibodies, evolocumab (AMG 145 and alirocumab (REGN727/SAR236553, have been extensively studied in a wide range of subjects, such as those with statin intolerance, as an add-on to statin therapy, as a monotherapy and in patients with familial hypercholesterolemia. PCSK9 antibodies result in a consistent and robust decrease in LDL-C plasma levels ranging from 40% to 70%, either on top of statins or as a monotherapy. If the safety data from the on-going phase 3 trials remain as reassuring as the data available till now, PCSK9 antibodies will offer a novel, powerful therapeutic option to decrease LDL-C plasma levels and, hopefully, cardiovascular risk.

  5. PCSK9 antibodies for the treatment of hypercholesterolemia.

    Science.gov (United States)

    Gouni-Berthold, Ioanna; Berthold, Heiner K

    2014-12-01

    The serine protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low-density lipoprotein (LDL) receptor (LDLR) and directs it to lysosomes for intracellular degradation. This results in decreased numbers of LDLR available on the hepatic cell surface to bind LDL particles and remove them from the circulation and therefore to a subsequent increase in circulating LDL-cholesterol (LDL-C) plasma levels. Since 2003, when the role of PCSK9 in LDL-C metabolism was discovered, there have been major efforts to develop efficient and safe methods to inhibit it. Amongst those, monoclonal antibodies against PCSK9 are the furthest in development, with multiple phase 3 trials already published and with cardiovascular endpoint trials currently underway. Two fully human monoclonal antibodies, evolocumab (AMG 145) and alirocumab (REGN727/SAR236553), have been extensively studied in a wide range of subjects, such as those with statin intolerance, as an add-on to statin therapy, as a monotherapy and in patients with familial hypercholesterolemia. PCSK9 antibodies result in a consistent and robust decrease in LDL-C plasma levels ranging from 40% to 70%, either on top of statins or as a monotherapy. If the safety data from the on-going phase 3 trials remain as reassuring as the data available till now, PCSK9 antibodies will offer a novel, powerful therapeutic option to decrease LDL-C plasma levels and, hopefully, cardiovascular risk.

  6. Mechanisms of resistance to HER family targeting antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Kruser, Tim J. [Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI (United States); Wheeler, Deric L., E-mail: dlwheeler@wisc.edu [Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI (United States)

    2010-04-15

    The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Receptor activation via ligand binding leads to downstream signaling that influence cell proliferation, angiogenesis, invasion and metastasis. Aberrant expression or activity of EGFR and HER2 have been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and breast cancer. With this, intense efforts have been made to inhibit the activity of the EGFR and HER2 by designing antibodies against the ligand binding domains (cetuximab, panitumumab and trastuzumab) or small molecules against the tyrosine kinase domains (erlotinib, gefitinib, and lapatinib). Both approaches have shown considerable clinical promise. However, increasing evidence suggests that the majority of patients do not respond to these therapies, and those who show initial response ultimately become refractory to treatment. While mechanisms of resistance to tyrosine kinase inhibitors have been extensively studied, resistance to monoclonal antibodies is less well understood, both in the laboratory and in the clinical setting. In this review, we discuss resistance to antibody-based therapies against the EGFR and HER2, similarities between these resistance profiles, and strategies to overcome resistance to HER family targeting monoclonal antibody therapy.

  7. Epigenetics of the antibody response.

    Science.gov (United States)

    Li, Guideng; Zan, Hong; Xu, Zhenming; Casali, Paolo

    2013-09-01

    Epigenetic marks, such as DNA methylation, histone post-translational modifications and miRNAs, are induced in B cells by the same stimuli that drive the antibody response. They play major roles in regulating somatic hypermutation (SHM), class switch DNA recombination (CSR), and differentiation to plasma cells or long-lived memory B cells. Histone modifications target the CSR and, possibly, SHM machinery to the immunoglobulin locus; they together with DNA methylation and miRNAs modulate the expression of critical elements of that machinery, such as activation-induced cytidine deaminase (AID), as well as factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1 (Blimp-1). These inducible B cell-intrinsic epigenetic marks instruct the maturation of antibody responses. Their dysregulation plays an important role in aberrant antibody responses to foreign antigens, such as those of microbial pathogens, and self-antigens, such as those targeted in autoimmunity, and B cell neoplasia.

  8. Molecular-specific urokinase antibodies

    Science.gov (United States)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  9. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2015-01-01

    into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive......Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...

  10. Autologous antibodies that bind neuroblastoma cells.

    Science.gov (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  11. Characterization of Inhibitory Anti-insulin-like Growth Factor Receptor Antibodies with Different Epitope Specificity and Ligand-blocking Properties: IMPLICATIONS FOR MECHANISM OF ACTION IN VIVOS⃞

    OpenAIRE

    Doern, Adam; Cao, Xianjun; Sereno, Arlene; Reyes, Christopher L.; Altshuler, Angelina; Huang, Flora; Hession, Cathy; Flavier, Albert; Favis, Michael; Tran, Hon; Ailor, Eric; Levesque, Melissa; MURPHY, TRACEY; Berquist, Lisa; Tamraz, Susan

    2009-01-01

    Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at es...

  12. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    Directory of Open Access Journals (Sweden)

    Gene S Tan

    2016-04-01

    Full Text Available In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9 virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  13. New-onset mediastinal and central nervous system sarcoidosis in a patient with metastatic melanoma undergoing CTLA4 monoclonal antibody treatment.

    LENUS (Irish Health Repository)

    Murphy, Kevin P

    2014-01-01

    Ipilimumab, a cytotoxic monoclonal antibody that inhibits cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), has been established as an effective therapy in the management of advanced melanoma. Immune-mediated adverse events are a common side effect.

  14. Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1.

    Science.gov (United States)

    Kubohara, Yuzuru; Kikuchi, Haruhisa; Nakamura, Koji; Matsuo, Yusuke; Oshima, Yoshiteru

    2010-05-28

    In the development of the cellular slime mold Dictyostelium discoideum, the differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) plays an important role in the regulation of cell differentiation and chemotaxis; however, the cellular signaling systems involving DIF-1 remain to be elucidated. To obtain a probe for DIF-1, we synthesized a DIF derivative (DIF-1-NH(2); 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), and prepared an anti-DIF-1 antibody using a DIF-1-NH(2)-conjugated macromolecule as the immunogen. A 100-fold dilution of the antibody bound to DIF-1-NH(2)-conjugated resin, and this binding was inhibited by co-addition of 20 microM DIF-1 or DIF-1-NH(2). In a monolayer culture of HM44 cells, a DIF-deficient D. discoideum strain, 0.5 nM exogenous DIF-1 induced stalk cell formation in approximately 60% of the cells; this induction was dose-dependently inhibited by the antibody (diluted 12.5- or 25-fold). Furthermore, this inhibition by the antibody was recovered by co-addition of 2.5 or10 nM DIF-1. The results indicate that the anti-DIF-1 antibody recognizes DIF-1 and neutralizes its function. PMID:20416278

  15. Osthole prevents anti-Fas antibody-induced hepatitis in mice by affecting the caspase-3-mediated apoptotic pathway.

    Science.gov (United States)

    Okamoto, Toshihiro; Kawasaki, Toru; Hino, Okio

    2003-02-15

    Fas (Apo-1/CD95) ligand, which is a type II membrane protein, is a major inducer of apoptosis. Osthole is a coumarin derivative present in medicinal plants. The effect of osthole on hepatitis induced by anti-Fas antibody in mice was studied. Pretreatment of mice with osthole (10, 50, and 100 mg/kg, i.p.) prevented the elevation of plasma alanine aminotransferase (ALT) caused by anti-Fas antibody (175 microg/kg, i.v.). Administration of osthole to mice even at a dose of 10 mg/kg significantly inhibited of anti-Fas antibody-induced elevation of plasma ALT. Capase-3 is a cysteine protease, and treatment of mice with anti-Fas antibody caused an elevation of caspase-3 activity at 3.5 and 6 hr. Pretreatment of mice with osthole (100 mg/kg, i.p.) inhibited the elevation of caspase-3 activity caused by anti-Fas antibody. However, the addition of osthole (up to 10(-4)M) to a liver cytosol fraction isolated from mice treated with anti-Fas antibody did not inhibit caspase-3 activity in vitro. Thus, treatment of mice with osthole inhibited caspase-3 activity by an effect upstream of caspase-3 activation. The livers of mice treated with anti-Fas antibody contained apoptotic and dead cells; osthole attenuated the development of this apoptosis and cell death. The present results show that osthole prevented anti-Fas antibody-induced hepatitis by inhibiting the Fas-mediated apoptotic pathway. PMID:12566097

  16. Radioimmunotherapy with engineered antibody fragments

    International Nuclear Information System (INIS)

    Authors have developed and begun evaluating radiometal-chelated (213Bi) engineered antibody fragments as radioimmunotherapy agents that target the HER2/neu (c-erbB-2) antigen. The diabody format was found to have 40-fold greater affinity for HER2/neu and to be associated with significantly greater tumor localization than is achieved with scFv molecule. It is shown that short-lived isotopes like 213Bi would be most effective when used in conjunction with antibodies that targeted diffuse malignancies (leukemia or lymphoma) or when used for very rapid pretargeted radioimmunotherapy application in which the radioisotope is conjugated to a very small ligand

  17. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  18. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A; Thompson, Vicki S

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  19. Antibody profiling sensitivity through increased reporter antibody layering

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  20. Antibody profiling sensitivity through increased reporter antibody layering

    International Nuclear Information System (INIS)

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  1. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

    Science.gov (United States)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

    1989-06-01

    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

  2. Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

    Science.gov (United States)

    Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

    2011-04-01

    We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. PMID:21215693

  3. Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera.

    Science.gov (United States)

    Wang, Qian; Ju, Huanyu; Li, Yanwei; Jing, Zhiqiang; Guo, Lu; Zhao, Yu; Ma, Bo; Gao, Mingchun; Zhang, Wenlong; Wang, Junwei

    2014-12-01

    An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n=225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV.

  4. Synthetic ganglioside analogues for sensitive biosensing : improved probes for antibodies and bacterial toxins

    NARCIS (Netherlands)

    Pukin, A.V.

    2010-01-01

    This thesis describes the synthesis of analogues of human gangliosides and applications thereof for the detection and inhibition of bacterial toxins and antibodies. An efficient glycosylation method was developed for the synthesis of ω-functionalized alkyl lactosides (Chapter 2). These lactosides we

  5. Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus

    NARCIS (Netherlands)

    Pal, Pankaj; Dowd, Kimberly A.; Brien, James D.; Edeling, Melissa A.; Gorlatov, Sergey; Johnson, Syd; Lee, Iris; Akahata, Wataru; Nabel, Gary J.; Richter, Mareike K. S.; Smit, Jolanda M.; Fremont, Daved H.; Pierson, Theodore C.; Heise, Mark T.; Diamond, Michael S.

    2013-01-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit

  6. Propylthiouracil induced anti-neutrophil cytoplasmic antibody-associated vasculitis with bone marrow plasmacytosis andgranulocytopenia

    Institute of Scientific and Technical Information of China (English)

    Abdullah Ozkok

    2009-01-01

    @@ Antithyroid drugs are molecules known as thionamides that inhibit thyroid hormone synthesis by interfering with thyroid peroxidase mediated iodination of tyrosine residues in thyroglobulin. These extensively used drugs are associated with a variety of well-known side effects such as anti-neutrophil cytoplasmic antibody (ANCA)-positive vasctilitis, granulocytopenia and aplastic anemia.

  7. Pharmacokinetics interactions of monoclonal antibodies.

    Science.gov (United States)

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

    2016-09-01

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

  8. Neural stem cells as a novel platform for tumor-specific delivery of therapeutic antibodies.

    Directory of Open Access Journals (Sweden)

    Richard T Frank

    Full Text Available BACKGROUND: Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS. Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors. METHODS AND FINDINGS: As proof-of-concept, we selected Herceptin (trastuzumab, a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice. CONCLUSIONS: Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically

  9. Induction of multi-epitope specific antibodies against HIV-1 by multi-epitope vaccines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strains in vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutation in vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutafions.

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

    Institute of Scientific and Technical Information of China (English)

    王慧琴; 林嘉友; 刘景生

    1995-01-01

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

  11. Isotype profiles of anti-influenza antibodies in mice bearing the xid defect

    International Nuclear Information System (INIS)

    The humoral response to influenza A/PR8 virus was examined in the CAB/N and C3J.xid strains of mice, both of which bear an X-linked genetic defect (xid), and in strains lacking this defect. Hemagglutination-inhibiting antibody titers and measurement of virus-specific antibodies by solid-phase radioimmunoassay indicated that the xid defect does not impair the production of an adequate anti-influenza antibody response. However, investigation of the isotopes of PR8 virus-specific antibodies disclosed a relative decrease in the levels of IgG3 and IgG1 in the xid-bearing strains. This was observed after both intraperitoneal immunization and aerosol infection. The isotope differences were not reflected in the susceptibility of these strains to influenza virus infection

  12. Anti-Self Phosphatidylserine Antibodies Recognize Uninfected Erythrocytes Promoting Malarial Anemia.

    Science.gov (United States)

    Fernandez-Arias, Cristina; Rivera-Correa, Juan; Gallego-Delgado, Julio; Rudlaff, Rachel; Fernandez, Clemente; Roussel, Camille; Götz, Anton; Gonzalez, Sandra; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel; Buffet, Pierre; Ndour, Papa Alioune; Rodriguez, Ana

    2016-02-10

    Plasmodium species, the parasitic agents of malaria, invade erythrocytes to reproduce, resulting in erythrocyte loss. However, a greater loss is caused by the elimination of uninfected erythrocytes, sometimes long after infection has been cleared. Using a mouse model, we found that Plasmodium infection induces the generation of anti-self antibodies that bind to the surface of uninfected erythrocytes from infected, but not uninfected, mice. These antibodies recognize phosphatidylserine, which is exposed on the surface of a fraction of uninfected erythrocytes during malaria. We find that phosphatidylserine-exposing erythrocytes are reticulocytes expressing high levels of CD47, a "do-not-eat-me" signal, but the binding of anti-phosphatidylserine antibodies mediates their phagocytosis, contributing to anemia. In human patients with late postmalarial anemia, we found a strong inverse correlation between the levels of anti-phosphatidylserine antibodies and plasma hemoglobin, suggesting a similar role in humans. Inhibition of this pathway may be exploited for treating malarial anemia.

  13. Isotype profiles of anti-influenza antibodies in mice bearing the xid defect

    Energy Technology Data Exchange (ETDEWEB)

    Reale, M.A.; Bona, C.A.; Schulman, J.L.

    1985-02-01

    The humoral response to influenza A/PR8 virus was examined in the CAB/N and C/sub 3/J.xid strains of mice, both of which bear an X-linked genetic defect (xid), and in strains lacking this defect. Hemagglutination-inhibiting antibody titers and measurement of virus-specific antibodies by solid-phase radioimmunoassay indicated that the xid defect does not impair the production of an adequate anti-influenza antibody response. However, investigation of the isotopes of PR8 virus-specific antibodies disclosed a relative decrease in the levels of IgG3 and IgG1 in the xid-bearing strains. This was observed after both intraperitoneal immunization and aerosol infection. The isotope differences were not reflected in the susceptibility of these strains to influenza virus infection.

  14. Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

    Directory of Open Access Journals (Sweden)

    Qi Zhao

    Full Text Available Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

  15. Protective antibody titre against Newcastle disease in ostriches (Struthio camelus

    Directory of Open Access Journals (Sweden)

    Darminto

    1998-12-01

    Full Text Available The aim of this study was to define an estimated antibody titre which was considered to be protective against Newcastle disease (ND virus infection in ostriches. Eighteen young ostriches of 4 days of age were divided into two groups each containing 9 birds. The first group was unvaccinated and the second group was vaccinated against ND virus twice at 4 and 14 days of age. Antibody titres were monitored at 1, 14, 28, 42, 56, 70 and 85 days of age by haemagglutination inhibition (HI test. All birds were then challenged with a velogenic strain of ND virus, Ita strain, at 42 days of age. The excretion of the challenge virus were monitored daily after challenge up to the end of this experiment. Several organs such as brain, trachea, lungs and spleen were collected from died birds for re-isolation of the challenged virus. Results indicated that all unvaccinated birds succumbed to the challenged virus, except one bird that survived challenged. In contrast to the unvaccinated birds, all vaccinated birds survived challenged, except two birds with low antibody titres succumbed challenged. All birds with antibody titres of 4 (HI-log2 or greater survived challenged. All challenged birds excreted the challenged virus through out their oropharyngs. Moreover, challenged virus can be successfully re-isolated from most organs of the died birds. This study concludes that : (a the estimated protective titre against ND in ostriches is 4 (HI-log2, (b the immune status for ostrich with antibody titre less the 4 (HI-log2 could not be defined, and (c vaccination against Newcastle disease in ostriches could successfully prevent birds from sick and died of ND, but unable to prevent virus infection and unable to stop carrier status after infection.

  16. Alternative affinity tools: more attractive than antibodies?

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.

    2011-01-01

    Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids

  17. 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.

  18. [Neuroimmunological diseases associated with VGKC complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-05-01

    Antibodies to voltage-gated potassium channels(VGKC) were first identified by radioimmunoassay of radioisotope labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were found only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in Morvan's syndrome and in a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins(for example LGI-1, Caspr-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now usually known as VGKC-complex antibodies. In general, LGI-1 antibodies are most common in limbic encephalitis with SIADH. Caspr-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability.

  19. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lin-Xu [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Mellon, Michael [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Bowder, Dane [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Quinn, Meghan [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Shea, Danielle; Wood, Charles [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Xiang, Shi-Hua, E-mail: sxiang2@unl.edu [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States)

    2015-01-15

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture.

  20. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

    Skottrup, Peter; 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...

  1. Production and Screening of Monoclonal Peptide Antibodies.

    Science.gov (United States)

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

    2015-01-01

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

  2. Virus Strain Discrimination Using Recombinant Antibodies

    OpenAIRE

    Boonham, N.; Barker, I.

    2002-01-01

    Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological faciliti...

  3. Sequence and structural analysis of antibodies

    OpenAIRE

    Raghavan, A. K.

    2009-01-01

    The work presented in this thesis focusses on the sequence and structural analysis of antibodies and has fallen into three main areas. First I developed a method to assess how typical an antibody sequence is of the expressed human antibody repertoire. My hypothesis was that the more \\humanlike" an antibody sequence is (in other words how typical it is of the expressed human repertoire), the less likely it is to elicit an immune response when used in vivo in humans. In practi...

  4. Matrix metalloproteinase inhibition in atherosclerosis and stroke.

    Science.gov (United States)

    Roycik, M D; Myers, J S; Newcomer, R G; Sang, Q-X A

    2013-09-01

    Matrix metalloproteinases (MMPs) are a family of tightly regulated, zinc-dependent proteases that degrade extracellular matrix (ECM), cell surface, and intracellular proteins. Vascular remodeling, whether as a function of normal physiology or as a consequence of a myriad of pathological processes, requires degradation of the ECM. Thus, the expression and activity of many MMPs are up-regulated in numerous conditions affecting the vasculature and often exacerbate vascular dysfunction. A growing body of evidence supports the rationale of using MMP inhibitors for the treatment of cardiovascular diseases, stroke, and chronic vascular dementia. This manuscript will examine promising targets for MMP inhibition in atherosclerosis and stroke, reviewing findings in preclinical animal models and human patient studies. Strategies for MMP inhibition have progressed beyond chelating the catalytic zinc to functional blocking antibodies and peptides that target either the active site or exosites of the enzyme. While the inhibition of MMP activity presents a rational therapeutic avenue, the multiplicity of roles for MMPs and the non-selective nature of MMP inhibitors that cause unintended side-effects hinder full realization of MMP inhibition as therapy for vascular disease. For optimal therapeutic effects to be realized, specific targets for MMP inhibition in these pathologies must first be identified and then attacked by potent and selective agents during the most appropriate timepoint.

  5. Immunoglobulin G4: an odd antibody

    NARCIS (Netherlands)

    R.C. Aalberse; S.O. Stapel; J. Schuurman; T. Rispens

    2009-01-01

    Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also

  6. Anti-DNA antibodies in SLE

    Energy Technology Data Exchange (ETDEWEB)

    Voss, E.W.

    1988-01-01

    This book contains 8 chapters. Some of the titles are: Anti-DNA Antibodies in SLE: Historical Perspective; Specificity of Anti-DNA Antibodies in Systemic Lupus Erythematosus; Monoclonial Autoimmune Anti-DNA Antibodies; and Structure--Function Analyses of Anti-DNA Autoantibodies.

  7. Contrasting Patterns of Serologic and Functional Antibody Dynamics to Plasmodium falciparum Antigens in a Kenyan Birth Cohort

    Science.gov (United States)

    Malhotra, Indu; Wang, Xuelie; Babineau, Denise; Yeo, Kee Thai; Anderson, Timothy; Kimmel, Rhonda J.; Angov, Evelina; Lanar, David E.; Narum, David; Dutta, Sheetij; Richards, Jack; Beeson, James G.; Crabb, Brendan S.; Cowman, Alan F.; Horii, Toshihiro; Muchiri, Eric; Mungai, Peter L.; King, Christopher L.; Kazura, James W.

    2015-01-01

    IgG antibodies to Plasmodium falciparum are transferred from the maternal to fetal circulation during pregnancy, wane after birth, and are subsequently acquired in response to natural infection. We examined the dynamics of malaria antibody responses of 84 Kenyan infants from birth to 36 months of age by (i) serology, (ii) variant surface antigen (VSA) assay, (iii) growth inhibitory activity (GIA), and (iv) invasion inhibition assays (IIA) specific for merozoite surface protein 1 (MSP1) and sialic acid-dependent invasion pathway. Maternal antibodies in each of these four categories were detected in cord blood and decreased to their lowest level by approximately 6 months of age. Serologic antibodies to 3 preerythrocytic and 10 blood-stage antigens subsequently increased, reaching peak prevalence by 36 months. In contrast, antibodies measured by VSA, GIA, and IIA remained low even up to 36 months. Infants sensitized to P. falciparum in utero, defined by cord blood lymphocyte recall responses to malaria antigens, acquired antimalarial antibodies at the same rate as those who were not sensitized in utero, indicating that fetal exposure to malaria antigens did not affect subsequent infant antimalarial responses. Infants with detectable serologic antibodies at 12 months of age had an increased risk of P. falciparum infection during the subsequent 24 months. We conclude that serologic measures of antimalarial antibodies in children 36 months of age or younger represent biomarkers of malaria exposure rather than protection and that functional antibodies develop after 36 months of age in this population. PMID:26656119

  8. Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Frankfurt, O.S. (Roswell Park Memorial Institute, Buffalo, NY (USA))

    1987-06-01

    A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.

  9. Development of a polyclonal competitive enzyme-linked immunosorbent assay for detection of antibodies to Ehrlichia ruminantium

    OpenAIRE

    Sumption, Keith J.; Paxton, Edith; Bell-Sakyi, Lesley

    2003-01-01

    A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnosti...

  10. Effects of Anti-Caries Antibodies on Lactobacillus GG in Its Fermentation and Storage Periods

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To investigate how antibodies influence the fermentation of Lactobacillus GG and how Lactobacillus GG influences the biological properties of antibodies during the fermentation and storage periods. Methods Anti-caries immune colostrum powder (IP) and control colostrum powder (CP), skimmed milk powder (SP) at concentrations of 1%, 2.5%, 5% and 10% (w/v) were added to MRS and 50mM Hepes buffer system was used in the milk, the growth curves of Lactobacillus GG including viable cells, lactic acid concentatrion and pH, and also the titer of specific antibodies were determined during the fermentation and storage periods. Results In MRS, SP could improve the growth of Lactobacillus GG in all periods of fermentation, especially at the concentrations of 5% and 10%. CP at the concentrations of 1% and 2.5% had a good initial velocity at the beginning and CP (1%, 2.5%, 5%, 10%) in all the groups could reach a high viable cell concentration at the end of fermentation, which suggested that there were some growth factors for Lactobacillus GG in CP overcoming the inhibition of unspecific antibodies. IP at 1%, 2.5%, 5% and 10% could inhibit the growth of Lactobacillus GG in all the fermentation periods, the critical concentration point was between 2.5%-5%, and there was a competition between growth factors and the inhibition of specific antibodies. In pasteurized milk, the influence of Hepes could help the fermentation start quickly and reach the log phase earlier than in the control group; however, when combined with 5% IP, the growth of Lactobacillus GG was strongly inhibited during all the fermentation periods. The fermentation of Lactobacillus GG had no significant effect on the titer change of anti-caries antibodies during the fermentation and storage periods. Conclusion SP and CP is beneficial to the growth of Lactobacillus GG in MRS, whereas there is a competition between growth factors and the inhibition of specific antibodies to the growth of Lactobacillus GG

  11. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    Science.gov (United States)

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  12. 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.

  13. Preparation and Characterization of Olaquindox Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    CHE,Huilian; SHI Baoxia; CHEN,Jinlan; ZHU,Xuejia; HE,Jiguo

    2009-01-01

    2-[N-(2-Hydroxyethyl)-carbamoyl]-3-methyl-oxoquinoxaline-l,4-oxides (OLA) was first mono-esterificated by succinic acid anhydride, and was then turned into a derivative with carboxyl. The synthetic product was purified by recrystallization with a yield of 52.47%. Its chemical structure was determined by NMR, IR and MS spectra. The determined structure was used to synthese OLA hapten, which had the molecular structural characteristics of OLA. The OLA hapten and bovine serum albumin (BSA) were conjugated using the activated ester method, while the hapten and ovalbumin (OVA) were coupled by the mixed anhydride method. The UV scanning and infrared spectrum results showed that the hapten and the carrier protein were successfully coupled to BSA and OVA, with the combined ratios of 3.8 : 1 and 5 : 1, respectively. OLA-BSA was used as the immunogen to immunize four New Zealand white rabbits, and the high titer anti-serum produced relatively. The titers were 1 " 6400, 1 : 1600, 1 : 12800 and 1 : 6400, respectively, determined by indirect ELISA OLA-OVA as the coating antigen. The intermediate inhibition concentration (IC50) of OLA in the indirect ELISA test was 743.3 ng/mL. The lowest detection limit (IC20) was 5.71 ng/mL. High-purity OLA polyclonal antibody has been obtained by saturated ammonium sulfate and ion-exchange chromatography.

  14. Low prevalence of antibodies and other plasma factors binding to CC chemokines and IL-2 in HIV-positive patients

    DEFF Research Database (Denmark)

    Meyer, C N; Svenson, M; Schade Larsen, C;

    2000-01-01

    Neutralizing cytokine antibodies are found in healthy and diseased individuals, including patients treated with recombinant cytokines. Identification of CCR-5 as co-receptor for HIV has focused interest on CC chemokines and their potential therapeutic use. Chemokine-binding components in plasma...... of HIV-infected patients were therefore assessed by radioimmunoassay and radioreceptor assay. IgG from 4/505 HIV patients and 9/2000 healthy controls (p>0.05) bound rMIP-1alpha and rMIP-1beta, but not rRANTES. No other plasma factors bound the chemokines. The antibodies inhibited receptor binding of both...... chemokines. There was no association between presence of antibodies and disease stage or HIV progression rate. Three of 11 patients treated with rIL-2 developed IgG antibodies suppressing cellular binding and growth promotion of rIL-2. Hence, circulating factors, including antibodies MIP-1alpha/MIP-1beta...

  15. The role of antibodies in myasthenia gravis.

    Science.gov (United States)

    De Baets, M; Stassen, M H W

    2002-10-15

    Myasthenia gravis is an autoimmune disease associated with antibodies directed to the postsynaptic acetylcholine receptor. These antibodies reduce the number of receptors. Autoantibodies against AChR and other muscle antigens can be used for the diagnosis of myasthenia gravis and related disorders. The origin and the role of these antibodies in the disease are discussed. Experimental autoimmune myasthenia gravis, an experimental model closely mimicking the disease, has provided answers to many questions about the role of antibodies, complement macrophages and AChR anchor proteins. Genetically modified anti-AChR antibodies may also be used in the future to treat myasthenia. PMID:12220686

  16. 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

  17. 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...

  18. Avian influenza in ovo vaccination with replication defective recombinant adenovirus in chickens: vaccine potency, antibody persistence, and maternal antibody transfer.

    Science.gov (United States)

    Mesonero, Alexander; Suarez, David L; van Santen, Edzard; Tang, De-Chu C; Toro, Haroldo

    2011-06-01

    Protective immunity against avian influenza (AI) can be elicited in chickens in a single-dose regimen by in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad)-vector encoding the AI virus (AIV) hemagglutinin (HA). We evaluated vaccine potency, antibody persistence, transfer of maternal antibodies (MtAb), and interference between MtAb and active in ovo or mucosal immunization with RCA-free recombinant Ad expressing a codon-optimized AIV H5 HA gene from A/turkey/WI/68 (AdTW68.H5(ck)). Vaccine coverage and intrapotency test repeatability were based on anti-H5 hemagglutination inhibition (HI) antibody levels detected in in ovo vaccinated chickens. Even though egg inoculation of each replicate was performed by individuals with varying expertise and with different vaccine batches, the average vaccine coverage of three replicates was 85%. The intrapotency test repeatability, which considers both positive as well as negative values, varied between 0.69 and 0.71, indicating effective vaccination. Highly pathogenic (HP) AIV challenge of chicken groups vaccinated with increasing vaccine doses showed 90% protection in chickens receiving > or = 10(8) ifu (infectious units)/bird. The protective dose 50% (PD50) was determined to be 10(6.5) ifu. Even vaccinated chickens that did not develop detectable antibody levels were effectively protected against HP AIV challenge. This result is consistent with previous findings ofAd-vector eliciting T lymphocyte responses. Higher vaccine doses significantly reduced viral shedding as determined by AIV RNA concentration in oropharyngeal swabs. Assessment of antibody persistence showed that antibody levels of in ovo immunized chickens continued to increase until 12 wk and started to decline after 18 wk of age. Intramuscular (IM) booster vaccination with the same vaccine at 16 wk of age significantly increased the antibody responses in breeder hens, and these responses were maintained at high

  19. Biomarkers for predicting the efficacy of anti-epidermal growth factor receptor antibody in the treatment of colorectal cancer.

    Science.gov (United States)

    Okada, Yasuyuki; Miyamoto, Hiroshi; Goji, Takahiro; Takayama, Tetsuji

    2014-01-01

    Anti-epidermal growth factor receptor (EGFR) antibodies have been widely utilized as a standard treatment for metastatic colorectal cancer (CRC). Anti-EGFR antibodies bind competitively to EGFRs to inhibit receptor activation and subsequent signal transduction of the RAS/RAF/MEK pathway and PI3K/AKT pathway. By inhibiting EGFR-mediated signal transduction, anti-EGFR antibodies inhibit cell growth, invasion, metastasis and angiogenesis, and they induce apoptosis. The IgG1-type antibody cetuximab is also capable of inducing antibody-dependent cellular cytotoxicity. Several studies have shown that KRAS mutation is a useful biomarker for predicting the efficacy of anti-EGFR agents, and the major guidelines for the treatment of CRC recommend the use of anti-EGFR antibody only for the cancers with wild-type KRAS. Alterations of other genes, including BRAF, NRAS, PTEN and AKT, and EGFR expression/gene copy number have also been reported to be candidate biomarkers for predicting the efficacy of anti-EGFR agents. The predictive values of these biomarkers are still controversial and further investigations are required.

  20. Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Yan-Da Lai

    2016-02-01

    Full Text Available Vascular endothelial growth factor (VEGF is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume on Day 21 vs. 435% with Avastin. This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

  1. Variable fragment of heavy chain antibody against the terminal protein region of hepatitis B virus polymerase introduced by Tat protein transduction domain inhibits the replication of hepatitis B virus in vitro%蛋白转导结构域介导乙型肝炎病毒聚合酶末端蛋白重链可变区抗体体外抑制病毒的复制

    Institute of Scientific and Technical Information of China (English)

    于俊岩; 兰林; 李俊刚; 张长江; 王宇明

    2009-01-01

    目的 蛋白转导结构域(TAT)介导HBV聚合酶末端蛋白(TP)重链可变区(VH)抗体,研究特异性TAT-VH抗体体外对HBV复制的影响.方法 将TAT-VH基因克隆入原核表达载体pET28a(+),在大肠埃希菌BL21(DE3)LysS内诱导融合蛋白表达并进行纯化.纯化的TAT-VH加入培养的HepG2.2.15细胞,间接免疫荧光法检测其导入HepG2.2.15细胞的效率,四甲基偶氮唑盐(MTT)法检测其对细胞生长代谢的影响,将TAT-VH加入培养的HepG2.2.15细胞,定量PCR法检测HBV DNA水平.数据行单因素方差分析和t检验.结果 成功制备了TAT-VH融合蛋白,间接免疫荧光及MTT证实TAT-VH可以跨膜导入HepG2.2.15细胞,且对细胞生长无影响;加入5 000 nmol/L TAT-VH的HepG2.2.15细胞培养上清液内HBV DNA为(1.211±0.132)lg拷贝/mL,对照组为(5.325±0.041)lg拷贝/mL(t=72.91,P<0.05);细胞内分别为(3.521±0.411)和(8.532±0.132)Ig拷贝/mL(t=28.41,P<0.05).结论 HBV聚合酶TP区特异性TAT-VH抗体在体外可抑制HBV复制,为应用细胞内抗体治疗HBV感染提供了良好的实验基础.%Objective To study a functional variable fragment of heavy chain(VH)antibody against the terminal protein(TP)region of hepatitis B virus(HBV)polymerase introduced by human immunodeficiency virus Tat protein transduction domain(TAT)and the inhibitive activity of TAT-VH on the replication of HBV in vitro.Methods The gene encoding TAT-VH was cloned into prokaryotic expression vector pET28a(+).Recombinant plasmid was transduced into E coli BL21(DE3)LysS,then the protein was expressed and purified.The purified TAT-VH fusion protein was added into HepG2.2.15 cell culture.The transduction efficiency was evaluated by indirect fluorescence assay(IFA).The cytotoxicity of TAT-VH was detected by Methabenzthiazuron(MTT)assay.HBV DNA level in HepG2.2.15 cell culture was measured using quantitative polymerase chain reaction(PCR).The data were analyzed by one-factor analysis of variance and t test.Results TAT

  2. 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...

  3. Antibody Peptide Based Antifungal Immunotherapy

    OpenAIRE

    Magliani, Walter; Conti, Stefania; Giovati, Laura; Zanello, Pier Paolo; Sperindè, Martina; Ciociola, Tecla; Polonelli, Luciano

    2012-01-01

    Fungal infections still represent relevant human illnesses worldwide and some are accompanied by unacceptably high mortality rates. The limited current availability of effective and safe antifungal agents makes the development of new drugs and approaches of antifungal vaccination/immunotherapy every day more needed. Among them, small antibody(Ab)-derived peptides are arousing great expectations as new potential antifungal agents. In this topic, the search path from the study of the yeast kill...

  4. The effect of anti-human plasminogen monoclonal antibodies on Glu-plasminogen activation by plasminogen activators

    Directory of Open Access Journals (Sweden)

    M. Akrami

    2006-07-01

    the A1D12 F(ab region to Glu-plasminogen increases the catalytic efficiency of plasminogen activation by plasminogen activators. Therefore, it may be useful to apply clinically A1D12 for the therapy of thromboembolic events such as myocardial infarction by humanizing the F(ab fragment of the A1D12 antibody. Inhibition pattern of antibody MC2B8 obey the mixed type of enzyme inhibition by binding the antibody probably at, or near, the cleavage site of Glu-plasminogen.

  5. T-cell activation. VI. Inhibitory and stimulatory effects of anti-major histocompatibility complex class I antibodies in allogeneic mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Röpke, M; Röpke, C; Claesson, Mogens Helweg

    1993-01-01

    Murine T splenocytes stimulated in primary allogeneic mixed lymphocyte culture (MLC) were incubated with soluble anti-major histocompatibility complex (MHC) class I monoclonal antibodies. These antibodies induced inhibition in the cytotoxicity of the responding population and this inhibition was ...... of the antibodies caused a reduction in generation of T-cell cytotoxicity, whereas low concentrations stimulated the same response. These results demonstrate that the MHC class I molecules of T cells are of significant importance in antigen-induced signal transduction.......Murine T splenocytes stimulated in primary allogeneic mixed lymphocyte culture (MLC) were incubated with soluble anti-major histocompatibility complex (MHC) class I monoclonal antibodies. These antibodies induced inhibition in the cytotoxicity of the responding population and this inhibition...... was not dependent on the domain on class I molecules recognized by the antibodies. Cross-reactivity of the antibodies between the responder and stimulating cell population caused a marked reduction in the inhibitory effect compared to systems where no such cross-reactivity was present. Saturating levels...

  6. 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

  7. C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

    Directory of Open Access Journals (Sweden)

    Xavier eCharest-Morin

    2013-09-01

    Full Text Available The C-C chemokine receptor-7 (CCR7 is a G protein coupled receptor that has a role in leukocyte homing, but that is also expressed in aggressive tumor cells. Preclinical research supports that CCR7 is a valid target in oncology. In view of the increasing availability of therapeutic monoclonal antibodies that carry cytotoxic cargoes, we studied the feasibility of forcing intact cells to internalize known monoclonal antibodies by exploiting the cycle of endocytosis and recycling triggered by the CCR7 agonist CCL19. Firstly, an anti-CCR7 antibody (CD197; clone 150503 labeled surface recombinant CCR7 expressed in intact HEK 293a cells and the fluorescent antibody was internalized following CCL19 treatment. Secondly, a recombinant myc-tagged CCL19 construction was exploited along the anti-myc monoclonal antibody 4A6. The myc-tagged ligand was produced as a conditioned medium of transfected HEK 293a cells that contained the equivalent of 430 ng/ml of immunoreactive CCL19 (average value, ELISA determination. CCL19-myc, but not authentic CCL19, carried the fluorophore-labeled antibody 4A6 into other recipient cells that expressed recombinant CCR7 (microscopy, cytofluorometry. The immune complexes were apparent in endosomal structures, colocalized well with the small GTPase Rab5 and progressed toward Rab7-positive endosomes. A dominant negative form of Rab5 (GDP-locked inhibited this endocytosis. Further, endosomes in CCL19-myc- or CCL19-stimulated cells were positive for β-arrestin2, but rarely for β-arrestin1. Following treatment with CCL19-myc and the 4A6 antibody, the melanoma cell line A375 that expresses endogenous CCR7 was specifically stained using a secondary peroxidase-conjugated antibody. Agonist-stimulated CCR7 can transport antibody-based cargoes, with possible therapeutic applications in oncology.

  8. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    International Nuclear Information System (INIS)

    Research highlights: → The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. → Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. → The universal antibodies cross-neutralize different influenza A subtypes. → The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  9. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    Energy Technology Data Exchange (ETDEWEB)

    Hashem, Anwar M. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Microbiology, Faculty of Medicine, King Abdulaziz University, Jeddah (Saudi Arabia); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada); Van Domselaar, Gary [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Li, Changgui; Wang, Junzhi [National Institute for the Control of Pharmaceutical and Biological Products, Beijing (China); She, Yi-Min; Cyr, Terry D. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Sui, Jianhua [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); He, Runtao [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Marasco, Wayne A. [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); Li, Xuguang, E-mail: Sean.Li@hc-sc.gc.ca [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada)

    2010-12-10

    Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  10. 125I-labeled protein A as a general tracer in immunoassay: suitability of goat and sheep antibodies

    International Nuclear Information System (INIS)

    The immunoassay method in which 125I-labeled staphylococcal Protein A ([125I]PA) serves as a general tracer has been extended to include goat and sheep IgG antibodies. Goat and sheep IgG normally do not react significantly with PA. However, once IgG antibody is bound to immobilized antigen or hapten, binding of [125I]PA is enhanced markedly. Binding efficiencies of [125I]PA to immune complexed goat anti-human IgM, human IgE, methotrexate and sheep anti-IgE were determined and compared quantitatively to rabbit IgG with the corresponding specificity. Immunoassays were developed based on the inhibition of [125I]PA binding as a measure of antibody inhibition by fluid-phase homologous ligand. Goat antibody to the monovalent hapten methotrexate behaved anomalously: for each concentration of IgG tested, there was an optimal amount of methotrexate beads that gave maximum binding of [125I]PA. In the other immune systems, for each antibody concentration maximum binding of tracer was a function only of the amount of immobilized anitgen added. In contrast to the results obtained with solid-phase antigen, solutions containing antibody and amounts of antigen ranging from large antigen excess to antibody excess failed to react significantly with PA or [125I]PA. (Auth.)

  11. S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance

    DEFF Research Database (Denmark)

    Grum-Schwensen, Birgitte; Klingelhöfer, Jörg; Beck, Mette;

    2015-01-01

    the mode of action of 6B12, a S100A4 neutralizing antibody. METHODS: The therapeutic effect of the 6B12 antibody was evaluated in two different mouse models. First, in a model of spontaneous breast cancer we assessed the dynamics of tumor growth and metastasis. Second, in a model of metastatic niche......, decreased vessel density and inhibition of metastases. CONCLUSION: The S100A4 blocking antibody (6B12) reduces tumor growth and metastasis in a model of spontaneous breast cancer. The 6B12 antibody treatment inhibits T cell accumulation at the primary and pre-metastatic tumor sites. The 6B12 antibody acts......BACKGROUND: The tumor microenvironment plays a determinative role in stimulating tumor progression and metastasis. Notably, tumor-stroma signals affect the pattern of infiltrated immune cells and the profile of tumor-released cytokines. Among the known molecules that are engaged in stimulating...

  12. Preexisting Antibody-Dependent Cellular Cytotoxicity-Activating Antibody Responses Are Stable Longitudinally and Cross-reactive Responses Are Not Boosted by Recent Influenza Exposure.

    Science.gov (United States)

    Valkenburg, Sophie A; Zhang, Yanyu; Chan, Ka Y; Leung, Kathy; Wu, Joseph T; Poon, Leo L M

    2016-10-15

    Cross-reactive influenza virus-specific antibody-dependent cellular cytotoxicity (ADCC)-activating antibodies are readily detected in healthy adults. However, little is known about the kinetics of these ADCC responses. We used retrospective serial blood samples from healthy donors to investigate this topic. All donors had ADCC responses against 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) and avian influenza A(H7N9) virus hemagglutinins (HAs) despite being seronegative for these viruses in standard hemagglutination inhibition and microneutralization serological assays. A(H1N1)pdm09 exposure did not boost ADCC responses specific for H7 HA antigens. H7 HA ADCC responses were variable longitudinally within donors, suggesting that these cross-reactive antibodies are unstable. We found no correlation between ADCC responses to the H7 HA and either influenza virus-specific immunoglobulin G1 concentration or age. PMID:27493238

  13. 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

  14. 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.

  15. Anti-idiotypic antibody: A new strategy for the development of a growth hormone receptor antagonist.

    Science.gov (United States)

    Lan, Hainan; Zheng, Xin; Khan, Muhammad Akram; Li, Steven

    2015-11-01

    In general, traditional growth hormone receptor antagonist can be divided into two major classes: growth hormone (GH) analogues and anti-growth hormone receptor (GHR) antibodies. Herein, we tried to explore a new class of growth hormone receptor (GHR) antagonist that may have potential advantages over the traditional antagonists. For this, we developed a monoclonal anti-idiotypic antibody growth hormone, termed CG-86. A series of experiments were conducted to characterize and evaluate this antibody, and the results from a competitive receptor-binding assay, Enzyme Linked Immunosorbent Assays (ELISA) and epitope mapping demonstrate that CG-86 behaved as a typical Ab2β. Next, we examined its antagonistic activity using in vitro cell models, and the results showed that CG-86 could effectively inhibit growth hormone receptor-mediated signalling and effectively inhibit growth hormone-induced Ba/F3-GHR638 proliferation. In summary, these studies show that an anti-idiotypic antibody (CG-86) has promise as a novel growth hormone receptor antagonist. Furthermore, the current findings also suggest that anti-idiotypic antibody may represent a novel strategy to produce a new class of growth hormone receptor antagonist, and this strategy may be applied with other cytokines or growth factors.

  16. Characterization of beta2-glycoprotein I-dependent and -independent "antiphospholipid" antibodies from lupus-prone NZW/BXSB F1 hybrid male mice.

    Science.gov (United States)

    Thiagarajan, P; Le, A; Shapiro, S S

    1997-10-01

    Male (NZW x BXSB)F1 (W/BF1) mice develop a systemic lupus-like syndrome characterized by thrombocytopenia, coronary vascular disease, nephritis, and anticardiolipin antibodies. Three stable hybridoma cell lines secreting monoclonal anticardiolipin antibodies were developed from these mice by fusing their splenic lymphocytes with nonsecreting myeloma cell line, NS-1. Monoclonal antibody A1.17 reacted with cardiolipin in a beta2-Glycoprotein I-dependent manner. The epitope for this antibody consisted of beta2-glycoprotein I bound to cardiolipin or immobilized on plastic plates. Other anionic phospholipid-binding proteins, such as prothrombin or annexin V, had no significant effect in the reactivity of these antibodies. The specificity is similar to the autoimmune anticardiolipin antibodies described in patients with systemic lupus erythematosus and other infectious diseases. In contrast, monoclonal antibodies A1.72 and A1.84 reacted with cardiolipin in the absence of beta2-glycoprotein I. Beta2-glycoprotein I, either in the fluid phase or bound to cardiolipin, inhibited the binding of these antibodies. The specificity of the latter two antibodies was similar to that described in patients with syphilis and allied disorders. Both types of antibodies had lupus anticoagulant properties. Thus lupus-prone male (NZW x BXSB)F1 (W/BF1) mice develop both beta2-glycoprotein I-dependent and beta2-glycoprotein I-independent anticardiolipin antibodies.

  17. Anti-Ro/SSA antibodies and cardiac arrhythmias in the adult: facts and hypotheses.

    Science.gov (United States)

    Lazzerini, P E; Capecchi, P L; Laghi-Pasini, F

    2010-09-01

    It is well established that the passive trans-placental passage of anti-Ro/SSA antibodies from mother to foetus is associated with the risk to develop an uncommon syndrome named neonatal lupus (NLE), where the congenital heart block represents the most severe clinical feature. Recent evidence demonstrated that also adult heart, classically considered invulnerable to the anti-Ro/SSA antibodies, may represent a target of the arrhythmogenicity of these autoantibodies. In particular, the prolongation of the QTc interval appears the most frequent abnormality observed in adults with circulating anti-Ro/SSA antibodies, with some data suggesting an association with an increased risk of ventricular arrhythmias, also life threatening. Moreover, even though the association between anti-Ro/SSA antibodies and conduction disturbances is undoubtedly less evident in adults than in infants, from the accurate dissection of the literature data the possibility arises that sometimes also the adult cardiac conduction tissue may be affected by such antibodies. The exact arrhythmogenic mechanisms involved in foetus/newborns and adults, respectively, have not been completely clarified as yet. However, increasing evidence suggests that anti-Ro/SSA antibodies may trigger rhythm disturbances through an inhibiting cross-reaction with several cardiac ionic channels, particularly the calcium channels (L-type and T-type), but also the potassium channel hERG, whose different expression and involvement in the cardiac electrophysiology during lifespan might account for the occurrence of age-related differences.

  18. Preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection.

    Science.gov (United States)

    Stevens, Natalie E; Hatjopolous, Antoinette; Fraser, Cara K; Alsharifi, Mohammed; Diener, Kerrilyn R; Hayball, John D

    2016-01-01

    Passive immunotherapy may have particular benefits for the treatment of severe influenza infection in at-risk populations, however little is known of the impact of passive immunotherapy on the formation of memory responses to the virus. Ideally, passive immunotherapy should attenuate the severity of infection while still allowing the formation of adaptive responses to confer protection from future exposure. In this study, we sought to determine if administration of influenza-specific ovine polyclonal antibodies could inhibit adaptive immune responses in a murine model of lethal influenza infection. Ovine polyclonal antibodies generated against recombinant PR8 (H1N1) hemagglutinin exhibited potent prophylactic capacity and reduced lethality in an established influenza infection, particularly when administered intranasally. Surviving mice were also protected against reinfection and generated normal antibody and cytotoxic T lymphocyte responses to the virus. The longevity of ovine polyclonal antibodies was explored with a half-life of over two weeks following a single antibody administration. These findings support the development of an ovine passive polyclonal antibody therapy for treatment of severe influenza infection which does not affect the formation of subsequent acquired immunity to the virus. PMID:27380890

  19. Preserved antiviral adaptive immunity following polyclonal antibody immunotherapy for severe murine influenza infection

    Science.gov (United States)

    Stevens, Natalie E.; Hatjopolous, Antoinette; Fraser, Cara K.; Alsharifi, Mohammed; Diener, Kerrilyn R.; Hayball, John D.

    2016-01-01

    Passive immunotherapy may have particular benefits for the treatment of severe influenza infection in at-risk populations, however little is known of the impact of passive immunotherapy on the formation of memory responses to the virus. Ideally, passive immunotherapy should attenuate the severity of infection while still allowing the formation of adaptive responses to confer protection from future exposure. In this study, we sought to determine if administration of influenza-specific ovine polyclonal antibodies could inhibit adaptive immune responses in a murine model of lethal influenza infection. Ovine polyclonal antibodies generated against recombinant PR8 (H1N1) hemagglutinin exhibited potent prophylactic capacity and reduced lethality in an established influenza infection, particularly when administered intranasally. Surviving mice were also protected against reinfection and generated normal antibody and cytotoxic T lymphocyte responses to the virus. The longevity of ovine polyclonal antibodies was explored with a half-life of over two weeks following a single antibody administration. These findings support the development of an ovine passive polyclonal antibody therapy for treatment of severe influenza infection which does not affect the formation of subsequent acquired immunity to the virus. PMID:27380890

  20. Iron as the Key Modulator of Hepcidin Expression in Erythroid Antibody-Mediated Hypoplasia

    Directory of Open Access Journals (Sweden)

    J. C. Fernandes

    2014-01-01

    Full Text Available Erythroid hypoplasia (EH is a rare complication associated with recombinant human erythropoietin (rHuEPO therapies, due to development of anti-rHuEPO antibodies; however, the underlying mechanisms remain poorly clarified. Our aim was to manage a rat model of antibody-mediated EH induced by rHuEPO and study the impact on iron metabolism and erythropoiesis. Wistar rats treated during 9 weeks with a high rHuEPO dose (200 IU developed EH, as shown by anemia, reduced erythroblasts, reticulocytopenia, and plasmatic anti-rHuEPO antibodies. Serum iron was increased and associated with mRNA overexpression of hepatic hepcidin and other iron regulatory mediators and downregulation of matriptase-2; overexpression of divalent metal transporter 1 and ferroportin was observed in duodenum and liver. Decreased EPO expression was observed in kidney and liver, while EPO receptor was overexpressed in liver. Endogenous EPO levels were normal, suggesting that anti-rHuEPO antibodies blunted EPO function. Our results suggest that anti-rHuEPO antibodies inhibit erythropoiesis causing anemia. This leads to a serum iron increase, which seems to stimulate hepcidin expression despite no evidence of inflammation, thus suggesting iron as the key modulator of hepcidin synthesis. These findings might contribute to improving new therapeutic strategies against rHuEPO resistance and/or development of antibody-mediated EH in patients under rHuEPO therapy.

  1. PREVALENCE OF ANTIBODIES AGAINST INFLUENZA VIRUS IN NON-VACCINATED EQUINES FROM THE BRAZILIAN PANTANAL

    Directory of Open Access Journals (Sweden)

    Lucas Gaíva E Silva

    2014-12-01

    Full Text Available The prevalence of antibodies against Equine Influenza Virus (EIV was determined in 529 equines living on ranches in the municipality of Poconé, Pantanal area of Brazil, by means of the hemagglutination inhibition test, using subtype H3N8 as antigen. The distribution and possible association among positive animal and ranches were evaluated by the chi-square test, spatial autoregressive and multiple linear regression models. The prevalence of antibodies against EIV was estimated at 45.2% (95% CI 30.2 - 61.1% with titers ranging from 20 to 1,280 HAU. Seropositive equines were found on 92.0% of the surveyed ranches. Equine from non-flooded ranches (66.5% and negativity in equine infectious anemia virus (EIAV (61.7% were associated with antibodies against EIV. No spatial correlation was found among the ranches, but the ones located in non-flooded areas were associated with antibodies against EIV. A negative correlation was found between the prevalence of antibodies against EIV and the presence of EIAV positive animals on the ranches. The high prevalence of antibodies against EIV detected in this study suggests that the virus is circulating among the animals, and this statistical analysis indicates that the movement and aggregation of animals are factors associated to the transmission of the virus in the region.

  2. Inhibition in multiclass classification

    OpenAIRE

    Huerta, Ramón; Vembu, Shankar; Amigó, José M.; Nowotny, Thomas; Elkan, Charles

    2012-01-01

    The role of inhibition is investigated in a multiclass support vector machine formalism inspired by the brain structure of insects. The so-called mushroom bodies have a set of output neurons, or classification functions, that compete with each other to encode a particular input. Strongly active output neurons depress or inhibit the remaining outputs without knowing which is correct or incorrect. Accordingly, we propose to use a classification function that embodies unselective inhibition and ...

  3. Carrier induced epitopic suppression of antibody responses induced by virus-like particles is a dynamic phenomenon caused by carrier-specific antibodies.

    Science.gov (United States)

    Jegerlehner, Andrea; Wiesel, Melanie; Dietmeier, Klaus; Zabel, Franziska; Gatto, Dominique; Saudan, Philippe; Bachmann, Martin F

    2010-07-26

    Pre-existing immunity against vaccine carrier proteins has been reported to inhibit the immune response against antigens conjugated to the same carrier by a process termed carrier induced epitopic suppression (CIES). Hence understanding the phenomenon of CIES is of major importance for the development of conjugate vaccines. Virus-like particles (VLPs) are a novel class of potent immunological carriers which have been successfully used to enhance the antibody response to virtually any conjugated antigen. In the present study we investigated the impact of a pre-existing VLP-specific immune response on the development of antibody responses against a conjugated model peptide after primary, secondary and tertiary immunization. Although VLP-specific immune responses led to reduced peptide-specific antibody titers, we showed that CIES against peptide-VLP conjugates could be overcome by high coupling densities, repeated injections and/or higher doses of conjugate vaccine. Furthermore we dissected VLP-specific immunity by adoptively transferring VLP-specific antibodies, B-cells or T(helper) cells separately into naïve mice and found that the observed CIES against peptide-VLP conjugates was mainly mediated by carrier-specific antibodies.

  4. A nylon ball solid-phase radioimmunoassay for specific antibodies in human sera. Application to measurement of IgG antibodies to pollen allergens

    International Nuclear Information System (INIS)

    The principle of the radioallergosorbent test (RAST) has been used to measure IgG antibodies to timothy grass pollen allergens in sera from desensitized allergic subjects. 125I-labeled goat anti-human IgG was used as detector protein. Non-specific binding was eliminated by use of a non-porous nylon ball an antigen carrier and by use of a special buffer with high ionic strength and pH, containing 1% bovine gamma globulin and 5% normal rabbit serum as 'balance proteins'. At dilution 1:80 non-specific binding was only 0.28% and the binding ratio for a high-titer serum was about 10. By inhibition experiments the assay was demonstrated to be specific for IgG antibodies to timothy grass pollen. The results obtained with this assay correlated statistically significantly with those found with a double-antibody method. Serum dilution curves were parallel, indicating that the assay is in allergen excess. The within-assay coefficient of variation ranged from 3.9 to 7.6%; the between-assay coefficient of variation from 8.4 to 19.5%. The assay is very simple to perform, requiring no centrifugation. The allergen-coated balls are stable for at least 3 months. The assay should be applicable to measurement of IgG antibodies and IgG subclass antibodies to any protein antigen of interest. (Auth.)

  5. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  6. Controlled delivery of antibodies from injectable hydrogels.

    Science.gov (United States)

    Fletcher, Nathan A; Babcock, Lyndsey R; Murray, Ellen A; Krebs, Melissa D

    2016-02-01

    Therapeutic antibodies are currently used for the treatment of various diseases, but large doses delivered systemically are typically required. Localized controlled delivery techniques would afford major benefits such as decreasing side effects and required doses. Injectable biopolymer systems are an attractive solution due to their minimally invasive potential for controlled release in a localized area. Here, alginate-chitosan hydrogels are demonstrated to provide controlled delivery of IgG model antibodies and also of Fab antibody fragments. Also, an alternate delivery system comprised of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with antibodies and encapsulated in alginate was shown to successfully provide another level of control over release. These biopolymer systems that offer controlled delivery for antibodies and antibody fragments will be promising for many applications in drug delivery and regenerative medicine.

  7. Factors determining antibody distribution in tumors

    Science.gov (United States)

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    The development of antibody therapies for cancer is increasing rapidly, primarily owing to their specificity. Antibody distribution in tumors is often extremely uneven, however, leading to some malignant cells being exposed to saturating concentrations of antibody, whereas others are completely untargeted. This is detrimental because large regions of cells escape therapy, whereas other regions might be exposed to suboptimal concentrations that promote a selection of resistant mutants. The distribution of antibody depends on a variety of factors, including dose, affinity, antigens per cell and molecular size. Because these parameters are often known or easily estimated, a quick calculation based on simple modeling considerations can predict the uniformity of targeting within a tumor. Such analyses should enable experimental researchers to identify in a straightforward way the limitations in achieving evenly distributed antibody, and design and test improved antibody therapeutics more rationally. PMID:18179828

  8. Stabilization of antibody fragments in adverse environments.

    Science.gov (United States)

    Dooley, H; Grant, S D; Harris, W J; Porter, A J

    1998-08-01

    Antibody fragments have the potential to be used as sensitive and specific binding agents in a broad range of industrial applications. Genetic manipulation has been used to design a series of antibody fragment configurations with a flexible linker and/or a disulphide bond between the heavy chain and light chain of an antibody fragment against the herbicide atrazine. The thermostability and stability to a range of denaturants, polar and non-polar solvents, surfactants and proteases have been compared. It has been found that a novel antibody fragment construct (STAB: stabilized antibody) containing both a flexible linker and a disulphide bond can be effectively produced and shows greatly improved stability in these diverse environments. These STABs should be useful in environmental diagnostics and remediation, and may provide a generic approach for stabilizing antibody fragments in formulations containing detergents and penetrants for topical application in the pharmaceutical and cosmetic industries.

  9. Phage displayed peptides and anti-idiotype antibodies recognised by a monoclonal antibody directed against a diagnostic antigen of Mycoplasma capricolum subsp. capripneumoniae.

    Science.gov (United States)

    Bengurić, D R; Dungu, B; Thiaucourt, F; du Plessis, D H

    2001-07-26

    A monoclonal antibody (Mab 4.52) raised against Mycoplasma capricolum subsp. capripneumoniae (Mccp) cell lysate was used as a template to obtain substitute antigens recognised by its paratope. Two approaches were investigated: a 17-mer random peptide library displayed on the surface of a filamentous phage was screened by panning on the immobilised Mab 4.52 and anti-idiotype antibodies were generated by immunising a chicken with the F(ab')(2) fragments of the antibody. Analysis of the peptide sequences displayed by the isolated phages identified two peptides. Both contained two cysteine residues and had identical or similar amino acids in positions 5 (P), 8 (I/L) and 13 (L). The fusion phages were also recognised by Mab 4.52 in enzyme-linked immunosorbent assay (ELISA) and binding was shown by surface plasmon resonance. One of the peptides was a markedly better inhibitor (67%) of the binding of Mab 4.52 to its original antigen than the other (20%) at 1mg/ml. After absorption, to remove isotypic and allotypic reactivities, the anti-idiotype IgY was specifically recognised by Mab 4.52 in ELISA and was able to inhibit its binding to the original antigen, whereas anti-idiotype antibodies raised against a bluetongue virus-specific antibody had no effect. In spite of unequivocal binding of the anti-idiotype antibodies and the fusion phages to the paratope of Mab 4.52, goat antisera appeared not to react with either of the surrogate antigens. In contrast, the test sera bound to the original antigen suggesting that Mab 4.52 does not recognise exactly the same antigenic site as antibodies in the goat antisera. PMID:11376960

  10. Snake venom antibodies in Ecuadorian Indians.

    Science.gov (United States)

    Theakston, R D; Reid, H A; Larrick, J W; Kaplan, J; Yost, J A

    1981-10-01

    Serum samples from 223 Waorani Indians, a tribe in eastern Ecuador, were investigated by enzyme-linked immunosorbent assay for antibodies to snake venom. Seventy-eight per cent were positive, confirming the highest incidence and mortality from snake bite poisoning yet recorded in the world. Most samples were positive for more than one venom antibody. Antibodies were found to venoms of Bothrops viper in 60% of positive cases, of Micrurus coral snake in 21%, and of the bushmaster, Lachesis muta, in 18%. Further studies are needed to determine whether high venom-antibody levels afford protection against further snake envenoming. PMID:7299877

  11. Sequence and structual analysis of antibodies.

    OpenAIRE

    Raghavan, A. K.

    2008-01-01

    The work presented in this thesis focuses on the sequence and structural analysis of antibodies and has fallen into three main areas. First I developed a method to assess how typical an antibody sequence is of the expressed human antibody repertoire. My hypothesis was that the more "human like" an antibody sequence is (in other words how typical it is of the expressed human repertoire), the less likely it is to elicit an immune response when used in vivo in humans. In practice, I found that, ...

  12. Antiphospholipid Antibodies and Systemic Scleroderma

    Directory of Open Access Journals (Sweden)

    Awa Oumar Touré

    2013-03-01

    Full Text Available Objective: Antiphospholipid antibodies (APLs could be associated with an increased risk of vascular pathologies in systemic scleroderma. The aim of our study was to search for APLs in patients affected by systemic scleroderma and to evaluate their involvement in the clinical manifestations of this disease. Materials and Methods: We conducted a cross-sectional descriptive study, from January 2009 until August 2010, with patients received at the Department of Dermatology (Dakar, Senegal. Blood samples were taken at the hematology laboratory and were analyzed for the presence of APLs. Results: Forty patients were recruited. Various types of either isolated or associated APLs were found in 23 patients, i.e. 57.5% of the study population. The most frequently encountered antibody was IgG anti-β2 GPI (37.5% of the patients, followed by anticardiolipins (17.5% and lupus anticoagulants (5%. No statistically significant association of positive antiphospholipid-related tests to any of the scleroderma complications could be demonstrated. Conclusion: A high proportion of patients showing association of systemic scleroderma and APLs suggests the presence of a morbid correlation between these 2 pathologies. It would be useful to follow a cohort of patients affected by systemic scleroderma in order to monitor vascular complications following confirmation of the presence of antiphospholipid syndrome.

  13. Therapeutic complement inhibition – from experimental to clinical medicine.

    Science.gov (United States)

    Lappegård, Knut Tore; Bjerre, Anna; Tjønnfjord, Geir Erland; Mollnes, Tom Eirik

    2015-10-20

    Internationally, the use of the C5-inhibiting monoclonal antibody eculizumab has in the course of just a few years become the first choice of treatment of atypical haemolytic uraemic syndrome and the most severe phenotypes of paroxysmal nocturnal haemoglobinuria. At present eculizumab is the only complement inhibitor in ordinary clinical use. This despite the fact that there only exists one randomised, placebo-controlled trial of eculizumab for paroxysmal nocturnal haemoglobinuria and none for atypical haemolytic uraemic syndrome, and that the therapy is very costly. There is reason to believe that complement inhibition as therapy will increase in the future, and that other drugs will also prove to be effective.

  14. Inhibition effects of parathyroid hormone on human medullary thyroid carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Yaqiong Ni; Qinjiang Liu; Shihong Ma; Ruihui Chen

    2014-01-01

    Objective:The purpose of the study was to investigate the ef ects of parathyroid hormone and parathyroid hor-mone receptor monoclonal antibody on in vitro growth and proliferation of human medul ary thyroid carcinoma celllines. Methods:The medul ary thyroid carcinoma cellline was cultured in vitro, with parathyroid hormone and parathyroid hormone receptor monoclonal antibody treatment intervention, the growth of the cells was observed under an inverted contrast micro-scope, the MTT assay was used to detect the cellgrowth inhibition rate. Results:Under the inverted contrast microscope, the cells changed significantly, the parathyroid hormone and parathyroid hormone receptor monoclonal antibodies can ef ectively inhibit the proliferation of medul ary thyroid cancer cells in a time and dose dependent. When parathyroid hormone concentra-tion reached a concentration of 2.0μmol/L, the parathyroid hormone receptor monoclonal antibody reached a concentration of 1.0μmol/L, the cellgrowth was most significantly inhibited (P<0.05). Conclusion:Parathyroid hormone and parathyroid hormone receptor monoclonal antibody were able to inhibit the proliferation of medul ary thyroid carcinoma cells and signifi-cantly reduce the proliferation index.

  15. Prolactin-like activity of anti-prolactin receptor antibodies on casein and DNA synthesis in the mammary gland.

    OpenAIRE

    Djiane, J; HOUDEBINE, L.M.; Kelly, P A

    1981-01-01

    Prolactin receptors were partially purified from rabbit mammary gland membranes by using an affinity chromatography technique. Antibodies against this prolactin receptor preparation were obtained in guinea pig and sheep. Both antisera were able to inhibit the binding of 125I-labeled ovine prolactin to rabbit mammary gland membranes. When added to culture media of rabbit mammary explants, the anti-prolactin receptor antiserum inhibited the capacity of prolactin to initiate casein synthesis and...

  16. Production and assay of forskolin antibodies

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

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

  17. Production and assay of forskolin antibodies

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2006-01-01

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

  19. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

    OpenAIRE

    Huie, Michael A.; Cheung, Mei-Chi; Muench, Marcus O.; Becerril, Baltazar; Kan, Yuet W.; Marks, James D.

    2001-01-01

    The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each pha...

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

    OpenAIRE

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

    2013-01-01

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

  1. 抗神经生长因子抗体对大鼠慢性坐骨神经压迫损伤模型的脊髓胶质细胞激活的抑制作用%Anti-nerve Growth Factor Antibody Inhibits the Spinal Cord Glial Cell Activation in Rats Model of Chronic Constriction Injury

    Institute of Scientific and Technical Information of China (English)

    李棋; 麻伟青; 王慧明; 董发团; 杨云丽; 李文锋

    2012-01-01

    Objective To investigate the effects of anti-nerve growth factor antibody (anti-NGF) on the pain behavior and the spinal astrocyte activation in rats model of chronic constriction sciatic nerve injury (CCI) . Methods Chronic sciatic nerve compression injury (chronic constriction injury, CCD model was prepared by surgery in rats. The experimental rats were randomly divided into four groups: the CCI + anti- NGF group (n = 8) . rats were given daily single intraperitoneal injection of anti-NGF (10 mg/kg) at 7th day after surgery; the CCI model + saline group (n = 8) : rats were given daily single intraperitoneal injection of saline at 7th day after surgery; sham operation anti-NGF group (n =8) and sham operation saline group (n =8) . Mechanical and thermal pain behavior were observed in every another day after surgery within 15 days. 15 days after surgery, rats in each group was perfused by 4% paraformaldehyde, the expression of astrocyte cell marker GFAP in rats'L4- 5- segment spinal cord was observed by immunohistochemical staining. Results Within 3 days after surgery, the CCI model + saline group's mechanical and thermal pain threshold decreased significantly and reached its peak after 7 days. At 7th day after surgery, a single dose of anti- NGF antibody intraperitoneal injection shortly alleviated the mechanical and thermal pain threshold decrease in CCI rats, continuously injection of anti-NGF 7 days significantly improved the mechanical and thermal pain threshold of CCI rats, which was higher than rats in CCI + saline group. The spinal GFAP expression in CCI + anti-NGF group was significantly lower than the CCI + saline group at 15th day after surgery. Conclusions Inrraperitoneal injection of anti-NGF antibody can effectively reverse the pain behavior of the CCI rat model. Continuous injection of anti-NGF can significantly improve the pain behavior in the CCI rat models. In the spinal cord of rats in CCI + anti- NGF group, GFAP expression is significantly lower

  2. Improvement of a low pH antigen-antibody dissociation procedure for ELISA measurement of circulating anti-Aβ antibodies

    Directory of Open Access Journals (Sweden)

    Ugen Kenneth E

    2007-03-01

    Full Text Available Abstract Background Prior work from our group found that acid dissociation (pH 2.5 incubation of serum from APP transgenic mice vaccinated against Aβ increased the apparent anti-Aβ titers, suggesting antibody masking by antigen in the ELISA assay. Subsequently, we found that pH 2.5 incubation of serum from unvaccinated non-transgenic mice showed antibody binding to Aβ1–42, but no increase when other proteins, including shorter Aβ peptides, coated the ELISA plate. To investigate further the effects of low pH incubation on apparent anti-Aβ1–42 signals, we examined normal sera from nonTg unvaccinated mice, nonTg mice vaccinated with Aβ peptide (to produce authentic anti-Aβ antibodies or a monoclonal antibody against Aβ (6E10 using competitive-inhibition ELISA and Aβ epitope mapping assays. In addition, we examined use of a less stringent low pH procedure at pH 3.5, to ascertain if it had the same effects as the pH 2.5 procedure. Results We believe there are three distinct effects of pH 2.5 incubation.; A an artifactual increase in binding to full length Aβ by mouse immunoglobulin which has low affinity for Aβ, B an inactivation of anti-Aβ antibodies that is time dependent and C unmasking of high affinity anti-Aβ antibodies when high levels of circulating Aβ is present in APP transgenic mice. All three reactions can interact to produce the final ELISA signal. Incubation of sera from unvaccinated nonTg mice at pH 2.5 enhanced ELISA signals by process A. Conversely, pH 2.5 incubation of sera from vaccinated nonTg mice with caused a time dependent reduction of antibody signal by process B (overcoming the increase caused by A. The artifactual anti-Aβ ELISA signal enhanced by pH 2.5 incubation of normal mouse sera could not be effectively competed by low to moderate concentrations of Aβ, nor bind to shorter Aβ peptides in a manner similar to authentic anti-Aβ antibodies. Incubation of mouse sera at pH 3.5 caused neither an apparent

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  4. Analyzing swine sera for functional antibody titers against influenza A neuraminidase proteins using an enzyme-linked lectin assay (ELLA).

    Science.gov (United States)

    Sandbulte, Matthew R; Eichelberger, Maryna C

    2014-01-01

    Neuraminidase (NA) is an envelope glycoprotein of influenza viruses, including swine-lineage influenza A viruses. NA possesses sialidase activity, which is functionally important at multiple points in viral replication, counter-balancing the sialic acid receptor binding activity of the hemagglutinin (HA), the other major envelope glycoprotein. The NA proteins of influenza A viruses have been classified into nine serological subtypes, and they undergo antigenic drift variation similar to that of HA. Antibodies to NA are analyzed much less often than antibodies to HA. The conventional assay for NA inhibition (NI) antibody titration, established decades ago, is widely considered unwieldy and inefficient for routine use. In recent years, a few new formats have been developed which still measure inhibition of NA enzymatic function, but more efficiently and with less chemical waste produced. Described here is the enzyme-linked lectin assay (ELLA), which is performed in 96-well plates and analyzed on a spectrophotometric plate reader. An important factor in adoption of the ELLA technique for animal studies, such as swine, is the choice of NA antigen, which may be purified protein or whole virus containing an antigenically irrelevant HA protein. This NI assay, in conjunction with the hemagglutination inhibiting (HI) antibody assay, offers a practical way to characterize viral isolates more fully and to quantify antibodies induced by infection or vaccination.

  5. ANTIBODI MONOKLONAL STREPTOKOKUS MUTANS 1(c 67 kDa DALAM PASTA GIGI BAHAN DASAR UNTUK MENGHAMBAT PERTUMBUHAN STREPTOKOKUS MUTANS

    Directory of Open Access Journals (Sweden)

    Rini Devijanti R.

    2015-08-01

    Full Text Available Prevention of dental caries is still continuing, because the prevalency caries is high. There was many methods to prevent dental caries etc. dental education, oral hygiene, special method on tooth brushing, water fluoridation, fissure sealant and later on the passive immunization with monoclonal antibodies. The purpose of this study was to investigate about monoclonal antibodies IgA, IgG1 and IgG3 against Streptococcus mutans 1(c in basic paste for inhibiting the growth Streptococcus mutans. The monoclonal antibodies were IgA Ab, IgG1 Ab and IgG3 Ab. Formula basic paste from PT “X” contained Aqua, Sorbitol, Nipagin, Dicalcium Phosphat, Titanium Dioxid, Sodium Carboxyl Methyl Sel. Sodium Lauryl Sulfate and Sacarin. Basic paste was mixed with monoclonal antibodies IgA, IgG1 and IgG3 in room temperature (27oC then to investigate zone of inhibition from these tooth paste with Wistreich and Lechman methods. The data obtained in this study was analyzed with one way Anova and LSD. The result showed that there was a significant differences between basic paste with or without monoclonal antibodies. From the data analyzed in this study it can be concluded that monoclonal antibodies against S. mutans 1( c could be formulation with basic paste.

  6. Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

    Science.gov (United States)

    Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

    1986-01-01

    Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

  7. Prevalence of antibodies to Japanese encephalitis virus among pigs in Bali and East Java, Indonesia, 2008.

    Science.gov (United States)

    Yamanaka, Atsushi; Mulyatno, Kris Cahyo; Susilowati, Helen; Hendrianto, Eryk; Utsumi, Takako; Amin, Mochamad; Lusida, Maria Inge; Soegijanto, Soegeng; Konishi, Eiji

    2010-01-01

    Japanese encephalitis virus (JEV) is a fatal disease in Asia. Pigs are considered to be the effective amplifying host for JEV in the peridomestic environment. Bali Island and Java Island in Indonesia provide a model to assess the effect of pigs on JEV transmission, since the pig density is nearly 100-fold higher in Bali than Java, while the geographic and climatologic environments are equivalent in these areas. We surveyed antibodies to JEV among 123 pigs in Mengwi (Bali) and 96 pigs in Tulungagung (East Java) in 2008 by the hemagglutination-inhibition (HAI) test. Overall prevalences were 49% in Bali and 6% in Java, with a significant difference between them (P Java. In addition, 2-mercaptoethanol-sensitive antibodies were found only from Bali samples. Further, the average HAI antibody titer obtained from positive samples was significantly higher in Bali (1:52) than Java (1:10; P Java.

  8. Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans.

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Dangy

    2016-06-01

    Full Text Available Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone.Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone.The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target.

  9. Anti-idiotypic antibodies that protect cells against the action of diphtheria toxin

    Energy Technology Data Exchange (ETDEWEB)

    Rolf, J.M.; Gaudin, H.M.; Tirrell, S.M.; MacDonald, A.B.; Eidels, L.

    1989-03-01

    An anti-idiotypic serum prepared against the combining site (idiotype) of specific anti-diphtheria toxoid antibodies was characterized with respect to its interaction with highly diphtheria toxin-sensitive Vero cells. Although the anti-idiotypic serum protected Vero cells against the cytotoxic action of diphtheria toxin, it did not prevent the binding of /sup 125/I-labeled diphtheria toxin to the cells but did inhibit the internalization and degradation of /sup 125/I-labeled toxin. This anti-idiotypic serum immunoprecipitated a cell-surface protein from radiolabeled Vero cells with an apparent Mr of approximately 15,000. These results are consistent with the hypothesis that the anti-idiotypic serum contains antibodies that carry an internal image of an internalization site on the toxin and that a cell-surface protein involved in toxin internalization possesses a complementary site recognized by both the toxin and the anti-idiotypic antibodies.

  10. Acquisition of Functional Antibodies That Block the Binding of Erythrocyte-Binding Antigen 175 and Protection Against Plasmodium falciparum Malaria in Children

    Science.gov (United States)

    Irani, Vashti; Ramsland, Paul A.; Guy, Andrew J.; Siba, Peter M.; Mueller, Ivo; Richards, Jack S.; Beeson, James G.

    2015-01-01

    Background. The targets and mechanisms of human immunity to malaria are poorly understood, which poses a major barrier to malaria vaccine development. Antibodies play a key role in human immunity and may act by inhibiting receptor-binding functions of key merozoite invasion ligands. Antibodies to the major invasion ligand and vaccine candidate, erythrocyte-binding antigen 175 (EBA-175), have been linked with protection, but how these antibodies function has not been established. Methods. We developed 2 new assays that quantify the ability of antibodies to inhibit binding of EBA-175 to its erythrocyte receptor, glycophorin A, using either native or recombinant EBA-175. Binding-inhibitory antibodies were evaluated in a longitudinal cohort study of Papua New Guinean children and related to risk of malaria, age, infection status, and markers of parasite exposure. Results. Binding-inhibition assays (BIAs) were reproducible, and the 2 assays had a high level of agreement. Inhibitory antibodies were common among children, acquired in association with markers of increasing parasite exposure, and high in those children with active infection. Inhibitory antibodies correlated with total immunoglobulin G levels to the EBA-175 binding domain (region II). Importantly, binding-inhibitory antibodies were significantly associated with protection from symptomatic malaria when measured using either BIA. Conclusions. Findings suggest that naturally acquired binding-inhibitory antibodies are an important functional mechanism that contributes to protection against malaria and further supports the potential of EBA-175 as a vaccine candidate. Identifying vaccines and approaches that induce potent binding-inhibitory antibodies may be a valuable strategy in the development of highly efficacious malaria vaccines. PMID:26136391

  11. Immunization of Macaca fascicularis (Macaca irus) monkeys with Streptococcus mutans: specificity of antibody responses in saliva.

    Science.gov (United States)

    Emmings, F G; Evans, R T; Genco, R J

    1976-04-01

    M fascicularis monkeys were immunized subcutaneously in the vicinity of the major salivary glands and by retrograde infusion into the parotid duct, with a vaccine containing Formalin-killed S mutans strain 6715 cells and culture-fluid antigens. Indirect immunofluorescent staining was used to titrate and classify antibodies. Subcutaneous immunization induced only a serum response, whereas intraductal infusion stimulated both an IgA antibody response in the parotid fluid and a serum response. Immunized and nonimmunized control groups were orally infected with S mutans strain 6715. The establishment in dental plaque was quantitated by recovery of the infecting organism on selective media and by immunofluorescent staining of plaque smears taken from individual tooth surfaces. The establishment of S mutans strain 6715 was noticeably inhibited in immune monkeys. Immunofluorescent assays for antibody also showed that serum and parotid fluid containing serum IgA antibodies cross reacted with other d serotype and a serotype strains but not representative b and c strains. Immune and control groups were then orally infected with S mutans strain GS-5, a c serotype strain, and no inhibition in establishment was detected of the non-cross-reacting type c organism in the immune group. A latter series of booster immunizations via the intraductal route resulted in a significant decrease in parotid fluid flow. Histological investigations showed inflammatory cell infiltration and replacement of epithelium by connective tissue in the glands from immunized monkeys. A separate group of monkeys, younger than the first, was immunized with the same vaccine via the duct only. In this group, immunizations were given at shorter intervals, but the immunization response was similar to that observed in the first group. The investigations reviewed here and new experiments reported show that immunization of monkeys with S mutan strain 6715 via the parotid duct elicited a reproducible IgA antibody

  12. TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations

    Science.gov (United States)

    Castoldi, Raffaella; Schanzer, Jürgen; Panke, Christian; Jucknischke, Ute; Neubert, Natalie J.; Croasdale, Rebecca; Scheuer, Werner; Auer, Johannes; Klein, Christian; Niederfellner, Gerhard; Kobold, Sebastian; Sustmann, Claudio

    2016-01-01

    Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development. PMID:27578890

  13. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  14. Photonic crystal fiber based antibody detection

    DEFF Research Database (Denmark)

    Duval, A; Lhoutellier, M; Jensen, J B;

    2004-01-01

    An original approach for detecting labeled antibodies based on strong penetration photonic crystal fibers is introduced. The target antibody is immobilized inside the air-holes of a photonic crystal fiber and the detection is realized by the means of evanescent-wave fluorescence spectroscopy...... and the use of a transversal illumination setup....

  15. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

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

    1989-01-01

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

  16. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.; Holdaway, Heather A.; Battisti, Anthony J.; Sukupolvi-Petty, Soila; Sedlak, Dagmar; Fremont, Daved H.; Chipman, Paul R.; Roehrig, John T.; Diamond, Michael S.; Kuhn, Richard J.; Rossmann, Michael G. (Purdue); (WU-MED); (CDC)

    2008-04-02

    The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

  17. Monoclonal antibody-based Surface Plasmon Resonance sensors for pathogen detection

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2007-01-01

    , that can detect and quantify specific plant pathogens and map these to defined positions within the field, would enable the farm manager to perform a precise and targeted application of pesticides and thereby reduce and optimise the use of agrochemicals. The ideal scenario for precision agriculture.......sp. tritici, the cause of wheat yellow rust and Phytophthora infestans, the cause of late blight disease in potato. As no antibody existed against urediniospores from P. striiformis, mouse monoclonal antibodies (mAbs) were produced and characterised. IgM-isotype mAbs from nine hybridoma cell lines were...... screened for cross-reactivity against representatives from common genera. Two specific mAbs were chosen for further characterisation and used to develop a competitive ELISA (using mAb4) and a subtractive inhibition ELISA (using mAb8). The subtractive inhibition ELISA was found to be more sensitive...

  18. Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Klausen, Joan; Barfod, Kristen;

    2002-01-01

    in samples of pig serum were detected by inhibition of the binding of polyclonal rabbit antibodies raised against Ap serotype 12. The assay was evaluated against sera from experimentally infected pigs, from pig herds naturally infected with Ap and from herds declared free of Ap serotypc 12 infection...

  19. Analysis of antigenic relationships among influenza virus strains using a taxonomic cluster procedure. Comparison of three kinds of antibody preparations.

    NARCIS (Netherlands)

    T.F. Weijers; A.D.M.E. Osterhaus (Albert); W.E.Ph. Beyer (Walter); J.A.A.M. van Asten (Jack); F.M. de Ronde-Verloop; K. Bijlsma (Klaas); J.C. de Jong (Jan)

    1985-01-01

    textabstractHemagglutination inhibiting (HI) monoclonal antibody preparations (MA) were raised against six influenza A (H3N2) strains from the period 1977-1982. Twenty-three hybridomas were selected and titrated in HI assays against these strains and against 18 influenza A (H3N2) viruses isolated in

  20. Epitopes on the peplomer protein of infectious bronchitis virus strain M41 as defined by monoclonal antibodies.

    NARCIS (Netherlands)

    N.M.C. Bleumink-Pluym; A.D.M.E. Osterhaus (Ab); M.C. Horzinek; B.A.M. van der Zeijst (Ben); H.G.M. Niesters (Bert)

    1987-01-01

    textabstractSixteen monoclonal antibodies (Mcabs) were prepared against infectious bronchitis virus strain M41, all of them reacting with the peplomer protein. One of them, Mcab 13, was able to neutralize the virus and to inhibit hemagglutination. Competition binding assays allowed the definition of

  1. GH and IGF-I induction by passive immunization of rainbow trout Oncorhynchus mykiss Walbaum using a somatostatin 14 antibody

    Science.gov (United States)

    Inhibition of the growth axis by somatostatin was studied in juvenile rainbow trout using passive immunization with a previously isolated somatostatin antibody (antiSS-14). Upon subcutaneously injection of laying hens (Gallus domesticus) with conjugated somatostatin-14 (SS-14), the antiSS-14 was iso...

  2. Synthetic Antibodies for Reversible Cell Recognition

    Science.gov (United States)

    Zhou, Jing Zhou

    2011-12-01

    Antibody-mediated cell recognition plays a critical role in various biological and biomedical applications. However, strong antibody-cell interactions can lead to the difficulty of separating antibodies from the bound cells in a simple and non-destructive manner, which is often necessary to numerous applications such as cell sorting or separation. Thus, this thesis research is aimed to create an antibody-like nanomaterial with the function of reversible cell recognition It was hypothesized that nucleic acid aptamer and dendrimer could be used as fundamental structural components to develop an antibody-like nanomaterial. The aptamer functions as the binding site of an antibody; the dendrimer is used as a robust, defined nano-scaffold to support the aptamer and to carry small molecules (e.g., fluorophores). To test this hypothesis, a novel method was first developed to discover the essential nucleotides of full-length aptamers to mimic the binding sites of antibodies. The essential nucleotides were further conjugated with a dendrimer to synthesize a monovalent aptamer-dendrimer nanomaterial. The results clearly showed that the essential nucleotides could maintain high affinity and specificity after tethered on dendrimer surface. To further test the hypothesis that antibody-like nanomaterials can be rationally designed to acquire the capability of reversible cell recognition, an aptamer that was selected at 0 °C was used as a model to synthesize a "Y-shaped" nanomaterial by conjugating two aptamers to the same dendrimer. The results showed that the nanomaterial-cell interaction could be affected by the distance between two binding aptamers. In addition, the "Y-shaped" antibody-like nanomaterial could bind target cells more strongly than its monovalent control. Importantly, the strong cell-nanomaterial interaction could be rapidly reversed when the temperature was shifted from 0 °C to 37 °C. In summary, we developed a synthetic antibody that can not only mimic the

  3. Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland

    International Nuclear Information System (INIS)

    The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL-like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity

  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. 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.

  6. Antiphospholipid antibody: laboratory, pathogenesis and clinical manifestations

    Directory of Open Access Journals (Sweden)

    T. Ziglioli

    2011-06-01

    Full Text Available Antiphospholipid antibodies (aPL represent a heterogeneous group of antibodies that recognize various antigenic targets including beta2 glycoprotein I (β2GPI, prothrombin (PT, activated protein C, tissue plasminogen activator, plasmin and annexin A2. The most commonly used tests to detect aPL are: lupus anticoagulant (LAC, a functional coagulation assay, anticardiolipin antibody (aCL and anti-β2GPI antibody (anti-β2GPI, which are enzyme-linked immunoassay (ELISA. Clinically aPL are associated with thrombosis and/or with pregnancy morbidity. Apparently aPL alone are unable to induce thrombotic manifestations, but they increase the risk of vascular events that can occur in the presence of another thrombophilic condition; on the other hand obstetrical manifestations were shown to be associated not only to thrombosis but mainly to a direct antibody effect on the trophoblast.

  7. Inhibition of the growth of hepatoma and hepatic metastasis by pingyangmycin conjugated with Fab′fragment of monoclonal antibody%应用平阳霉素与单克隆抗体Fab′片段偶联物抑制肝癌生长与肿瘤肝转移

    Institute of Scientific and Technical Information of China (English)

    刘小云; 尚伯扬; 刘秀均; 甄永苏

    2001-01-01

    目的 研制一种具有抑制肝癌生长和肿瘤肝转移作用的单克隆抗体Fab′片段与平阳霉素(PYM)偶联物。方法 以胃蛋白酶酶解结合二巯基苏糖醇(DTT)还原法制备单抗Fab′片段;以葡聚糖T-40(dextran T-40)为中介体制备Fab′片段和PYM偶联物Fab′-PYM;用间接ELISA法、2,3,5-氯化三苯基四氮唑 (TTC)抑菌效价检测法,克隆形成法及动物移植性肿瘤模型测定Fab′-PYM偶联物在体外的生物学活性与在小鼠体内的抗肿瘤作用。结果 Fab′-PYM与BEL-7402、肝癌22(H22)及HT-29细胞呈免疫学阳性反应, 但与KB细胞无反应;偶联物中PYM的抑菌活性为游离PYM的15%;Fab′-PYM对体外培养的BEL-7402细胞、HT-29细胞和KB细胞的50%抑制浓度(IC50)分别为0.02、0.08及0.50 μmol/L;静脉注射10 mg/kg Fab′-PYM偶联物和PYM皮下接种对小鼠肝癌22的抑瘤率分别为86%和62%;腹腔注射,10 mg/kg Fab′-PYM偶联物和PYM对小鼠脾内接种的结肠癌26肝转移的抑制率分别为91%和62%。结论 Fab′-PYM偶联物具有比游离平阳霉素更强的抑制肝癌生长与肿瘤肝转移作用。%Objective To develop an immunoconjugate with inhibitory effect on the growth of hepatoma and hepatic metastasis of tumor by linking Fab′fragment of monoclona antibody (McAb) to pingyangmycin (PYM). Methods Monoclonal antibody Fab′ fragment was obtained by enzymolysis with pepsin and DTT reduction. Linking of the Fab′ fragment and PYM was mediated by dextran T40. Indirect ELISA, TTC bacteriostatic titer test, clonogenic assay and animal models transplanted with tumor were used to assess the biological activities and antitumor effects of Fab′PYM conjugate within and without the bodies of 10 mice. Results The Fab′PYM conjugate showed immunoreactivity to BEL7402, hepatoma 22 (H22) and HT29 cells, but no reaction to KB cells. The bacteriostatic activity of PYM in the conjugate was 15

  8. A radioimmunoassay that sandwiches human interleukin-2 between radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line

    International Nuclear Information System (INIS)

    Two monoclonal antibodies were raised against human interleukin-2 (IL-2) produced by E. coli harboring recombinant complemental DNA. Both antibodies did not neutralize its activity, nor did they inhibit the binding of IL-2 to the receptor on target cells. Taking advantage of the ability of monoclonal antibodies to detect IL-2 that had bound to the receptor, a radioimmunoassay was developed that sandwiched IL-2 between the radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line infected with human T cell leukemia virus Type I. The assay has the advantage of detecting only IL-2 with the ability to bind to the receptor, and display a linear dose-response relationship over concentrations ranging from 5 to 100 ng/ml. (Auth.)

  9. Antibodies to variant antigens on the surfaces of infected erythrocytes are associated with protection from malaria in Ghanaian children

    DEFF Research Database (Denmark)

    Dodoo, D; Staalsoe, T; Giha, H;

    2001-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand...... to endothelial cells and enables the parasites to avoid splenic passage. PfEMP1 antibodies may protect from disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen. In this study, we have measured antibodies in Ghanaian children to a conserved region of PfEMP...... during the season. The prevalences of antibodies to both the conserved PfEMP1 peptide and the variant epitopes were greater than 50%, and the levels of immunoglobulin G (IgG) correlated with age. The levels of antibodies to both the conserved peptide and the variant epitopes were higher in protected than...

  10. Vector-Mediated In Vivo Antibody Expression.

    Science.gov (United States)

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. PMID:26104192

  11. A latex slide agglutination test for rapid detection of antimyeloperoxidase antibody.

    OpenAIRE

    Ko, K.H.; S. S. Lee; Lawton, J W

    1999-01-01

    AIM: To develop and test a new latex slide agglutination test (MPO-LSAT) to detect antimyeloperoxidase (anti-MPO) antibody in serum. METHODS: Latex bead coating was adjusted to give maximum sensitivity by attending to latex size, MPO to latex ratio for coupling, ratio of diluted serum to MPO-latex, reaction time and temperature for coupling, and reaction time for agglutination. Inhibition studies were performed using MPO, proteinase 3, bactericidal/permeability increasing protein, and lactofe...

  12. Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization

    International Nuclear Information System (INIS)

    Immobilization of antibodies and enzyme-labeled antibodies by radiation polymerization at low temperatures was studied. The antibody activity of antibody was not affected by irradiation at an irradiation dose of below 8 MR and low temperatures. Immobilization of peroxidase-labeled anti-rabbit IgG goat IgG, anti-peroxidase, peroxidase, and anti-alpha-fetoprotein was carried out with hydrophilic and hydrophobic monomers. The activity of the immobilized enzyme-labeled antibody membranes varied with the thickness of the membranes and increased with decreasing membrane thickness. The activity of the immobilized antibody particles was varied by particle size. Immobilized anti-alpha-fetoprotein particles and membranes can be used for the assay of alpha-fetoprotein by the antigen-antibody reaction, such as a solid-phase sandwich method with high sensitivity

  13. Radiohalogenated half-antibodies and maleimide intermediate therefor

    Science.gov (United States)

    Kassis, A.I.; Khawli, L.A.

    1991-02-19

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings

  14. Structural and functional analysis of CR2/EBV receptor by means of monoclonal antibodies and limited tryptic digestion.

    Science.gov (United States)

    Petzer, A L; Schulz, T F; Stauder, R; Eigentler, A; Myones, B L; Dierich, M P

    1988-01-01

    The receptor for the C3d fragment of the third component of complement, CR2, has recently been shown also to act as the receptor for the Epstein-Barr virus (EBV) and to be involved in the control of B-cell proliferation. In order to define functionally important domains on this molecule, we produced monoclonal antibodies to several distinct epitopes. CR2 was purified from a NP-40 lysate of human tonsils by a new method involving sequential chromatography on lentil lectin Sepharose 4B and DEAE-Sephadex and used to immunize mice. After fusion we obtained four stable hybridoma lines producing antibody to CR2. Specificity of these antibodies for CR2 was ascertained by immunofluorescence analysis on a panel of various cells known to possess CR2, by their reactivity in a recently described ELISA for C3 receptors, by Western blotting with purified CR2 and immunoprecipitation from 125I-labelled Raji cells. These four antibodies were found to recognize three distinct epitopes localized on the same fragments of 95,000, 72,000, 50,000, 32,000 and 28,000 MW obtained after mild tryptic digestion of CR2. The 72,000 MW fragment contains the binding site for C3d. Two monoclonal antibodies recognizing the same epitope did not inhibit the binding of C3d-coated sheep erythrocytes to Raji cells, whereas the other two antibodies against distinct epitopes did inhibit in the presence of a second antibody. All four monoclonal antibodies stimulated the proliferation of human peripheral blood B cells. PMID:2448232

  15. Methods of Telomerase Inhibition

    OpenAIRE

    Andrews, Lucy G.; Tollefsbol, Trygve O.

    2008-01-01

    Telomerase is central to cellular immortality and is a key component of most cancer cells although this enzyme is rarely expressed to significant levels in normal cells. Therefore, the inhibition of telomerase has garnered considerable attention as a possible anticancer approach. Many of the methods applied to telomerase inhibition focus on either of the two major components of the ribonucleoprotein holoenzyme, that is, the telomerase reverse transcriptase (TERT) catalytic subunit or the telo...

  16. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  17. Monoclonal anti-vasopressin (VP) antibodies penetrate into VP neurons, in vivo.

    Science.gov (United States)

    Burlet, A J; Leon-Henri, B P; Robert, F R; Arahmani, A; Fernette, B M; Burlet, C R

    1987-01-01

    The fate of monoclonal anti-vasopressin antibodies (VP-MAbs) injected in vivo into the paraventricular nucleus (PVN) of the rat brain was studied by immunocytochemistry. Depending on the post survival time, VP-MAbs contained in an ascites fluid were stained at different levels of the VP neurons: the cytoplasm of the PVN neurons, the fibres of the median eminence and the granular layer of the Gyrus Dentatus. The identification of endogenous peptides synthesized by PVN neurons showed that the VP-MAbs uptake was specific: it did not appear either in the oxytocinergic neurons or in the non immunoreactive neurons of the Brattleboro rat brain, this rat being genetically incapable of synthesizing central VP. Conversely, VP-MAbs only penetrated into the VP neurons: ascites fluid containing monoclonal antibodies prepared against bovine thyroglobulin (the carrier conjugated to VP in our immunizations) was neither stained in magnocellular neurons nor carried in nerve fibres. The neuronal uptake and transport of VP-MAbs occurred in vivo: they were totally inhibited by heating of the ascites fluid at 56 degrees C for 30 min; this treatment did not alter the VP-MAbs themselves but probably destroyed some thermic sensitive component essential to the macromolecule internalization. The biological effects of antibodies injected in vivo have been reported. The results described here suggest that some specific antibodies passively transferred into the brain could act directly on the peptide synthesis recognized by the antibodies. PMID:3556490

  18. Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus.

    Science.gov (United States)

    Trinh, Dai Quang; Ogawa, Haruko; Bui, Vuong Nghia; Nguyen, Tham Thi Hong; Gronsang, Dulyatad; Baatartsogt, Tugsbaatar; Kizito, Mugimba Kahoza; AboElkhair, Mohammed; Yamaguchi, Shigeo; Nguyen, Viet Khong; Imai, Kunitoshi

    2015-09-01

    A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology.

  19. Anti-idiotypic antibodies function as a surrogate surface epitope of Brugia malayi infective larvae.

    Science.gov (United States)

    Carlow, C K; Busto, P; Storey, N; Philipp, M

    1990-07-01

    Anti-idiotypic (AB2) antibodies were generated in rabbits following immunization with a murine IgM monoclonal antibody (AB1) recognizing a surface determinant of Brugia malayi infective stage larvae. AB2 specifically inhibited the binding of AB1 to B. malayi larvae. Furthermore, AB2 had the ability to mimic the original antigen since mice immunized with AB2 possessed serum antibodies (AB3) specific for the B. malayi surface determinant. The presence of anti-surface antibodies (AB3 and AB1) induced either by AB2 immunization or by administration of AB1, did not alter the outcome of an intraperitoneal infection of B. malayi larvae in BABL/c mice when compared to untreated animals. AB3 antibodies like AB1, were IgM, thus indicating an isotype restricted response to the B. malayi epitope. There were no detectable cell mediated responses to the surface determinant in mice immunized with AB2, assessed by lymphocyte blastogenesis or IL3 production in vitro in response to the idiotope as presented by living larvae. The lack of cellular responses and/or the previously demonstrated rapid shedding of the epitope may explain the inability of AB1 or AB2 to protect mice against larval challenge in this study.

  20. Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus.

    Science.gov (United States)

    Trinh, Dai Quang; Ogawa, Haruko; Bui, Vuong Nghia; Nguyen, Tham Thi Hong; Gronsang, Dulyatad; Baatartsogt, Tugsbaatar; Kizito, Mugimba Kahoza; AboElkhair, Mohammed; Yamaguchi, Shigeo; Nguyen, Viet Khong; Imai, Kunitoshi

    2015-09-01

    A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology. PMID:25952731

  1. 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

  2. Chelate chase of radiopharmaceuticals reversibly bound to monoclonal antibodies improves dosimetry

    International Nuclear Information System (INIS)

    One hundred micrograms of monoclonal antibody (MoAb) CHA 255 with a binding constant Kb of 4 x 109 was complexed with indium-111 labeled BLEDTA II, GLEDTA IV, benzyl EDTA, and an EDTA conjugate of Fab. The 24-hour tumor and organ distribution in BALB/c mice bearing KHJJ tumors was studied for each compound alone, the antibody complex, and 3 hours following a chelate chase of the antibody complex. Whole-body biological half-life was measured for 7 days with and without a chelate chase for each antibody complex. The 24-hour whole-body counts dropped 20-60% within 3 hours of administering the chelate chase. Blood concentration fell over 89% within 3 hours of administering the chase and there was a decrease in concentration in all organs, except the kidneys, of 10 to 85%. Theoretical equivalent human doses were calculated from the 24-hour organ concentrations, effective half-life, and MIRD 11 S values (absorbed dose per cumulated activity). Liver and spleen were the target organs, with the dose ranging from 0.50 to 3.91 rads per millicurie. The reduction in organ radiation dose varied up to 95% following the chelate chase. Rapid selective renal clearance of chelate labeled radiopharmaceuticals by competitive inhibition (chelate chase) of their reversible binding to monoclonal antibodies, greatly improves the radiation dosimetry of tumor imaging agents. 28 references, 5 figures, 5 tables

  3. Comparative efficacy of antigen and antibody detection tests for human trichinellosis

    International Nuclear Information System (INIS)

    Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation

  4. Epitope Mapping of Anti-Interleukin-13 Neutralizing Antibody CNTO607

    Energy Technology Data Exchange (ETDEWEB)

    Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O' Neil, Karyn; Gilliland, Gary L.; (Centocor)

    2009-06-24

    CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

  5. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    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.

  6. Yops of Yersinia enterocolitica Inhibit Receptor-Dependent Superoxide Anion Production by Human Granulocytes

    OpenAIRE

    Visser, L.G.; Seijmonsbergen, E.; Nibbering, P H; van den Broek, P J; van Furth, R

    1999-01-01

    The virulence plasmid-borne genes encoding Yersinia adhesin A (YadA) and several Yersinia secreted proteins (Yops) are involved in the inhibition of phagocytosis and killing of Yersinia enterocolitica by human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis of Y. enterocolitica by human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing ...

  7. A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available BACKGROUND: Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant. METHODS AND FINDINGS: Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation. CONCLUSION: This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.

  8. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality

    DEFF Research Database (Denmark)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J;

    2016-01-01

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perfor...

  9. Combinatorial antibody libraries: new advances, new immunological insights.

    Science.gov (United States)

    Lerner, Richard A

    2016-08-01

    Immunochemists have become quite proficient in engineering existing antibody molecules to control their pharmacological properties. However, in terms of generating new antibodies, the combinatorial antibody library has become a central feature of modern immunochemistry. These libraries are essentially an immune system in a test tube and enable the selection of antibodies without the constraints of whole animal or cell-based systems. This Review provides an overview of how antibody libraries are constructed and discusses what can be learnt from these synthetic systems. In particular, the Review focuses on new biological insights from antibody libraries - such as the concept of 'SOS antibodies' - and the growing use of intracellular antibodies to perturb cellular functions.

  10. Antiphospholipid Antibodies in Lupus Nephritis.

    Science.gov (United States)

    Parodis, Ioannis; Arnaud, Laurent; Gerhardsson, Jakob; Zickert, Agneta; Sundelin, Birgitta; Malmström, Vivianne; Svenungsson, Elisabet; Gunnarsson, Iva

    2016-01-01

    Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE). It remains unclear whether antiphospholipid antibodies (aPL) alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204) or without (n = 294) LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous), before and after induction treatment (short-term outcomes). Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR) and the Chronic Kidney Disease (CKD) stage, after a median follow-up of 11.3 years (range: 3.3-18.8). Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all), but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1-2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are affected by

  11. Antiphospholipid Antibodies in Lupus Nephritis.

    Directory of Open Access Journals (Sweden)

    Ioannis Parodis

    Full Text Available Lupus nephritis (LN is a major manifestation of systemic lupus erythematosus (SLE. It remains unclear whether antiphospholipid antibodies (aPL alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204 or without (n = 294 LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous, before and after induction treatment (short-term outcomes. Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR and the Chronic Kidney Disease (CKD stage, after a median follow-up of 11.3 years (range: 3.3-18.8. Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all, but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1-2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are

  12. Structure Based Antibody-Like Peptidomimetics

    Directory of Open Access Journals (Sweden)

    Mark I. Greene

    2012-02-01

    Full Text Available Biologics such as monoclonal antibodies (mAb and soluble receptors represent new classes of therapeutic agents for treatment of several diseases. High affinity and high specificity biologics can be utilized for variety of clinical purposes. Monoclonal antibodies have been used as diagnostic agents when coupled with radionuclide, immune modulatory agents or in the treatment of cancers. Among other limitations of using large molecules for therapy the actual cost of biologics has become an issue. There is an effort among chemists and biologists to reduce the size of biologics which includes monoclonal antibodies and receptors without a reduction of biological efficacy. Single chain antibody, camel antibodies, Fv fragments are examples of this type of deconstructive process. Small high-affinity peptides have been identified using phage screening. Our laboratory used a structure-based approach to develop small-size peptidomimetics from the three-dimensional structure of proteins with immunoglobulin folds as exemplified by CD4 and antibodies. Peptides derived either from the receptor or their cognate ligand mimics the functions of the parental macromolecule. These constrained peptides not only provide a platform for developing small molecule drugs, but also provide insight into the atomic features of protein-protein interactions. A general overview of the reduction of monoclonal antibodies to small exocyclic peptide and its prospects as a useful diagnostic and as a drug in the treatment of cancer are discussed.

  13. Next generation of antibody therapy for cancer

    Institute of Scientific and Technical Information of China (English)

    Zhenping Zhu; Li Yan

    2011-01-01

    Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective altematives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-basod regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics.

  14. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  15. Production of soluble recombinant proteins with Kell, Duffy and Lutheran blood group antigen activity, and their use in screening human sera for Kell, Duffy and Lutheran antibodies.

    Science.gov (United States)

    Ridgwell, K; Dixey, J; Scott, M L

    2007-10-01

    The aim of this study was to show that soluble recombinant (sr) proteins can mimic blood group antigens and be used to screen human sera for blood-group-specific antibodies. The blood of all pregnant women and pretransfusion patients should be screened for blood-group-specific antibodies to identify and monitor pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN), and to prevent haemolytic transfusion reactions. Current antibody screening and identification methods use human red blood cell panels, which can complicate antibody identification if more than one antibody specificity is present. COS-7 cells were transfected to produce sr forms of the extracellular domains of the red blood cell membrane proteins that express Kell, Duffy or Lutheran blood group antigens. These sr proteins were used to screen for and identify anti-Kell, anti-Duffy or anti-Lutheran blood-group-specific allo-antibodies in human sera by haemagglutination inhibition and in solid-phase enzyme-linked immunosorbent assays (ELISAs). There is a positive correlation (correlation coefficient 0.605, P value 0.002) between antibody titre by standard indirect antiglobulin test (IAT) and signal intensity in the ELISA test. This work shows that sr proteins can mimic blood group antigens and react with human allogeneic antibodies, and that such proteins could be used to develop solid-phase, high-throughput blood group antibody screening and identification platforms. PMID:17725551

  16. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    Energy Technology Data Exchange (ETDEWEB)

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Marco, Ario de, E-mail: ario.demarco@ung.si [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Dept. Environmental Sciences, University of Nova Gorica (UNG), Vipavska 13, P.O. Box 301-SI-5000, Rozna Dolina, Nova Gorica (Slovenia)

    2011-05-20

    Highlights: {yields} Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. {yields} These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. {yields} The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  17. Role of anti-vimentin antibodies in allograft rejection.

    Science.gov (United States)

    Rose, Marlene L

    2013-11-01

    Production of anti-vimentin antibodies (AVA) after solid organ transplantation are common. Although classically thought to be expressed mainly within the cytosol, recent evidence demonstrates that extracellular or cell surface expression of vimentin is not unusual. This review examines the evidence to assess whether AVA contribute to allograft pathology. Clinical studies suggest that AVA are associated with cardiac allograft vasculopathy in heart transplant recipients. Studies in non-human primates confirm that production of AVA after renal and heart transplantation are not inhibited by Cyclosporine. Experimental studies have demonstrated that mice pre-immunised with vimentin undergo accelerated acute rejection and vascular intimal occlusion of cardiac allografts. Adoptive transfer of hyperimmune sera containing AVA into B-cell-knock-out mice caused accelerated rejection of allografted hearts, this is clear evidence that antibodies to vimentin accelerate rejection. AVA act in concert with the alloimmune response and AVA do not damage syngeneic or native heart allografts. Confocal microscopy of allografted organs in vimentin immunised mice shows extensive expression of vimentin on endothelial cells, apoptotic leukocytes and platelet/leukocyte conjugates, co-localising with C4d. One explanation for the ability of AVA to accelerate rejection would be fixation of complement within the graft and subsequent pro-inflammatory effects; there may also be interactions with platelets within the vasculature.

  18. [Interferon alpha antibodies show no cross reactions with typical autoantibodies].

    Science.gov (United States)

    Görg, S; Klouche, M; Wilhelm, D; Kirchner, H

    1993-04-01

    Patients treated with natural human interferon alpha develop anti-interferon antibodies (IFN-AB) only in very rare cases. By contrast, patients with autoimmune disorders are able to generate high-titered IFN-AB against endogenous interferon alpha. One explanation for the development of auto-IFN-AB could be cross-reactivity with typical autoimmune antigens. We investigated the cross-reactivity of 3 high-titered IgG IFN-AB of female autoimmune patients (aged 32, 36, 74 years; two severe cases of SLE, one case of autoimmune thyroiditis) as well as 25 low-titered natural IgM IFN-AB of healthy blood donors (aged 19-48 years). Typical autoimmune antigens including dsDNA, ENA, as well as natural interferon beta and recombinant interferon gamma are not able to inhibit binding of IFN-AB to interferon alpha in an ELISA test system. Preincubation of sera containing either dsDNA antibodies (dsDNA-AB) (24 patients), thyroid peroxidase (TPO-AB) (9 patients) or thyroglobulin (TG-AB) (12 patients) with interferon alpha resulted in no change in the respective autoantibody titer. These data suggest that there is no cross-reactivity between IFN-alpha-AB and dsDNA-AB, TPO-AB or TG-AB. Thus, an explanation for the occurrence of IFN-AB in autoimmune disorders cannot be found in a cross-reaction between interferon alpha with typical autoimmune antigens.

  19. 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

  20. Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity.

    OpenAIRE

    Turnbull, P. C.; Broster, M G; Carman, J A; Manchee, R J; Melling, J

    1986-01-01

    A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in serum to the protective antigen (PA) and lethal factor (LF) components of anthrax toxin. Current human vaccination schedules with an acellular vaccine induce predictable and lasting antibody titers to PA and, when present in the vaccine, to LF. Live spore vaccine administered to guinea pigs in a single dose conferred significantly better protection than the human vaccines (P less than 0.00...