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Sample records for binding antigen eba-175

  1. Safety and Immunogenicity of EBA-175 RII-NG Malaria Vaccine Administered Intramuscularly in Semi-Immune Adults: A Phase 1, Double-Blinded Placebo Controlled Dosage Escalation Study.

    Directory of Open Access Journals (Sweden)

    Kwadwo A Koram

    Full Text Available The erythrocyte binding antigen region II (EBA-175 RII is a Plasmodium falciparum ligand that mediates erythrocyte invasion and is considered an important malaria vaccine candidate. A phase Ia trial in malaria naïve adults living in the United States found the recombinant non-glycosylated vaccine antigen, EBA-175 RII-NG adjuvanted with aluminium phosphate to be safe, immunogenic and capable of inducing biologically active antibodies that can inhibit parasite growth in vitro. The aim of the current study was to assess the safety and immunogenicity of this vaccine in malaria exposed semi-immune healthy adults living in a malaria endemic country, Ghana. In this double-blinded, placebo controlled, dose escalation phase I trial, eighteen subjects per group received ascending dose concentrations (5 μg, 20 μg or 80 μg of the vaccine intramuscularly at 0, 1 and 6 months, while 6 subjects received placebo (normal saline. The primary end point was the number of subjects experiencing Grade 3 systemic or local adverse events within 14 days post-vaccination. Serious adverse events were assessed throughout the study period. Blood samples for immunological analyses were collected at days 0, 14, 28, 42, 180 and 194. A total of 52 subjects received three doses of the vaccine in the respective groups. No serious adverse events were reported. The majority of all adverse events reported were mild to moderate in severity, with local pain and tenderness being the most common. All adverse events, irrespective of severity, resolved without any sequelae. Subjects who received any of the EBA-175 RII-NG doses had high immunoglobulin G levels which moderately inhibited P. falciparum growth in vitro, compared to those in the placebo group. In conclusion, the EBA-175 RII-NG vaccine was safe, well tolerated and immunogenic in malaria semi-immune Ghanaian adults. Its further development is recommended.ClinicalTrials.gov. Identifier: NCT01026246.

  2. Safety and Immunogenicity of EBA-175 RII-NG Malaria Vaccine Administered Intramuscularly in Semi-Immune Adults: A Phase 1, Double-Blinded Placebo Controlled Dosage Escalation Study.

    Science.gov (United States)

    Koram, Kwadwo A; Adu, Bright; Ocran, Josephine; Karikari, Yaa S; Adu-Amankwah, Susan; Ntiri, Michael; Abuaku, Benjamin; Dodoo, Daniel; Gyan, Ben; Kronmann, Karl C; Nkrumah, Francis

    2016-01-01

    The erythrocyte binding antigen region II (EBA-175 RII) is a Plasmodium falciparum ligand that mediates erythrocyte invasion and is considered an important malaria vaccine candidate. A phase Ia trial in malaria naïve adults living in the United States found the recombinant non-glycosylated vaccine antigen, EBA-175 RII-NG adjuvanted with aluminium phosphate to be safe, immunogenic and capable of inducing biologically active antibodies that can inhibit parasite growth in vitro. The aim of the current study was to assess the safety and immunogenicity of this vaccine in malaria exposed semi-immune healthy adults living in a malaria endemic country, Ghana. In this double-blinded, placebo controlled, dose escalation phase I trial, eighteen subjects per group received ascending dose concentrations (5 μg, 20 μg or 80 μg) of the vaccine intramuscularly at 0, 1 and 6 months, while 6 subjects received placebo (normal saline). The primary end point was the number of subjects experiencing Grade 3 systemic or local adverse events within 14 days post-vaccination. Serious adverse events were assessed throughout the study period. Blood samples for immunological analyses were collected at days 0, 14, 28, 42, 180 and 194. A total of 52 subjects received three doses of the vaccine in the respective groups. No serious adverse events were reported. The majority of all adverse events reported were mild to moderate in severity, with local pain and tenderness being the most common. All adverse events, irrespective of severity, resolved without any sequelae. Subjects who received any of the EBA-175 RII-NG doses had high immunoglobulin G levels which moderately inhibited P. falciparum growth in vitro, compared to those in the placebo group. In conclusion, the EBA-175 RII-NG vaccine was safe, well tolerated and immunogenic in malaria semi-immune Ghanaian adults. Its further development is recommended. ClinicalTrials.gov. Identifier: NCT01026246.

  3. Assessment of the quality and quantity of naturally induced antibody responses to EBA175RIII-V in Ghanaian children living in two communities with varying malaria transmission patterns

    DEFF Research Database (Denmark)

    Abagna, Hamza B; Acquah, Festus K; Okonu, Ruth

    2018-01-01

    of malaria parasites, which can enhance immune responses against parasite antigens. This study determined the prevalence and relative avidities of naturally induced antibodies to EBA175RIII-VLl in asymptomatic children living in two communities with varying malaria transmission patterns. METHODS: An asexual...... each study site however, children living in Obom had significantly higher EBA175RIII-VLl antibody concentrations than children living in Abura (P 0.05, Mann-Whitney test). Over the course of the study, the relative antibody avidities of EBA175RIII-VLl IgG antibodies were similar within and between......BACKGROUND: Recent global reports on malaria suggest significant decrease in disease severity and an increase in control interventions in many malaria endemic countries, including Ghana. However, a major driving force sustaining malaria transmission in recent times is the asymptomatic carriage...

  4. Binding of hydrophobic antigens to surfaces

    DEFF Research Database (Denmark)

    2017-01-01

    A first aspect of the present invention is a method of detecting antibodies comprising the steps of: i) providing a first group of beads comprising a surface modified with C1-C10 alkyl groups comprising amine, ammonium, ether and/or hydroxyl groups, ii) contacting said first group of beads......-antigen-antibody conjugates, and v) detecting said bead-antigen-antibody conjugates. Further aspects include an antibody detection kit, a bead-antigen conjugate and a composition comprising at least two different groups of bead-antigen-conjugates....

  5. Autologous peptides constitutively occupy the antigen binding site on Ia

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1988-01-01

    Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype...

  6. Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

    Science.gov (United States)

    Lynch, D M; Howe, S E

    1985-11-01

    Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

  7. Specific binding of antigen-antibody in physiological environments: Measurement, force characteristics and analysis

    Science.gov (United States)

    Gu, Xin; Zhou, Jun; Zhou, Lu; Xie, Shusen; Petti, Lucia; Wang, Shaomin; Wang, Fuyan

    2018-05-01

    The specific recognition of the antigen by the antibody is the crucial step in immunoassays. Measurement and analysis of the specific recognition, including the ways in which it is influenced by external factors are of paramount significance for the quality of the immunoassays. Using prostate-specific antigen (PSA)/anti-PSA antibody and α-fetoprotein (AFP) /anti-AFP antibody as examples, we have proposed a novel solution for measuring the binding forces between the antigens and their corresponding antibodies in different physiological environments by combining laminar flow control technology and optical tweezers technology. On the basis of the experimental results, the different binding forces of PSA/anti-PSA antibody and AFP/anti-AFP antibody in the same phosphate-buffered saline (PBS) environments are analysed by comparing the affinity constant of the two antibodies and the number of antigenic determinants of the two antigens. In different electrolyte environments, the changes of the binding force of antigens-antibodies are explained by the polyelectrolyte effect and hydrophobic interaction. Furthermore, in different pH environments, the changes of binding forces of antigens-antibodies are attributed to the role of the denaturation of protein. The study aims to recognise the antigen-antibody immune mechanism, thus ensuring further understanding of the biological functions of tumour markers, and it promises to be very useful for the clinical diagnosis of early-stage cancer.

  8. Antigen-binding radioimmunoassays for human IgG antibodies to bovine ν-lactoglobulin

    International Nuclear Information System (INIS)

    Turner, M.W.; Paganelli, R.; Levinsky, R.J.; Williams, A.

    1983-01-01

    A double antibody antigen-binding assay for the detection of human IgG antibodies to the bovine milk allergen ν-lactoglobulin is described. The levels of such antibodies in patients with established cows' milk protein intolerance were significantly higher than the levels observed in a healthy control group (P<0.01). The assay showed excellent correlation with a solid phase antigen binding assay (rsub(s) = 0.8, P<0.001). (Auth.)

  9. Identification of a peptide binding protein that plays a role in antigen presentation

    International Nuclear Information System (INIS)

    Lakey, E.K.; Margoliash, E.; Pierce, S.K.

    1987-01-01

    The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from [ 35 S]methionine-labeled cells, appears as two discrete bands of ≅72 and 74 kDa after NaDodSO 4 /PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation

  10. Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

    Energy Technology Data Exchange (ETDEWEB)

    Kennel, S J

    1982-01-01

    Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

  11. Computational identification of antigen-binding antibody fragments.

    Science.gov (United States)

    Burkovitz, Anat; Leiderman, Olga; Sela-Culang, Inbal; Byk, Gerardo; Ofran, Yanay

    2013-03-01

    Determining which parts of the Ab are essential for Ag recognition and binding is crucial for understanding B cell-mediated immunity. Identification of fragments of Abs that maintain specificity to the Ag will also allow for the development of improved Ab-based therapy and diagnostics. In this article, we show that structural analysis of Ab-Ag complexes reveals which fragments of the Ab may bind the Ag on their own. In particular, it is possible to predict whether a given CDR is likely to bind the Ag as a peptide by analyzing the energetic contribution of each CDR to Ag binding and by assessing to what extent the interaction between that CDR and the Ag depends on other CDRs. To demonstrate this, we analyzed five Ab-Ag complexes and predicted for each of them which of the CDRs may bind the Ag on its own as a peptide. We then show that these predictions are in agreement with our experimental analysis and with previously published experimental results. These findings promote our understanding of the modular nature of Ab-Ag interactions and lay the foundation for the rational design of active CDR-derived peptides.

  12. B700, a murine melanoma-specific antigen, binds Vitamin D3; conservation of binding among albuminoid molecules

    International Nuclear Information System (INIS)

    Farzaneh, N.K.; Walden, T.L. Jr.; Hearing, V.J.; Gersten, D.M.

    1990-01-01

    B700, a murine melanoma-specific antigen, is a member of the serum albumin protein family. Other members of this family include serum albumin (SMA), a-fetoprotein (AFP), vitamin D binding protein (DBP), and C700. The primary structure and biochemical functions of B700, as well as its in vivo metabolic fate are largely unknown. The authors examined the functional characteristics of MSA, AFP, and DBP, and for their ability to specifically bind [ 3 H]-1,25-dihydroxy-vitamin D 3 . Scatchard analysis revealed a single binding site for B700 with a Kd of 51,000 M and a Bmax of 4.51 x 10 -7 . There is no significant difference between the Kd and Bmax values among the albuminoid proteins. However, differences in the binding sites could be distinguished by competition of the 1,25-dihydroxy vitamin D 3 with other steroids. 2nM of vitamin D 3 , vitamin D 2 , or estrogen competed for the specific binding of 1,25-dihydroxy vitamin D 3 by B700 but not by DBP. The MSA binding site for 1,25 dihydroxy vitamin D 3 more closely resembles that of DBP than B700. These data indicate that the binding function of the albuminoid proteins has been conserved in the B700 melanoma antigen

  13. Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

    Directory of Open Access Journals (Sweden)

    Christine Bee

    Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

  14. Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

    DEFF Research Database (Denmark)

    Kirkeby, Svend; André, Sabine; Gabius, Hans-Joachim

    2004-01-01

    OBJECTIVES: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic alphagalactose (Galalpha) antigens in the doner tissue. DESIGN AND METHODS: We have measured the binding potentials of Galalpha detecting lectins and antibodies, including...

  15. Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles.

    Science.gov (United States)

    Kocher, Jacob F; Lindesmith, Lisa C; Debbink, Kari; Beall, Anne; Mallory, Michael L; Yount, Boyd L; Graham, Rachel L; Huynh, Jeremy; Gates, J Edward; Donaldson, Eric F; Baric, Ralph S

    2018-05-22

    Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens. IMPORTANCE Caliciviruses are

  16. Dynamics behind affinity maturation of an anti-HCMV antibody family influencing antigen binding

    KAUST Repository

    Di Palma, Francesco; Tramontano, Anna

    2017-01-01

    The investigation of antibody affinity maturation and its effects on antigen binding is important with respect to understanding the regulation of the immune response. To shed light on this crucial process, we analyzed two Igs neutralizing the human cytomegalovirus: the primary germline antibody M2J1 and its related mature antibody 8F9. Both antibodies target the AD-2S1 epitope of the gB envelope protein and are considered to establish similar interactions with the cognate antigen. We used molecular dynamics simulations to understand the effect of mutations on the antibody–antigen interactions. The results provide a qualitative explanation for the increased 8F9 peptide affinity compared with that of M2J1. The emerging atomistic-detailed description of these complexes reveals the molecular effects of the somatic hypermutations occurring during affinity maturation.

  17. Foreign or Domestic CARs: Receptor Ligands as Antigen-Binding Domains

    Directory of Open Access Journals (Sweden)

    Donald R. Shaffer

    2014-01-01

    Full Text Available Chimeric antigen receptors (CARs are increasingly being used in clinical trials to treat a variety of malignant conditions and recent results with CD19-specific CARs showing complete tumor regressions has sparked the interest of researchers and the public alike. Traditional CARs have been generated using single-chain variable fragments (scFv, often derived from murine monoclonal antibodies, for antigen specificity. As the clinical experience with CAR T cells grows, so does the potential for unwanted immune responses against the foreign transgene. Strategies that may reduce the immunogenicity of CAR T cells are humanization of the scFv and the use of naturally occurring receptor ligands as antigen-binding domains. Herein, we review the experience with alternatively designed CARs that contain receptor ligands rather than scFv. While most of the experiences have been in the pre-clinical setting, clinical data is also emerging.

  18. Dynamics behind affinity maturation of an anti-HCMV antibody family influencing antigen binding

    KAUST Repository

    Di Palma, Francesco

    2017-08-03

    The investigation of antibody affinity maturation and its effects on antigen binding is important with respect to understanding the regulation of the immune response. To shed light on this crucial process, we analyzed two Igs neutralizing the human cytomegalovirus: the primary germline antibody M2J1 and its related mature antibody 8F9. Both antibodies target the AD-2S1 epitope of the gB envelope protein and are considered to establish similar interactions with the cognate antigen. We used molecular dynamics simulations to understand the effect of mutations on the antibody–antigen interactions. The results provide a qualitative explanation for the increased 8F9 peptide affinity compared with that of M2J1. The emerging atomistic-detailed description of these complexes reveals the molecular effects of the somatic hypermutations occurring during affinity maturation.

  19. Functional antigen binding by the defective B cells of CBA/N mice.

    Science.gov (United States)

    Snippe, H; Merchant, B; Lizzio, E F; Inman, J K

    1982-01-01

    CBA/N mice have an X-linked B cell defect which prevents them from responding to nonmitogenic thymic independent (TI-2) antigens such as dinitrophenylated DNP-Ficoll (1,2). The F1 male progeny of CBA/N female mice express the same defect. Spleen cell suspensions from such defective mice (CBA/N X C3H/HeN F1 males) could not respond to DNP-Ficoll following in vitro immunization and subsequent transfer into irradiated, syngeneic, F1 male recipients as expected. In contrast, normal CBA/N X C3H/HeN F1 female spleen cells could respond and effect a "rescue"; they mounted strong plaque-forming cell responses 7 days after in vitro exposure to DNP-Ficoll and subsequent transfer into irradiated F1 male recipients. Defective F1 male spleen cells, however, could bind significant quantities of 125I-DNP-Ficoll after in vitro exposure. Extensive washing of these spleen cells could not reverse this binding. Such DNP-Ficoll-exposed and washed F1 male spleen cells could, after transfer, aid normal untreated F1 female cells in their rescue function. The defective F1 male spleen cells could convey immunogenic quantities of DNP-Ficoll to the "rescuing" F1 female cells. Mitomycin treatment of F1 male cells did not interfere with their conveyor function. Goat anti-mouse mu serum impeded the passive antigen conveyor function of defective F1 male cells as did prior exposure to high concentrations of free DNP hapten. Our data support the view that the B cell defect of CBA/N X C3H/HeN F1 male mice does not relate to antigen binding, but rather to an inability to be effectively triggered by certain cell-bound polymeric antigens.

  20. Cell-mediated immunity against human retinal extract, S-antigen, and interphotoreceptor retinoid binding protein in onchocercal chorioretinopathy

    NARCIS (Netherlands)

    van der Lelij, A.; Rothova, A.; Stilma, J. S.; Hoekzema, R.; Kijlstra, A.

    1990-01-01

    Autoimmune mechanisms are thought to be involved in the pathogenesis of onchocercal chorioretinopathy. Cell-mediated immune responses to human retinal S-antigen, interphotoreceptor retinoid binding protein (IRBP), and crude retinal extract were investigated in patients with onchocerciasis from

  1. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

    2015-01-01

    Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells ofte...... or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/....

  2. Concanavalin A immobilized magnetic poly(glycidyl methacrylate) beads for prostate specific antigen binding.

    Science.gov (United States)

    Idil, Neslihan; Perçin, Işık; Karakoç, Veyis; Yavuz, Handan; Aksöz, Nilüfer; Denizli, Adil

    2015-10-01

    The aim of this study was to prepare Concanavalin A (Con A) immobilized magnetic poly(glycidyl methacrylate) (mPGMA) beads for prostate specific antigen (PSA) binding and to study binding capacities of the beads using lectin-glycoprotein interactions. Firstly, iron oxide nanoparticles were synthesized by co-precipitation method and then, beads were synthesized by dispersion polymerization in the presence of iron oxide nanoparticles. Con A molecules were both covalently immobilized onto the beads directly and through the spacer arm (1,6-diaminohexane-HDMA). The total PSA and free PSA binding onto the mPGMA-HDMA-Con A beads were higher than that of the mPGMA-Con A beads. Maximum PSA binding capacity was observed as 91.2 ng/g. Approximately 45% of the bound PSA was eluted by using 0.1 M mannose as elution agent. The mPGMA-HDMA-Con A beads could be reused without a remarkable decrease in the binding capacities after 5 binding-desorption cycles. Serum fractions were analyzed using SDS-PAGE. The mPGMA-HDMA-Con A beads could be useful for the detection of PSA and suggested as a model system for other glycoprotein biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Native Mass Spectrometry, Ion mobility, and Collision-Induced Unfolding Categorize Malaria Antigen/Antibody Binding

    Science.gov (United States)

    Huang, Yining; Salinas, Nichole D.; Chen, Edwin; Tolia, Niraj H.; Gross, Michael L.

    2017-09-01

    Plasmodium vivax Duffy Binding Protein (PvDBP) is a promising vaccine candidate for P. vivax malaria. Recently, we reported the epitopes on PvDBP region II (PvDBP-II) for three inhibitory monoclonal antibodies (2D10, 2H2, and 2C6). In this communication, we describe the combination of native mass spectrometry and ion mobility (IM) with collision induced unfolding (CIU) to study the conformation and stabilities of three malarial antigen-antibody complexes. These complexes, when collisionally activated, undergo conformational changes that depend on the location of the epitope. CIU patterns for PvDBP-II in complex with antibody 2D10 and 2H2 are highly similar, indicating comparable binding topology and stability. A different CIU fingerprint is observed for PvDBP-II/2C6, indicating that 2C6 binds to PvDBP-II on an epitope different from 2D10 and 2H2. This work supports the use of CIU as a means of classifying antigen-antibody complexes by their epitope maps in a high throughput screening workflow. [Figure not available: see fulltext.

  4. A novel method for radiolabeling antigen-binding receptors of lymphocytes

    International Nuclear Information System (INIS)

    Choi, Y.S.; Lee, M.S.; Rosenspire, A.J.

    1983-01-01

    Antigen-binding receptor (ABR) molecules have been selectively radiolabeled and isolated from immunized chicken spleen cells. The specific radiolabeling of the receptors has been accomplished by utilizing a novel technique employing lactoperoxidase (LPO) covalently linked to antigen (Ag) for which human gammaglobulin was used. The cell surface ABRs were first bound to the Ag-LPO conjugates through specific recognition sites on the Ag portion of the conjugates. The bound LPO portions were then allowed to catalyze the radioiodination of the ABRs. After radiolabeling, cells were solubilized with detergents, ABRs still bound to Ag-LPO conjugates were directly isolated from the lysates via immunoaffinity chromatography utilizing an immunoaffinity reagent directed toward the antigen portion of the ABR-Ag-LPO complex. The radioactive materials were then analyzed via SDS-PAGE under reducing conditions. Most of the specifically-labeled and isolated materials were immunoglobulin (Ig). Both the membrane-bound form of the heavy chain as well as the secreted form were detected, along with the light chain. An additional polypeptide was also selectively labeled and isolated along with the Ig. This may be a molecule closely associated with the membrane immunoglobulin on the B-cell surface. (author)

  5. Isolation of a peptide binding protein and its role in antigen presentation

    International Nuclear Information System (INIS)

    Lakey, E.; Pierce, S.K.; Margoliash, E.

    1986-01-01

    A mouse T cell hybrid, TPc9.1, recognizes pigeon cytochrome c (Pc) as processed and presented by histocompatible antigen presenting cells (APC). When paraformaldehyde fixed APC are employed, only a peptide fragment of Pc, Pc 81-104, and not the native Pc, is capable of stimulating TPc9.1 cells. Pc 81-104 appears to associate tightly with the APC surface since paraformaldehyde fixed APC which have been incubated with Pc 81-104 remain stimulatory following extensive washing. When APC are surface labeled with 125 I, solubilized and affinity purified on Pc 81-104-Sepharose 4B columns, two predominant polypeptides of approximately 72 and 74 kd are isolated. Little or no immunoglobulin, Class I or Class II proteins are obtained under these conditions. Antisera from rabbits immunized with the affinity purified material, but not preimmune sera, block the activation of TPc 9.1 cells by Pc as well as Pc 81-104 when presented by live APC. Furthermore, these antisera are even more effective in blocking the activation of TPc9.1 cells by either APC which had been pulsed with Pc and then paraformaldehyde fixed, or by Pc 81-104 when added to paraformaldehyde fixed APC, suggesting that these antisera were not affecting antigen processing. Thus, these peptide binding proteins may play a role in antigen presentation, and they are being further characterized

  6. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition.

    Directory of Open Access Journals (Sweden)

    Yves Nominé

    Full Text Available Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15 derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F was equally well tolerated as the wild type glutamine (Q at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv-peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology. Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.

  7. Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

    Science.gov (United States)

    Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

    2000-03-01

    MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

  8. A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

    Science.gov (United States)

    Miller, A

    1977-01-01

    The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented.

  9. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  10. Characterisation of peptide microarrays for studying antibody-antigen binding using surface plasmon resonance imagery.

    Directory of Open Access Journals (Sweden)

    Claude Nogues

    Full Text Available BACKGROUND: Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65 and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van't Hoff type analyses to provide comparative DeltaG, DeltaS and DeltaH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding. CONCLUSIONS: The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen

  11. The Leptospiral Antigen Lp49 is a Two-Domain Protein with Putative Protein Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira Giuseppe,P.; Oliveira Neves, F.; Nascimento, A.; Gomes Guimaraes, B.

    2008-01-01

    Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 Angstroms resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date.

  12. Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

    Science.gov (United States)

    Usui, Daiki; Inaba, Satomi; Kamatari, Yuji O; Ishiguro, Naotaka; Oda, Masayuki

    2017-09-02

    The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

    Science.gov (United States)

    Kainer, Manuela; Antes, Bernhard; Wiederkum, Susanne; Wozniak-Knopp, Gordana; Bauer, Anton; Rüker, Florian; Woisetschläger, Max

    2012-10-15

    Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Radioimmunoassay for Epstein-Barr Virus (EBV)-associated Nuclear Antigen (EBNA). Binding of iodinated antibodies to antigen immobilized in polyacrylamide gel

    International Nuclear Information System (INIS)

    Dolken, G.; Klein, G.

    1977-01-01

    A solid-phase radioimmunoassay was developed for the EBV-associated nuclear antigen (EBNA). Total homogenates of EBV-DNA and EBNA positive or negative cells were polymerized in polyacrylamide gel and compared for their ability to bind 125 I-IgG prepared from anti-EBNA positive and anti-EBNA negative sera. EBNA specific binding was demonstrated and confirmed by serological and cellular specificity controls. The assay allows the quantitation of antigen or antibody even in the presence of detergents and is suitable for biochemical characterization of the antigen. Reciprocal blocking studies with extracts from different cell lines showed quantitative and qualitative differences. One part of the EBNA specificiti(es) present in the human Burkitt lymphoma derived lines RAJI, DAUDI and AW-RAMOS was lacking in B96-8, a marmoset line carrying EBV derived from a human infectious mononucleosis line. This result may reflect differences in the viral genomes derived from Burkitt lymphoma and infectious mononucleosis lines or differences in the host cells. (author)

  15. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

    2015-01-01

    Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells often...... target diverse regions in highly variable viral pathogens and this diversity may need to be addressed through redefinition of suitable peptide targets. Methods: We have developed a method for antigen assessment and target selection for polyvalent vaccines, with which we identified immune epitopes from...... variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results: We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased...

  16. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms.

    Science.gov (United States)

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-04-17

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments' ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

  17. Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Rui; McBride, Ryan; Paulson, James C.; Basler, Christopher F.; Wilson, Ian A. (Sinai); (Scripps)

    2010-03-04

    The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.

  18. Analysis of Swine Leukocyte Antigen Peptide Binding Profiles and the Identification of T cell Epitopes by Tetramer Staining

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers

    class I peptide binding characteristics in relation to immune responses to vaccination or infection. Applying proven technologies to newly produced, recombinant swine leukocyte antigen (SLA) class I proteins yielded a body of data for peptide:SLA:β2m (pSLA) complex affinity and stability. Mapping...... system to specifically identify and react upon non-self peptide fragments unique only to the foreign intruder. The polymorphism of the MHC molecule effectively individualizes the immune response of each member of any given species. Moreover, responding T cells recognize antigen ligands, only...... in the context of peptide:MHC:β2m (pMHC) complex. The gene encoding the MHC is one of the most polymorphic regions of the genome known. Despite thousands of different human leukocyte antigen (HLA) variants identified, each member of a species only inherits and expresses a few of these MHC alleles. The “MHC...

  19. Human Leukocyte Antigen (HLA) Class I Restricted Epitope Discovery in Yellow Fewer and Dengue Viruses: Importance of HLA Binding Strength

    DEFF Research Database (Denmark)

    Lund, Ole; Nascimento, Eduardo J. M.; Maciel, Milton, Jr

    2011-01-01

    Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV...... inoculated twice with the 17DD YFV vaccine strain. Three of the YFV A*02:01 restricted peptides activated T-cells from the infected mice in vitro. All three peptides that elicited responses had an HLA binding affinity of 2 nM or less. The results indicate the importance of the strength of HLA binding...

  20. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-09-08

    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  1. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2011-01-01

    Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

  2. HLA-A*0201-restricted CTL epitope of a novel osteosarcoma antigen, papillomavirus binding factor

    Directory of Open Access Journals (Sweden)

    Tsukahara Tomohide

    2009-06-01

    Full Text Available Abstract Background To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF as a CTL-defined osteosarcoma antigen in the context of HLA-B55. However, clinical application of PBF-based immunotherapy requires identification of naturally presented CTL epitopes in osteosarcoma cells in the context of more common HLA molecules such as HLA-A2. Methods Ten peptides with the HLA-A*0201 binding motif were synthesized from the amino acid sequence of PBF according to the BIMAS score and screened with an HLA class I stabilization assay. The frequency of CTLs recognizing the selected PBF-derived peptide was determined in peripheral blood of five HLA-A*0201+ patients with osteosarcoma using limiting dilution (LD/mixed lymphocyte peptide culture (MLPC followed by tetramer-based frequency analysis. Attempts were made to establish PBF-specific CTL clones from the tetramer-positive CTL pool by a combination of limiting dilution and single-cell sorting. The cytotoxicity of CTLs was assessed by 51Cr release assay. Results Peptide PBF A2.2 showed the highest affinity to HLA-A*0201. CD8+ T cells reacting with the PBF A2.2 peptide were detected in three of five patients at frequencies from 2 × 10-7 to 5 × 10-6. A tetramer-positive PBF A2.2-specific CTL line, 5A9, specifically lysed allogeneic osteosarcoma cell lines that expressed both PBF and either HLA-A*0201 or HLA-A*0206, autologous tumor cells, and T2 pulsed with PBF A2.2. Five of 12 tetramer-positive CTL clones also lysed allogeneic osteosarcoma cell lines expressing both PBF and either HLA-A*0201 or HLA-A*0206 and T2 pulsed with PBF A2.2. Conclusion These findings indicate that PBF A2.2 serves as a CTL epitope on osteosarcoma cells in the context of HLA-A*0201, and potentially, HLA-A*0206. This extends the availability of PBF-derived therapeutic peptide vaccines for patients with osteosarcoma.

  3. Characteristics Studies of 125I- and total PSA antibody's Binding with prostate specific antigen (PSA) in Human Uterus Tumors

    International Nuclear Information System (INIS)

    Al-Mudaffar, S.; Al-Salihi, J.

    2005-01-01

    Two groups of uterus tumors (benign and malignant) postmenopausal patients were used to investigate the presence of prostate specific antigen (PSA). Preliminary experiments were performed to follow the binding of '1 25 I-anti total PSA antibody with PSA in uterus tissues homogenates of the two groups with their corresponding antigen and found to be (8.8,7.1%) for benign and malignant tumors, respectively. An Immuno Radio Metric Assay (IRMA) procedure was developed for measuring PSA in benign and malignant uterus tumors homogenates. The optimum conditions of the binding of 125 I-anti total PSA antibody with PSA were as follows: PSA concentration (150,200 μg protein),tracer antibody concentration (125,250 μg protein), p H (7.6,7.2), temp (15,25?C) and time (1.5 hrs) for postmenopausal benign and malignant uterus tumors tissue homogenates, respectively. The use of different concentrations of Na + and Mg 2+ ions were shown to cause an increase in the binding at concentration of (125,75 mΜ) of Na 1+ ions (75,225 mΜ) of Mg 2+ ions for benign and malignant uterus tumors homogenates, respectively, while the use of different concentrations of urea and polyethylene glycol (PEG) Caused a decrease in the binding with the increase in the concentration of each of urea and PEG in the both cases

  4. A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules

    DEFF Research Database (Denmark)

    Pandya, Mital; Rasmussen, Michael; Hansen, Andreas

    2015-01-01

    Major histocompatibility complex (MHC) class I molecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8+ T cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presentation......, and different antigen peptide motifs are associated with specific genetic sequences of class I molecules. Understanding bovine leukocyte antigen (BoLA), peptide-MHC class I binding specificities may facilitate development of vaccines or reagents for quantifying the adaptive immune response to intracellular...... pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network...

  5. The HLA-B*5101 molecule-binding capacity to antigens used in animal models of Behçet's disease: a bioinformatics study.

    Science.gov (United States)

    Baharav, Ehud; Weinberger, Abraham

    2012-07-01

    The human lymphocyte antigen (HLA) molecule B*5101 is a functioning receptor of the immune system and is generally accepted as a genetic marker for Behçet disease (BD), a multi-organ, chronic inflammatory disorder. The role of the HLA-B*5101 in the pathogenesis of BD is elusive. The assumption that HLA-B*5101 has an active role in BD is suggestive, but no antigen has yet been identified. To evaluate the potential binding capacity of various antigens to the HLA-B*5101 molecule. Using bioinformatics programs, we studied the binding capacity of HLA-B*5101 and its corresponding rat molecule RT.A1 to the following antigens: heatshock protein-60 (HSP60), major histocompatibility complex class I chain-related gene A (MICA), retinal S-antigen (S-Ag), HLA-B27 molecule and its peptide (PD) and tropomyosin (TPM), all of which serve as antigens in animal models corresponding to BD. In each protein including the B*5101 molecule itself, the computerized programs revealed several short sequences with potential high binding capacity to HLA-B*5101 with the exception of B-27PD. The rat MHC RT1. Al. had no binding capacity to S-Ag. The evaluated proteins have the potential to bind to and to serve as potential antigens to the HLA-B*5101 and the rat MHC RT1.Al. molecules. The pathogenicity of these suggested short peptides should be evaluated in animal models of BD.

  6. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    Abstract Background The surface glycoprotein (SU, gp120) of the human immunodeficiency virus (HIV) must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP) to bind the Duffy Antigen Receptor for Chemokines (DARC) and invade reticulocytes. Results Variable loop 3 (V3) of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, ...

  7. Carcinoma-specific Ulex europaeus agglutinin-I binding glycoproteins of human colorectal carcinoma and its relation to carcinoembryonic antigen.

    Science.gov (United States)

    Matsushita, Y; Yonezawa, S; Nakamura, T; Shimizu, S; Ozawa, M; Muramatsu, T; Sato, E

    1985-08-01

    Glycoproteins binding to Ulex europaeus agglutinin-I (UEA-I) lectin, which recognizes the terminal alpha-L-fucose residue, were analyzed in 18 cases of human colorectal carcinoma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by the Western blotting method. In the distal large bowel (descending and sigmoid colon and rectum), high-molecular-weight glycoproteins binding to UEA-I existed in carcinoma tissue but not in normal mucosa. In the proximal large bowel (ascending and transverse colon), high-molecular-weight glycoproteins binding to UEA-I were found both in normal mucosa and in carcinoma tissue, whereas those from the carcinoma tissue had an apparently lower molecular weight as compared to the weight of those from the normal mucosa. Thus there is a biochemical difference in UEA-I binding glycoproteins between the normal mucosa and the carcinoma tissue, although in our previous histochemical study no difference was observed in UEA-I binding glycoproteins of the proximal large bowel between the carcinoma tissue and the normal mucosa. Furthermore, carcinoembryonic antigen from the carcinoma tissue was found to have the same electrophoretical mobility as the UEA-I binding glycoproteins.

  8. Competitor analogs for defined T cell antigens: peptides incorporating a putative binding motif and polyproline or polyglycine spacers.

    Science.gov (United States)

    Maryanski, J L; Verdini, A S; Weber, P C; Salemme, F R; Corradin, G

    1990-01-12

    We describe a new approach for modeling antigenic peptides recognized by T cells. Peptide A24 170-182 can compete with other antigenic peptides that are recognized by H-2kd-restricted cytolytic T cells, presumably by binding to the Kd molecule. By comparing substituted A24 peptides as competitors in a functional competition assay, the A24 residues Tyr-171, Thr-178, and Leu-179 were identified as possible contact residues for Kd. A highly active competitor peptide analog was synthesized in which Tyr was separated from the Thr-Leu pair by a pentaproline spacer. The choice of proline allowed the prediction of a probable conformation for the analog when bound to the Kd molecule. The simplest conformation of the A24 peptide that allows the same spacing and orientation of the motif as in the analog would be a nearly extended polypeptide chain incorporating a single 3(10) helical turn or similar structural kink.

  9. MHC class II-derived peptides can bind to class II molecules, including self molecules, and prevent antigen presentation

    DEFF Research Database (Denmark)

    Rosloniec, E F; Vitez, L J; Buus, S

    1990-01-01

    the alpha k-3 peptide binds slightly less well. These combined data, suggesting that class II-derived peptides can bind to MHC class II molecules, including the autologous molecule from which they are derived, have important implications for the molecular basis of alloreactivity and autoreactivity. Further...... found in the first and third polymorphic regions (PMR) of the A alpha k chain (alpha k-1 and alpha k-3) were capable of inhibiting the presentation of three different HEL-derived peptide antigens to their appropriate T cells. In addition, the alpha k-1 peptide inhibited the presentation of the OVA(323......-339) immunodominant peptide to the I-Ad-restricted T cell hybridomas specific for it. Prepulsing experiments demonstrated that the PMR peptides were interacting with the APC and not with the T cell hybridomas. These observations were confirmed and extended by the demonstration that the alpha k-1 and alpha k-3...

  10. Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction

    International Nuclear Information System (INIS)

    Rebelo, Tânia S.C.R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M.H.; Noronha, João P.; Sales, M. Goreti F.

    2014-01-01

    Graphical abstract: EF13-201, Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction. - Abstract: This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10 −11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower

  11. Structural analysis of peptides capable of binding to more than one Ia antigen

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Colon, S

    1989-01-01

    The Ia binding regions were analyzed for three unrelated peptide Ag (sperm whale myoglobin 106-118, influenza hemagglutinin 130-142, and lambda repressor protein 12-26) for which binding to more than one Ia molecule has previously been demonstrated. By determining the binding profile of three...... separate series of truncated synthetic peptides, it was found that in all three cases the different Ia reactivities mapped to largely overlapping regions of the peptides; although, for two of the peptides, the regions involved in binding the different Ia specificities were distinct. Moreover, subtle...... differences were found to dramatically influence some, but not other, Ia reactivities. Using a large panel of synthetic peptides it was found that a significant correlation exists between the capacity of peptides to interact with different alleles of the same molecule (i.e., IAd and IAk), but no correlation...

  12. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1

    Directory of Open Access Journals (Sweden)

    Wang Pu

    2010-10-01

    Full Text Available Abstract The Epstein-Barr Virus (EBV Nuclear Antigen 1 (EBNA1 protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP, regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP combined with massively parallel deep-sequencing (ChIP-Seq was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA

  13. Diagnostic value of soluble receptor-binding cancer antigen expressed on SiSo cells and carcinoembryonic antigen in differentiating malignant from benign pleural effusion.

    Science.gov (United States)

    Dong, Jiahui; Sun, Gengyun; Zhu, Hongbin

    2016-03-01

    Diagnosis of malignant pleural effusion (MPE) remains a major clinical challenge. The aim of this study was to evaluate the diagnostic value of combined detection of receptor-binding cancer antigen expressed on SiSo cells (RCAS1) and carcinoembryonic antigen (CEA) in patients with MPE and benign pleural effusion (BPE). The serum and pleural fluid samples were collected from 53 patients diagnosed with MPE and 49 patients with BPE. Enzyme-linked immunosorbent assay was used to detect the concentration of RCAS1 in serum and pleural effusion. The clinical data and laboratory information, including CEA levels, were gathered from these cases. The concentration of RCAS1 in MPE was significantly higher than that of BPE (P < 0.001). There was no significant difference between the two serum groups. The diagnostic sensitivity and specificity of pleural fluid RCAS1 were 67.92 and 81.63 %, respectively, at the optimized cutoff value of 7.326 U/mL; meanwhile, the sensitivity and specificity of pleural fluid CEA were 83.02 and 91.84 % at the cutoff value of 3.93 ng/mL. The specificity could be elevated to 98.50 % in serial detection, while the sensitivity may be improved to 94.55 % in parallel detection. Serum RCAS1 concentration was only detected in 53 serum samples out of the 102 samples, indicating that serum RCAS1 may not be a better option in differential diagnosis of malignancies compared with serum CEA, of which the diagnostic sensitivity and specificity were 64.15 and 83.67 % at the cutoff value of 3.90 ng/mL. No significant differences were found in pleural fluid RCAS1 concentration in MPE patients with different ages, gender, and pathological types of lung cancers. The detection of RCAS1 concentration in pleural fluid is informative for the diagnosis of MPE. Joint detection of RCAS1 and CEA can improve the diagnostic sensitivity and specificity. However, the diagnostic value of RCAS1 is not higher than that of CEA.

  14. Cytotoxic T-lymphocyte Antigen-4 Binding to SHP2 Interacting ...

    African Journals Online (AJOL)

    in 1% Triton X-100 lysis buffer, subjected to SDS-PAGE, and immunoblotted with anti-pTyr Ab (upper panel) or anti-SIT Ab (lower panel). Positions of molecular mass markers (kDa) are indicated. C, Densitometric analysis using the Scantjet laser scanner (Hewlett-Packard) of anti-pTyr binding to SIT (B, upper panel). Values ...

  15. Carbon-13 NMR study of switch variant anti-dansyl antibodies: Antigen binding and domain-domain interactions

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Koichi; Matsunaga, Chigusa; Odaka, Asano; Yamato, Sumie; Takaha, Wakana; Shimada, Ichio; Arata, Yoji (Univ. of Tokyo (Japan))

    1991-07-02

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of the antibodies has been selectively labeled with {sup 13}C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by folowing the procedure described previously for the switch variant antibodies labeled with (1-{sup 13}C)Met. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, the authors discuss how {sup 13}C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule.

  16. Carbon-13 NMR study of switch variant anti-dansyl antibodies: Antigen binding and domain-domain interactions

    International Nuclear Information System (INIS)

    Kato, Koichi; Matsunaga, Chigusa; Odaka, Asano; Yamato, Sumie; Takaha, Wakana; Shimada, Ichio; Arata, Yoji

    1991-01-01

    A 13 C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical V H , V L , and C L domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of the antibodies has been selectively labeled with 13 C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by folowing the procedure described previously for the switch variant antibodies labeled with [1- 13 C]Met. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, the authors discuss how 13 C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule

  17. Frequency of Helicobacter pylori blood-group antigen-binding adhesion 2 and sialic acid binding adhesion genes among dyspeptic patients in Tabriz, Iran

    Directory of Open Access Journals (Sweden)

    Leila Yousefi

    2015-06-01

    Full Text Available Introduction: The purpose of this research was to analyze blood-group antigen-binding adhesion (babA2 and sialic acid binding adhesion (sabA genotypes status in Helicobacter pylori (H. pylori isolates and their relationship with clinical outcomes. Methods: Gastric biopsy specimens were homogenized and placed in Brucella agar medium supplemented with 5% sheep blood and 3 antibiotics and were cultured at 37 °C under microaerophilic conditions and incubated for 4-7 days. H. pylori was identified by typical morphology, gram-staining and urease tests, and babA2 and sabA genes were detected by polymerase chain reaction (PCR. Results: From a total of 100 H. pylori isolates; babA2 and sabA genes were detected in 23.0 and 26.4%, respectively. There was a significant relationship between these genes and clinical outcomes (P < 0.050. Conclusion: We found that the babA2 status was not related to clinical outcomes in Tabriz, Iran. However, sabA was a promoting determinant for disease, and multivariate analysis disclosed sabA to be an independent marker of non-ulcer diseases in our subjects.

  18. Genotoxic effect and antigen binding characteristics of SLE auto-antibodies to peroxynitrite-modified human DNA.

    Science.gov (United States)

    Khan, Md Asad; Alam, Khursheed; Mehdi, Syed Hassan; Rizvi, M Moshahid A

    2017-12-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by auto-antibodies against native deoxyribonucleic acid after modification and is one of the reasons for the development of SLE. Here, we have evaluated the structural perturbations in human placental DNA by peroxynitrite using spectroscopy, thermal denaturation and high-performance liquid chromatography (HPLC). Peroxynitrite is a powerful potent bi-functional oxidative/nitrative agent that is produced both endogenously and exogenously. In experimental animals, the peroxynitrite-modified DNA was found to be highly immunogenic. The induced antibodies showed cross-reactions with different types of DNA and nitrogen bases that were modified with peroxynitrite by inhibition ELISA. The antibody activity was inhibited by approximately 89% with its immunogen as the inhibitor. The antigen-antibodies interaction between induced antibodies with peroxynitrite-modified DNA showed retarded mobility as compared to the native form. Furthermore, significantly increased binding was also observed in SLE autoantibodies with peroxynitrite-modified DNA than native form. Moreover, DNA isolated from lymphocyte of SLE patients revealed significant recognition of anti-peroxynitrite-modified DNA immunoglobulin G (IgG). Our data indicates that DNA modified with peroxynitrite presents unique antigenic determinants that may induce autoantibody response in SLE. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. An analytical approach to reduce between-plate variation in multiplex assays that measure antibodies to Plasmodium falciparum antigens.

    Science.gov (United States)

    Fang, Rui; Wey, Andrew; Bobbili, Naveen K; Leke, Rose F G; Taylor, Diane Wallace; Chen, John J

    2017-07-17

    Antibodies play an important role in immunity to malaria. Recent studies show that antibodies to multiple antigens, as well as, the overall breadth of the response are associated with protection from malaria. Yet, the variability and reliability of antibody measurements against a combination of malarial antigens using multiplex assays have not been well characterized. A normalization procedure for reducing between-plate variation using replicates of pooled positive and negative controls was investigated. Sixty test samples (30 from malaria-positive and 30 malaria-negative individuals), together with five pooled positive-controls and two pooled negative-controls, were screened for antibody levels to 9 malarial antigens, including merozoite antigens (AMA1, EBA175, MSP1, MSP2, MSP3, MSP11, Pf41), sporozoite CSP, and pregnancy-associated VAR2CSA. The antibody levels were measured in triplicate on each of 3 plates, and the experiments were replicated on two different days by the same technician. The performance of the proposed normalization procedure was evaluated with the pooled controls for the test samples on both the linear and natural-log scales. Compared with data on the linear scale, the natural-log transformed data were less skewed and reduced the mean-variance relationship. The proposed normalization procedure using pooled controls on the natural-log scale significantly reduced between-plate variation. For malaria-related research that measure antibodies to multiple antigens with multiplex assays, the natural-log transformation is recommended for data analysis and use of the normalization procedure with multiple pooled controls can improve the precision of antibody measurements.

  20. Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.

    Science.gov (United States)

    Kramers, C; Hylkema, M N; van Bruggen, M C; van de Lagemaat, R; Dijkman, H B; Assmann, K J; Smeenk, R J; Berden, J H

    1994-01-01

    Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis. Images PMID:8040312

  1. Human leukocyte antigen (HLA class I restricted epitope discovery in yellow fewer and dengue viruses: importance of HLA binding strength.

    Directory of Open Access Journals (Sweden)

    Ole Lund

    Full Text Available Epitopes from all available full-length sequences of yellow fever virus (YFV and dengue fever virus (DENV restricted by Human Leukocyte Antigen class I (HLA-I alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV epitopes were selected using the EpiSelect algorithm to allow for optimal coverage of viral strains. The selected predicted epitopes were synthesized and approximately 75% were found to bind the predicted restricting HLA molecule with an affinity, K(D, stronger than 500 nM. The immunogenicity of 25 HLA-A*02:01, 28 HLA-A*24:02 and 28 HLA-B*07:02 binding peptides was tested in three HLA-transgenic mice models and led to the identification of 17 HLA-A*02:01, 4 HLA-A*2402 and 4 HLA-B*07:02 immunogenic peptides. The immunogenic peptides bound HLA significantly stronger than the non-immunogenic peptides. All except one of the immunogenic peptides had K(D below 100 nM and the peptides with K(D below 5 nM were more likely to be immunogenic. In addition, all the immunogenic peptides that were identified as having a high functional avidity had K(D below 20 nM. A*02:01 transgenic mice were also inoculated twice with the 17DD YFV vaccine strain. Three of the YFV A*02:01 restricted peptides activated T-cells from the infected mice in vitro. All three peptides that elicited responses had an HLA binding affinity of 2 nM or less. The results indicate the importance of the strength of HLA binding in shaping the immune response.

  2. I-Ad-binding peptides derived from unrelated protein antigens share a common structural motif

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Colon, S

    1988-01-01

    on the I-Ad binding of the immunogenic peptide OVA 323-339. The results obtained demonstrated the very permissive nature of Ag-Ia interaction. We also showed that unrelated peptides that are good I-Ad binders share a common structural motif and speculated that recognition of such motifs could represent...... that I-Ad molecules recognize a large library of Ag by virtue of common structural motifs present in peptides derived from phylogenetically unrelated proteins....

  3. Using molecular principal axes for structural comparison: determining the tertiary changes of a FAB antibody domain induced by antigenic binding

    Directory of Open Access Journals (Sweden)

    Silverman B David

    2007-11-01

    Full Text Available Abstract Background Comparison of different protein x-ray structures has previously been made in a number of different ways; for example, by visual examination, by differences in the locations of secondary structures, by explicit superposition of structural elements, e.g. α-carbon atom locations, or by procedures that utilize a common symmetry element or geometrical feature of the structures to be compared. Results A new approach is applied to determine the structural changes that an antibody protein domain experiences upon its interaction with an antigenic target. These changes are determined with the use of two different, however comparable, sets of principal axes that are obtained by diagonalizing the second-order tensors that yield the moments-of-geometry as well as an ellipsoidal characterization of domain shape, prior to and after interaction. Determination of these sets of axes for structural comparison requires no internal symmetry features of the domains, depending solely upon their representation in three-dimensional space. This representation may involve atomic, Cα, or residue centroid coordinates. The present analysis utilizes residue centroids. When the structural changes are minimal, the principal axes of the domains, prior to and after interaction, are essentially comparable and consequently may be used for structural comparison. When the differences of the axes cannot be neglected, but are nevertheless slight, a smaller relatively invariant substructure of the domains may be utilized for comparison. The procedure yields two distance metrics for structural comparison. First, the displacements of the residue centroids due to antigenic binding, referenced to the ellipsoidal principal axes, are noted. Second, changes in the ellipsoidal distances with respect to the non-interacting structure provide a direct measure of the spatial displacements of the residue centroids, towards either the interior or exterior of the domain

  4. Detection of a nuclear, EBNA-type antigen in apparently EBNA-negative Herpesvirus papio (HVP)-transformed lymphoid lines by the acid-fixed nuclear binding technique.

    Science.gov (United States)

    Ohno, S; Luka, J; Falk, L; Klein, G

    1977-12-15

    In agreement with the findings of previous authors, we could not detect a virally determined nuclear antigen in Herpesvirus papio (HVP)-transformed baboon lymphoid lines by anticomplementary staining in situ, as for EBNA. However, by means of our recently developed acid-fixed nuclear binding technique an EBNA-like antigen could be readily demonstrated, after extraction from both producer and non-producer lines. We propose to designate the antigen as HUPNA. It can be detected by a human anti-EBNA antibody, suggesting cross-reactivity, if not identity, between EBNA and HUPNA. HVP-DNA carrying non-producer lines, negative for in situ ACIF stainability but capable of yielding HUPNA by the nuclear binding technique, can be superinfected with EBV, with brilliant EBNA expression as the result, suggesting that the defective in situ staining is a property associated with the baboon HVP, rather than the baboon lymphoid cell per se.

  5. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains

    International Nuclear Information System (INIS)

    LingNah Su; Little, J.B.

    1992-01-01

    A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D o 192 ± 13 vs 127 ± 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. (author)

  6. Humoral markers of active Epstein-Barr virus infection associate with anti-extractable nuclear antigen autoantibodies and plasma galectin-3 binding protein in systemic lupus erythematosus.

    Science.gov (United States)

    Rasmussen, N S; Nielsen, C T; Houen, G; Jacobsen, S

    2016-12-01

    We investigated if signs of active Epstein-Barr virus and cytomegalovirus infections associate with certain autoantibodies and a marker of type I interferon activity in patients with systemic lupus erythematosus. IgM and IgG plasma levels against Epstein-Barr virus early antigen diffuse and cytomegalovirus pp52 were applied as humoral markers of ongoing/recently active Epstein-Barr virus and cytomegalovirus infections, respectively. Plasma galectin-3 binding protein served as a surrogate marker of type I interferon activity. The measurements were conducted in 57 systemic lupus erythematosus patients and 29 healthy controls using ELISAs. Regression analyses and univariate comparisons were performed for associative evaluation between virus serology, plasma galectin-3 binding protein and autoantibodies, along with other clinical and demographic parameters. Plasma galectin-3 binding protein concentrations were significantly higher in systemic lupus erythematosus patients (P = 0.009) and associated positively with Epstein-Barr virus early antigen diffuse-directed antibodies and the presence of autoantibodies against extractable nuclear antigens in adjusted linear regressions (B = 2.02 and 2.02, P = 0.02 and P = 0.002, respectively). Furthermore, systemic lupus erythematosus patients with anti-extractable nuclear antigens had significantly higher antibody levels against Epstein-Barr virus early antigen diffuse (P = 0.02). Our study supports a link between active Epstein-Barr virus infections, positivity for anti-extractable nuclear antigens and increased plasma galectin-3 binding protein concentrations/type I interferon activity in systemic lupus erythematosus patients. © The Author(s) 2016.

  7. Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses.

    Science.gov (United States)

    Parker, Lauren; Wharton, Stephen A; Martin, Stephen R; Cross, Karen; Lin, Yipu; Liu, Yan; Feizi, Ten; Daniels, Rodney S; McCauley, John W

    2016-06-01

    Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

  8. Antigen-binding properties of monoclonal antibodies reactive with EBNA1 and use in immunoaffinity chromatography.

    Directory of Open Access Journals (Sweden)

    Sarah J Duellman

    Full Text Available Epstein-Barr virus (EBV nuclear antigen 1 (EBNA1 was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG(1 and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6 fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.

  9. Quantitative analysis of rat Ig (sub)classes binding to cell surface antigens

    International Nuclear Information System (INIS)

    Nilsson, R.; Brodin, T.; Sjoegren, H.-O.

    1982-01-01

    An indirect 125 I-labeled protein A assay for detection of cell surface-bound rat immunoglobulins is presented. The assay is quantitative and rapid and detects as little as 1 ng of cell surface-bound Ig. It discriminates between antibodies belonging to different IgG subclasses, IgM and IgA. The authors describe the production and specificity control of the reagents used and show that the test can be used for quantitative analysis. A large number of sera from untreated rats are tested to evaluate the frequency of falsely positive responses and variation due to age, sex and strain of rat. With this test it is relatively easy to quantitate the binding of classes and subclasses of rat immunoglobulins in a small volume (6 μl) of untreated serum. (Auth.)

  10. An Extended Surface Loop on Toxoplasma gondii Apical Membrane Antigen 1 (AMA1 Governs Ligand Binding Selectivity.

    Directory of Open Access Journals (Sweden)

    Michelle L Parker

    Full Text Available Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs on the parasite surface and rhoptry neck 2 (RON2 proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1ΔDIIloop. Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2 peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1ΔDIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1ΔDIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON

  11. Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

    Science.gov (United States)

    Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

    2015-02-02

    We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

  12. Proliferating cell nuclear antigen binds DNA polymerase-β and mediates 1-methyl-4-phenylpyridinium-induced neuronal death.

    Directory of Open Access Journals (Sweden)

    Zhentao Zhang

    Full Text Available The mechanisms leading to dopaminergic neuronal loss in the substantia nigra of patients with Parkinson disease (PD remain poorly understood. We recently reported that aberrant DNA replication mediated by DNA polymerase-β (DNA pol-β plays a causal role in the death of postmitotic neurons in an in vitro model of PD. In the present study, we show that both proliferating cell nuclear antigen (PCNA and DNA pol-β are required for MPP(+-induced neuronal death. PCNA binds to the catalytic domain of DNA pol-β in MPP(+-treated neurons and in post-mortem brain tissues of PD patients. The PCNA-DNA pol-β complex is loaded into DNA replication forks and mediates DNA replication in postmitotic neurons. The aberrant DNA replication mediated by the PCNA-DNA pol-β complex induces p53-dependent neuronal cell death. Our results indicate that the interaction of PCNA and DNA pol-β contributes to neuronal death in PD.

  13. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    International Nuclear Information System (INIS)

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-01-01

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

  14. Humoral markers of active Epstein-Barr virus infection associate with anti-extractable nuclear antigen autoantibodies and plasma galectin-3 binding protein in systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Rasmussen, N S; Nielsen, C T; Houen, G

    2016-01-01

    We investigated if signs of active Epstein-Barr virus and cytomegalovirus infections associate with certain autoantibodies and a marker of type I interferon activity in patients with systemic lupus erythematosus. IgM and IgG plasma levels against Epstein-Barr virus early antigen diffuse...... and cytomegalovirus pp52 were applied as humoral markers of ongoing/recently active Epstein-Barr virus and cytomegalovirus infections, respectively. Plasma galectin-3 binding protein served as a surrogate marker of type I interferon activity. The measurements were conducted in 57 systemic lupus erythematosus patients...... concentrations were significantly higher in systemic lupus erythematosus patients (P = 0.009) and associated positively with Epstein-Barr virus early antigen diffuse-directed antibodies and the presence of autoantibodies against extractable nuclear antigens in adjusted linear regressions (B = 2.02 and 2.02, P...

  15. Specific T-cell recognition of the merozoite proteins rhoptry-associated protein 1 and erythrocyte-binding antigen 1 of Plasmodium falciparum

    DEFF Research Database (Denmark)

    Jakobsen, P H; Hviid, L; Theander, T G

    1993-01-01

    The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living i...... by individuals living in an area with a high transmission rate of malaria. Most of the donor plasma samples tested contained immunoglobulin G (IgG) and IgM antibodies recognizing the merozoite proteins, while only a minority showed high IgG reactivity to the synthetic peptides.......The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living...

  16. RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

    Science.gov (United States)

    Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

    2013-01-01

    T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

  17. Binding Properties of Streptococcus gordonii SspA and SspB (Antigen I/II Family) Polypeptides Expressed on the Cell Surface of Lactococcus lactis MG1363

    OpenAIRE

    Holmes, Ann R.; Gilbert, Christophe; Wells, Jeremy M.; Jenkinson, Howard F.

    1998-01-01

    The oral bacterium Streptococcus gordonii expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as Actinomyces naeslundii. To determine if SspA and SspB have differential binding propert...

  18. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    Abstract Background The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infectio...

  19. Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain

    International Nuclear Information System (INIS)

    Meinke, Gretchen; Phelan, Paul; Fradet-Turcotte, Amélie; Archambault, Jacques; Bullock, Peter A.

    2011-01-01

    With the aim of forming the ‘lock-washer’ conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner

  20. The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance.

    Science.gov (United States)

    Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

    2003-07-05

    Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.

  1. The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance

    International Nuclear Information System (INIS)

    Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

    2003-01-01

    Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable

  2. Use of thermodynamic coupling between antibody-antigen binding and phospholipid acyl chain phase transition energetics to predict immunoliposome targeting affinity.

    Science.gov (United States)

    Klegerman, Melvin E; Zou, Yuejiao; Golunski, Eva; Peng, Tao; Huang, Shao-Ling; McPherson, David D

    2014-09-01

    Thermodynamic analysis of ligand-target binding has been a useful tool for dissecting the nature of the binding mechanism and, therefore, potentially can provide valuable information regarding the utility of targeted formulations. Based on a consistent coupling of antibody-antigen binding and gel-liquid crystal transition energetics observed for antibody-phosphatidylethanolamine (Ab-PE) conjugates, we hypothesized that the thermodynamic parameters and the affinity for antigen of the Ab-PE conjugates could be effectively predicted once the corresponding information for the unconjugated antibody is determined. This hypothesis has now been tested in nine different antibody-targeted echogenic liposome (ELIP) preparations, where antibody is conjugated to dipalmitoylphosphatidylethanolamine (DPPE) head groups through a thioether linkage. Predictions were satisfactory (affinity not significantly different from the population of values found) in five cases (55.6%), but the affinity of the unconjugated antibody was not significantly different from the population of values found in six cases (66.7%), indicating that the affinities of the conjugated antibody tended not to deviate appreciably from those of the free antibody. While knowledge of the affinities of free antibodies may be sufficient to judge their suitability as targeting agents, thermodynamic analysis may still provide valuable information regarding their usefulness for specific applications.

  3. Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs.

    Science.gov (United States)

    Juhl, David; Marget, Matthias; Hallensleben, Michael; Görg, Siegfried; Ziemann, Malte

    2017-03-01

    Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Circulating immune complexes in the serum in systemic lupus erythematosus and in carriers of hepatitis B antigen: quantitation by binding to radiolabeled Cl/sub q/

    International Nuclear Information System (INIS)

    Nydegger, U.E.; Lambert, P.H.; Gerber, H.; Miescher, P.A.

    1974-01-01

    A sensitive and reproducible procedure fr the detection of souble immune complexes in sera from patients with various immunopathological disorders is reported. Radiolabeled Clq is reacted with sera containing immune complexes. Separation of free from complex bound [ 125 I]Clq is achieved by selective precipitation with polyethylene glycol (PEG). The minimal amount of aggregated immunoglobulins thus detected is about 10 μg and that of soluble human IgG-anti-IgG complexes is about 3 μg of complexed antibody. Some immune complexes formed in large antigen excess (Ag 2 Ab) can still be detected by this radiolabeled Clq bining assay. In a second step, this radiolabeled Clq binding assay was applied to an experimental model of immune complex disease and was shown to be efficient for the detection of in vivo formed immune complexes. Finally, the technique could be applied to the study of sera from patients with systemic lupus erythematosus (SLE) or to carriers of the Hepatitis B antigen (HB-Ag). Particularly high values were seen in active disease, a finding which was confirmed by follow-up studies performed with four SLE patients. No increased [ 125 I]Clq binding was seen in 18 healthy carriers of the HB-Ag; whereas, sera from carriers with hepatitis appear to precipitate increased [ 125 I]Clq percentages: 7/24 cases with acute transient and 4/7 cases with chronic persistent hepatitis were found to increasingly bind [ 125 I]Clq. The results were also used for a correlative study of [ 125 I]Clq binding to IgG levels in the sera but increased [ 125 I]Clq binding could not be attributed to high serum IgG levels which are likely to account for the evaluation of immune complex diseases in human pathology. (U.S.)

  5. Multivalent Soluble Antigen Arrays Exhibit High Avidity Binding and Modulation of B Cell Receptor-Mediated Signaling to Drive Efficacy against Experimental Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Hartwell, Brittany L; Pickens, Chad J; Leon, Martin; Berkland, Cory

    2017-06-12

    A pressing need exists for antigen-specific immunotherapies (ASIT) that induce selective tolerance in autoimmune disease while avoiding deleterious global immunosuppression. Multivalent soluble antigen arrays (SAgA PLP:LABL ), consisting of a hyaluronic acid (HA) linear polymer backbone cografted with multiple copies of autoantigen (PLP) and cell adhesion inhibitor (LABL) peptides, are designed to induce tolerance to a specific multiple sclerosis (MS) autoantigen. Previous studies established that hydrolyzable SAgA PLP:LABL , employing a degradable linker to codeliver PLP and LABL, was therapeutic in experimental autoimmune encephalomyelitis (EAE) in vivo and exhibited antigen-specific binding with B cells, targeted the B cell receptor (BCR), and dampened BCR-mediated signaling in vitro. Our results pointed to sustained BCR engagement as the SAgA PLP:LABL therapeutic mechanism, so we developed a new version of the SAgA molecule using nonhydrolyzable conjugation chemistry, hypothesizing it would enhance and maintain the molecule's action at the cell surface to improve efficacy. "Click SAgA" (cSAgA PLP:LABL ) uses hydrolytically stable covalent conjugation chemistry (Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC)) rather than a hydrolyzable oxime bond to attach PLP and LABL to HA. We explored cSAgA PLP:LABL B cell engagement and modulation of BCR-mediated signaling in vitro through flow cytometry binding and calcium flux signaling assays. Indeed, cSAgA PLP:LABL exhibited higher avidity B cell binding and greater dampening of BCR-mediated signaling than hydrolyzable SAgA PLP:LABL . Furthermore, cSAgA PLP:LABL exhibited significantly enhanced in vivo efficacy compared to hydrolyzable SAgA PLP:LABL , achieving equivalent efficacy at one-quarter of the dose. These results indicate that nonhydrolyzable conjugation increased the avidity of cSAgA PLP:LABL to drive in vivo efficacy through modulated BCR-mediated signaling.

  6. Glycosphingolipid antigens from Leishmania (L. amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    A.H. Straus

    1997-03-01

    Full Text Available Specific glycosphingolipid antigens of Leishmania (L. amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC. Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L. amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues

  7. Improved Prediction of Bovine Leucocyte Antigens (BoLA) Presented Ligands by Use of Mass-Spectrometry-Determined Ligand and in Vitro Binding Data

    DEFF Research Database (Denmark)

    Nielsen, Morten; Connelley, Tim; Ternette, Nicola

    2018-01-01

    Peptide binding to MHC class I molecules is the single most selective step in antigen presentation and the strongest single correlate to peptide cellular immunogenicity. The cost of experimentally characterizing the rules of peptide presentation for a given MHC-I molecule is extensive, and predic...... available at http://www.cbs.dtu.dk/services/NetMHCpan/NetBoLApan . The approach has here been applied to the BoLA-I system, but the pipeline is readily applicable to MHC systems in other species....

  8. High throughput testing of the SV40 Large T antigen binding to cellular p53 identifies putative drugs for the treatment of SV40-related cancers

    International Nuclear Information System (INIS)

    Carbone, Michele; Rudzinski, Jennifer; Bocchetta, Maurizio

    2003-01-01

    SV40 has been linked to some human malignancies, and the evidence that this virus plays a causative role in mesothelioma and brain tumors is mounting. The major SV40 oncoprotein is the Large tumor antigen (Tag). A key Tag transforming activity is connected to its capability to bind and inactivate cellular p53. In this study we developed an effective, high throughput, ELISA-based method to study Tag-p53 interaction in vitro. This assay allowed us to screen a chemical library and to identify a chemical inhibitor of the Tag binding to p53. We propose that our in vitro assay is a useful method to identify molecules that may be used as therapeutic agents for the treatment of SV40-related human cancers

  9. A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules

    Czech Academy of Sciences Publication Activity Database

    Zhang, A.X.; Murelli, R.P.; Bařinka, Cyril; Michel, J.; Cocleaza, A.; Jorgensen, W.L.; Lubkowski, J.; Spiegel, D.A.

    2010-01-01

    Roč. 132, č. 36 (2010), s. 12711-12716 ISSN 0002-7863 Institutional research plan: CEZ:AV0Z50520701 Keywords : Prostate -specific membrane antigen * antibody recruiting molecules * Structure-activity relationship Subject RIV: CE - Biochemistry Impact factor: 9.019, year: 2010

  10. Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2

    International Nuclear Information System (INIS)

    Katagiri, Yohko U.; Kiyokawa, Nobutaka; Nakamura, Kyoko; Takenouchi, Hisami; Taguchi, Tomoko; Okita, Hajime; Umezawa, Akihiro; Fujimoto, Junichiro

    2005-01-01

    We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [ 14 C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP

  11. Role of N-terminal His6-Tags in binding and efficient translocation of polypeptides into cells using anthrax protective antigen (PA.

    Directory of Open Access Journals (Sweden)

    Christoph Beitzinger

    Full Text Available It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB(7/8-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His(6-tag to different proteins increased their binding affinity to the protective antigen (PA PA(63-channels, irrespective if they are related (C2I or unrelated (gpJ, EDIN to the AB(7/8-family of toxins. His(6-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to -70 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA(63. Cell-based experiments demonstrated that addition of an N-terminal His(6-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA(63. Our results revealed that addition of His(6-tags to several factors increase their binding properties to PA(63 and enhance the property to intoxicate cells.

  12. The binding parameters of radiolabelled monoclonal F (ab')2 and Fab' fragments relative to immunoglobulin G in reactions with surface-bound antigens

    International Nuclear Information System (INIS)

    Fjeld, J.G.; Nustad, K.; Michaelsen, T.E.

    1992-01-01

    The binding parameters of iodine-125-labelled intact monoclonal immunoglobulin G (IgG), F(ab') 2 and Fab' fragments were compared. The study was carried out with the two monoclonal antibodies (MoAbs) K13 and K16 specific for human Ig light chains κ and λ, respectively. When testing the 125 I-MoAbs against monodisperse polymer particles coated with the specific antigens, the K a for the F(ab') 2 fragments were similar to that for IgG, while the K a for the Fab' fragments were reduced to 10%-20% of that for IgG. The number N of effective target sites revealed with Fab' was higher than with F(ab') and IgG, presumably because less surface area is occupied by the small Fab' molecules. The immunoreactive fraction F ranged according to IgG>F(ab') 2 >Fab'. The explanation of the moderate difference between the K a of the monoclonal Fab' and the divalent IgG and F(ab') 2 was that the divalent molecules were not divalently attached to the particles. When testing the same antibody preparations against humanlymphoma cells producing Ig with light chains κ or λ, the binding results were less reliable than when particles were utilised, presumably due to antigen shedding. Different MoAbs vary in their loss of immunoreactivity due to enzymatic degradation and the radiolabelling procedure. The preparation of the radiolabelled fragments should therefore be optimized for each MoAb, and evaluation is necessary before injection. Artificial targets with a low leakage of antigen, like the monodisperse polymer particles here applied, are recommended for the in vitro evaluation of the immunoreactivity of labelled MoAb preparations. (orig.)

  13. Timing of the human prenatal antibody response to Plasmodium falciparum antigens.

    Directory of Open Access Journals (Sweden)

    Samuel Tassi Yunga

    Full Text Available Plasmodium falciparum (Pf-specific T- and B-cell responses may be present at birth; however, when during fetal development antibodies are produced is unknown. Accordingly, cord blood samples from 232 preterm (20-37 weeks of gestation and 450 term (≥37 weeks babies were screened for IgM to Pf blood-stage antigens MSP1, MSP2, AMA1, EBA175 and RESA. Overall, 25% [95% CI = 22-28%] of the 682 newborns were positive for IgM to ≥1 Pf antigens with the earliest response occurring at 22 weeks. Interestingly, the odds of being positive for cord blood Pf IgM decreased with gestational age (adjusted OR [95% CI] at 20-31 weeks = 2.55 [1.14-5.85] and at 32-36 weeks = 1.97 [0.92-4.29], with ≥37 weeks as reference; however, preterm and term newborns had similar levels of Pf IgM and recognized a comparable breadth of antigens. Having cord blood Pf IgM was associated with placental malaria (adjusted OR [95% CI] = 2.37 [1.25-4.54]. To determine if in utero exposure occurred via transplacental transfer of Pf-IgG immune complexes (IC, IC containing MSP1 and MSP2 were measured in plasma of 242 mother-newborn pairs. Among newborns of IC-positive mothers (77/242, the proportion of cord samples with Pf IC increased with gestational age but was not associated with Pf IgM, suggesting that fetal B cells early in gestation had not been primed by IC. Finally, when cord mononuclear cells from 64 term newborns were cultured in vitro, only 11% (7/64 of supernatants had Pf IgM; whereas, 95% (61/64 contained secreted Pf IgG. These data suggest fetal B cells are capable of making Pf-specific IgM from early in the second trimester and undergo isotype switching to IgG towards term.

  14. Predicted MHC peptide binding promiscuity explains MHC class I 'hotspots' of antigen presentation defined by mass spectrometry eluted ligand data.

    Science.gov (United States)

    Jappe, Emma Christine; Kringelum, Jens; Trolle, Thomas; Nielsen, Morten

    2018-02-15

    Peptides that bind to and are presented by MHC class I and class II molecules collectively make up the immunopeptidome. In the context of vaccine development, an understanding of the immunopeptidome is essential, and much effort has been dedicated to its accurate and cost-effective identification. Current state-of-the-art methods mainly comprise in silico tools for predicting MHC binding, which is strongly correlated with peptide immunogenicity. However, only a small proportion of the peptides that bind to MHC molecules are, in fact, immunogenic, and substantial work has been dedicated to uncovering additional determinants of peptide immunogenicity. In this context, and in light of recent advancements in mass spectrometry (MS), the existence of immunological hotspots has been given new life, inciting the hypothesis that hotspots are associated with MHC class I peptide immunogenicity. We here introduce a precise terminology for defining these hotspots and carry out a systematic analysis of MS and in silico predicted hotspots. We find that hotspots defined from MS data are largely captured by peptide binding predictions, enabling their replication in silico. This leads us to conclude that hotspots, to a great degree, are simply a result of promiscuous HLA binding, which disproves the hypothesis that the identification of hotspots provides novel information in the context of immunogenic peptide prediction. Furthermore, our analyses demonstrate that the signal of ligand processing, although present in the MS data, has very low predictive power to discriminate between MS and in silico defined hotspots. © 2018 John Wiley & Sons Ltd.

  15. Structure and calcium binding activity of LipL32, the major surface antigen of pathogenic Leptospira sp

    International Nuclear Information System (INIS)

    Hauk, Pricila; Roman-Ramos, Henrique; Ho, Paulo Lee; Guzzo, Cristiane R.; Farah, Chuck S.

    2009-01-01

    Leptospirosis, caused by the spirochaete Leptospira is an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospira. It is highly immunogenic and has been shown to bind to host extracellular matrix components, including collagens, fibronectin and laminin. In this work we crystallized recombinant LipL32 protein and determined its structure to 2.25 A resolution. Initial phases were determined using the multi-wavelength anomalous dispersion technique with data collected from selenomethionine-containing crystals at the MX2 beamline at the LNLS. The LipL32 monomer is made of a jelly-roll fold core from which protrude several peripheral secondary structures. Some structural features suggested that LipL32 could bind Ca 2+ ions and indeed, spectroscopic data (circular (dichroism. intrinsic tryptophan fluorescence and extrinsic 1-amino-2-anaphthol-4-sulfonic acid fluorescence) confirmed the calcium binding properties of LipL32. (author)

  16. An integrated top-down and bottom-up proteomic approach to characterize the antigen-binding fragment of antibodies.

    Science.gov (United States)

    Dekker, Lennard; Wu, Si; Vanduijn, Martijn; Tolić, Nikolai; Stingl, Christoph; Zhao, Rui; Luider, Theo; Paša-Tolić, Ljiljana

    2014-05-01

    We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens toward which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step toward the identification of disease specific antibodies in patient samples with potentially significant clinical impact. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Autoantibodies from patients with systemic lupus erythematosus bind a shared sequence of SmD and Epstein-Barr virus-encoded nuclear antigen EBNA I.

    Science.gov (United States)

    Sabbatini, A; Bombardieri, S; Migliorini, P

    1993-05-01

    SmD is one of the small nuclear ribonucleoproteins frequently targeted by autoantibodies in systemic lupus erythematosus. We isolated and characterized the antibodies present in lupus sera that are specific for the C-terminal region of SmD (sequence 95-119). This region is highly homologous to sequence 35-58 of the EBNA I antigen, one of the nuclear antigens induced by infection with Epstein-Barr virus. Antibodies affinity purified over a peptide 95-119 column were able to recognize this sequence in the context of the whole SmD molecule, as they reacted with blotted recombinant SmD. Anti-SmD 95-119 antibodies bound also the EBNA I 35-58 peptide and detected the EBNA I molecule in a total cell extract from Epstein-Barr virus-infected lines. A population of anti-SmD antibodies is, therefore, able to bind an epitope shared by the autoantigen and the viral antigen EBNA I. To investigate the involvement of this shared epitope in the generation of anti-SmD antibodies, we immunized mice with the EBNA I 35-58 peptide. Sera from immunized animals displayed the same pattern of reactivity of spontaneously produced anti-SmD antibodies. They reacted in fact with the EBNA peptide as well as with SmD 95-119 and recombinant SmD. These data suggest that molecular mimicry may play a role in the induction of anti-SmD autoantibodies.

  18. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Seho [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Lim, Chunghun [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Lee, Jae Young [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Song, Yoon-Jae [Department of Life Science, Kyungwon University, Seongnam-Si, Kyeonggi-Do 461-701 (Korea, Republic of); Park, Junsoo [Division of Biological Science and Technology, Yonsei University, Wonju 220-100 (Korea, Republic of); Choe, Joonho [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Seo, Taegun, E-mail: tseo@dongguk.edu [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)

    2010-04-16

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  19. DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication

    International Nuclear Information System (INIS)

    Cha, Seho; Lim, Chunghun; Lee, Jae Young; Song, Yoon-Jae; Park, Junsoo; Choe, Joonho; Seo, Taegun

    2010-01-01

    During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

  20. A FRET-based high throughput screening assay to identify inhibitors of anthrax protective antigen binding to capillary morphogenesis gene 2 protein.

    Directory of Open Access Journals (Sweden)

    Michael S Rogers

    Full Text Available Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA, a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2 protein and tumor endothelial marker 8 (TEM8. Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein-protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.

  1. Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity.

    Science.gov (United States)

    Dosenovic, Pia; Kara, Ervin E; Pettersson, Anna-Klara; McGuire, Andrew T; Gray, Matthew; Hartweger, Harald; Thientosapol, Eddy S; Stamatatos, Leonidas; Nussenzweig, Michel C

    2018-04-16

    The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.

  2. Structure and calcium binding activity of LipL32, the major surface antigen of pathogenic Leptospira sp

    Energy Technology Data Exchange (ETDEWEB)

    Hauk, Pricila; Roman-Ramos, Henrique; Ho, Paulo Lee [Instituto Butantan, Sao Paulo, SP (Brazil). Centro de Biotecnologia; Guzzo, Cristiane R.; Farah, Chuck S. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica. Dept. de Bioquimica

    2009-07-01

    Leptospirosis, caused by the spirochaete Leptospira is an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospira. It is highly immunogenic and has been shown to bind to host extracellular matrix components, including collagens, fibronectin and laminin. In this work we crystallized recombinant LipL32 protein and determined its structure to 2.25 A resolution. Initial phases were determined using the multi-wavelength anomalous dispersion technique with data collected from selenomethionine-containing crystals at the MX2 beamline at the LNLS. The LipL32 monomer is made of a jelly-roll fold core from which protrude several peripheral secondary structures. Some structural features suggested that LipL32 could bind Ca{sup 2+} ions and indeed, spectroscopic data (circular (dichroism. intrinsic tryptophan fluorescence and extrinsic 1-amino-2-anaphthol-4-sulfonic acid fluorescence) confirmed the calcium binding properties of LipL32. (author)

  3. Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

    OpenAIRE

    Welfringer, Frédéric; D'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise

    2005-01-01

    International audience; Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab...

  4. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

    2016-07-27

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  5. Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Hamada, Junichi; Shoda, Katsutoshi; Masuda, Kiyoshi; Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

    2016-03-29

    T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC.

  6. Differential protein expression, DNA binding and interaction with SV40 large tumour antigen implicate the p63-family of proteins in replicative senescence.

    Science.gov (United States)

    Djelloul, Siham; Tarunina, Marina; Barnouin, Karin; Mackay, Alan; Jat, Parmjit S

    2002-02-07

    P53 activity plays a key role in mammalian cells when they undergo replicative senescence at their Hayflick limit. To determine whether p63 proteins, members of the family of p53-related genes, are also involved in this process, we examined their expression in serially passaged rat embryo fibroblasts. Upon senescence, two truncated DeltaNp63 proteins decreased in abundance whereas two TAp63 isoforms accumulated. 2-D gel analysis showed that the DeltaNp63 proteins underwent post-translational modifications in both proliferating and senescent cells. Direct binding of DeltaNp63 proteins to a p53 consensus motif was greater in proliferating cells than senescent cells. In contrast p63alpha isoforms bound to DNA in a p53 dependent manner and this was higher in senescent cells than proliferating cells. An interaction of p63alpha proteins with SV40 large tumour antigen was also detected and ectopic expression of DeltaNp63alpha can extend the lifespan of rat embryo fibroblasts. Taken together the results indicate that p63 proteins may play a role in replicative senescence either by competition for p53 DNA binding sites or by direct interaction with p53 protein bound to DNA.

  7. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

    Energy Technology Data Exchange (ETDEWEB)

    Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N. (NIH); (Kyoto)

    2008-07-29

    Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

  8. Characterization of highly frequent epitope-specific CD45RA+/CCR7+/- T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors

    Directory of Open Access Journals (Sweden)

    Zajac Paul

    2006-11-01

    Full Text Available Abstract Human polyomavirus BK (BKV has been implicated in oncogenic transformation. Its ability to replicate is determined by the binding of its large tumor antigen (LTag to products of tumor-suppressor genes regulating cell cycle, as specifically p53. We investigated CD8+ T immune responses to BKV LTag portions involved in p53 binding in HLA-A*0201+ BKV LTag experienced individuals. Peptides selected from either p53-binding region (LTag351–450 and LTag533–626 by current algorithms and capacity to bind HLA-A*0201 molecule were used to stimulate CD8+ T responses, as assessed by IFN-γ gene expression ex vivo and detected by cytotoxicity assays following in vitro culture. We observed epitope-specific immune responses in all HLA-A*0201+ BKV LTag experienced individuals tested. At least one epitope, LTag579–587; LLLIWFRPV, was naturally processed in non professional antigen presenting cells and induced cytotoxic responses with CTL precursor frequencies in the order of 1/20'000. Antigen specific CD8+ T cells were only detectable in the CD45RA+ subset, in both CCR7+ and CCR7- subpopulations. These data indicate that widespread cellular immune responses against epitopes within BKV LTag-p53 binding regions exist and question their roles in immunosurveillance against tumors possibly associated with BKV infection.

  9. Fasciola spp: Mapping of the MF6 epitope and antigenic analysis of the MF6p/HDM family of heme-binding proteins.

    Directory of Open Access Journals (Sweden)

    Victoria Martínez-Sernández

    Full Text Available MF6p/FhHDM-1 is a small cationic heme-binding protein which is recognized by the monoclonal antibody (mAb MF6, and abundantly present in parenchymal cells and secreted antigens of Fasciola hepatica. Orthologs of this protein (MF6p/HDMs also exist in other causal agents of important foodborne trematodiasis, such as Clonorchis sinensis, Opisthorchis viverrini and Paragonimus westermani. Considering that MF6p/FhHDM-1 is relevant for heme homeostasis in Fasciola and was reported to have immunomodulatory properties, this protein is expected to be a useful target for vaccination. Thus, in this study we mapped the epitope recognized by mAb MF6 and evaluated its antigenicity in sheep. The sequence of the MF6p/FhHDM-1 ortholog from F. gigantica (MF6p/FgHDM-1 was also reported. By means of ELISA inhibitions with overlapping synthetic peptides, we determined that the epitope recognized by mAb MF6 is located within the C-terminal moiety of MF6p/FhHDM-1, which is the most conserved region of MF6p/HDMs. By immunoblotting analysis of parasite extracts and ELISA inhibitions with synthetic peptides we also determined that mAb MF6 reacted with the same intensity with F. hepatica and F. gigantica, and in decreasing order of intensity with C. sinensis, O.viverrini and P. westermani orthologs. On the contrary, mAb MF6 showed no reactivity against Dicrocoelium dendriticum and Schistosoma mansoni. The study of the recognition of peptides covering different regions of MF6p/FhHDM-1 by sera from immunized sheep revealed that the C-terminal moiety is the most antigenic, thus being of potential interest for vaccination. We also demonstrated that the production of antibodies to MF6p/FhHDM-1 in sheep infected by F. hepatica occurs relatively early and follows the same pattern as those produced against L-cathepsins.

  10. Heat shock factor 1 upregulates transcription of Epstein–Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

    International Nuclear Information System (INIS)

    Wang, Feng-Wei; Wu, Xian-Rui; Liu, Wen-Ju; Liao, Yi-Ji; Lin, Sheng; Zong, Yong-Sheng; Zeng, Mu-Sheng; Zeng, Yi-Xin; Mai, Shi-Juan; Xie, Dan

    2011-01-01

    Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the − 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

  11. Heat shock factor 1 upregulates transcription of Epstein-Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng-Wei [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China); Wu, Xian-Rui [Department of Surgery, Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou (China); Liu, Wen-Ju; Liao, Yi-Ji [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China); Lin, Sheng [Laboratory of Integrated Biosciences, School of Life Science, Sun Yat-sen University, Guangzhou (China); Zong, Yong-Sheng; Zeng, Mu-Sheng; Zeng, Yi-Xin [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China); Mai, Shi-Juan, E-mail: maishj@sysucc.org.cn [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China); Xie, Dan, E-mail: xied@mail.sysu.edu.cn [The State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou (China)

    2011-12-20

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the - 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

  12. Antibodies against PfEMP1, RIFIN, MSP3 and GLURP are acquired during controlled Plasmodium falciparum malaria infections in naïve volunteers

    DEFF Research Database (Denmark)

    Turner, Louise; Wang, Christian W; Lavstsen, Thomas

    2011-01-01

    Antibodies to polymorphic antigens expressed during the parasites erythrocytic stages are important mediators of protective immunity against P. falciparum malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and GLURP and variant surface antigens PfEMP1 and RIFIN are considered...

  13. An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM).

    Science.gov (United States)

    Chue, B; Ferguson, T A; Beaman, K D; Rosenman, S J; Cone, R E; Flood, P M; Green, D R

    1989-01-01

    A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.

  14. Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules.

    Science.gov (United States)

    Hilton, H G; Parham, P

    2013-04-01

    Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. © 2013 John Wiley & Sons A/S.

  15. Use of JH4 joining segment gene by an anti-arsonate antibody that bears the major A-strain cross-reactive idiotype but displays diminished antigen binding.

    Science.gov (United States)

    Slaughter, C A; Jeske, D J; Kuziel, W A; Milner, E C; Capra, J D

    1984-06-01

    One of the antibody families utilized by the A/J mouse in its response to p-azophenylarsonate (Ars) is characterized by the expression of the major anti-arsonate cross-reactive idiotype (CRI) of the A strain. This family has been termed the Ars-A family. A hybridoma antibody (HP 101F11 ) obtained after immunization of an A/J mouse with Ars was identified initially as displaying the CRI, but was subsequently found to bind antigen at a level much lower than most members of the Ars-A family. The results of binding studies suggested that HP 101F11 possesses reduced avidity for antigen. When isolated light and heavy chains were allowed to recombine with the heavy and light chains of a strongly antigen-binding, strongly CRI-positive antibody of the Ars-A family (HP 93G7 ), the low level of antigen binding by HP 101F11 was found to be due to a structurally variant heavy chain. Whereas antibodies of the Ars-A family with normal avidity for antigen had been shown to use the JH2 joining segment gene, amino acid sequence analysis of HP 101F11 revealed that this antibody has a JH segment with a sequence identical to that encoded by a portion of a different JH gene, JH4 . The implication that 101F11 uses the JH4 gene instead of JH2 was supported by the observation that the productively rearranged gene is associated with an Eco R1 restriction fragment 0.95 Kb smaller than the corresponding fragments of Ars-A hybridomas with normal avidity for antigen. The size difference of 0.95 Kb corresponds exactly to the known distance between the JH2 and JH4 genes in BALB/c germline DNA. In addition to the structural differences immediately attributable to the use of JH4 , HP 101F11 has shown an amino acid interchange in the DH segment, and a single amino acid deletion at the DH-JH boundary. These results show that variation among members of the Ars-A family in the DH and/or JH segments provides alternative structural forms of Ars-A antibodies upon which selective processes can operate

  16. Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet light (UVL): Implications for the pathogenesis of photosensitive cutaneous lupus

    International Nuclear Information System (INIS)

    Furukawa, F.; Kashihara-Sawami, M.; Lyons, M.B.; Norris, D.A.

    1990-01-01

    Autoantibodies to the non-histone nucleoprotein antigens SS-A/Ro, SS-B/La, and RNP are highly associated with photosensitive cutaneous lupus erythematosus (LE). In order to better understand the potential mechanisms of ultraviolet (UV) light on photosensitivity in patients with cutaneous LE, we designed immunopathologic in vitro and in vivo experiments to evaluate the effects of UV on the binding of such autoantibodies to the surface of human keratinocytes, one major target of immunologic damage in photosensitive LE. Short-term 2% paraformaldehyde fixation of suspensions of cultured human keratinocytes previously incubated with monospecific antiserum probes enabled the detection of ENA expression on the cell surface by flow-cytometry analysis. UVB light (280-320 nm) induced the binding of monospecific antibody probes for SS-A/Ro and SS-B/La on keratinocytes in a dose-dependent pattern with maximal induction observed at the dose of 200 mJ/cm2 UVB. Binding of SS-A/Ro, SS-B/La, and RNP antibody was augmented strongly, but binding of anti-Sm was very weak. In contrast, UVA (320-400 nm) light had no effect on the induction of binding of these antibody probes. Identical results were seen by standard immunofluorescence techniques. Hydroxyurea-treated keratinocytes showed similar induction of those antigens by UVB irradiation, which suggested that ENA expression on cultured keratinocytes by UVB were cell-cycle independent. Tunicamycin, an inhibitor of glycosylation of proteins, reduced UVB light effect on the SS-A/Ro and SS-B/La antigen's expression. These in vitro FACS analyses revealed that ENA augmentation on the keratinocyte cell surface was dose dependent, UVB dependent, glycosylation dependent, and cell-cycle independent. In vivo ENA augmentation on the keratinocyte surface was examined in suction blister epidermal roofs

  17. Tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the Middle East respiratory coronavirus (MERS-CoV receptor-binding domain as an antigen.

    Directory of Open Access Journals (Sweden)

    Jiaming Lan

    Full Text Available The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV pandemic. Some studies have indicated the receptor-binding domain (RBD protein of MERS-CoV spike (S is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m. with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum alone, alum and polyriboinosinic acid (poly I:C or alum and cysteine-phosphate-guanine (CpG oligodeoxynucleotides (ODN. The immune responses of mice vaccinated with RBD, incomplete Freund's adjuvant (IFA and CpG ODN by a subcutaneous (s.c. route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production. Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting

  18. β-endorphin antigen

    International Nuclear Information System (INIS)

    1981-01-01

    This invention relates to the production of antigens comprising β-endorphin, βsub(h)-endorphin, or βsub(c)-endorphin, in covalent conjugation with human gammaglobulin as immunogenic carrier material, and an antibody having the property of specifically binding β-endorphin or fragments thereof, containing the (6-15) residue sequence. (U.K.)

  19. Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.

    Science.gov (United States)

    Welfringer, Frédéric; d'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise

    2005-12-20

    Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.

  20. Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

    Science.gov (United States)

    Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

    2017-01-01

    CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

  1. Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B isolectin to alphagalactosyl carbohydrate antigens in the surface phase

    DEFF Research Database (Denmark)

    Kirkeby, Svend; Winter, Harry C; Goldstein, Irwin J

    2004-01-01

    The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4) (GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and lam...

  2. A study on antigenicity and receptor-binding ability of fragment 450-650 of the spike protein of SARS coronavirus

    International Nuclear Information System (INIS)

    Zhao Jincun; Wang Wei; Yuan Zhihong; Jia Rujing; Zhao Zhendong; Xu Xiaojun; Lv Ping; Zhang Yan; Jiang Chengyu; Gao Xiaoming

    2007-01-01

    The spike (S) protein of SARS coronavirus (SARS-CoV) is responsible for viral binding with ACE2 molecules. Its receptor-binding motif (S-RBM) is located between residues 424 and 494, which folds into 2 anti-parallel β-sheets, β5 and β6. We have previously demonstrated that fragment 450-650 of the S protein (S450-650) is predominantly recognized by convalescent sera of SARS patients. The N-terminal 60 residues (450-510) of the S450-650 fragment covers the entire β6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the β6 fragment, while the mouse antisera, induced by immunization of BALB/c mice with recombinant S450-650, mainly recognized the β6 strand-containing region. Unlike patient sera, however, the mouse antisera were unable to inhibit the infectivity of S protein-expressing (SARS-CoV-S) pseudovirus. Fusion protein between green fluorescence protein (GFP) and S450-650 (S450-650-GFP) was able to stain Vero E6 cells and deletion of the β6 fragment rendered the fusion product (S511-650-GFP) unable to do so. Similarly, recombinant S450-650, but not S511-650, was able to block the infection of Vero E6 cells by the SARS-CoV-S pseudovirus. Co-precipitation experiments confirmed that S450-650 was able to specifically bind with ACE2 molecules in lysate of Vero E6 cells. However, the ability of S450-510, either alone or in fusion with GFP, to bind with ACE2 was significantly poorer compared with S450-650. Our data suggest a possibility that, although the β6 strand alone is able to bind with ACE2 with relatively high affinity, residues outside the S-RBM could also assist the receptor binding of SARS-CoV-S protein

  3. Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I.

    Science.gov (United States)

    Unverzagt, K L; Martinson, J; Lee, W; Stiff, P J; Williams, S; Bender, J G

    1996-01-01

    Two and three color flow cytometry of normal human bone marrow was used to identify CD34+ progenitor cells and examine their binding to the plant lectin Ulex europaeus I (Ulex). In normal bone marrow, 48.48 +/- 17.4% of the CD34+ cells bind to Ulex. Two color flow cytometry was used to sort CD34 + cells, and subsets of CD34+ cells, CD34+ Ulex+ and CD34+ Ulex-. These populations were sorted into colony assays to assess myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The CD34+ Ulex+ subset was 84 +/- 14% BFU-E colonies (mean +/- S.D.) and had the highest cloning efficiency of 28 +/- 13%. Three color analysis of CD34+ Ulex+ cells showed staining with other erythroid (CD71, GlyA) antibodies and lack of stain. ing with myeloid (CD13, CD45RA) antibodies. These studies confirmed the erythroid characteristics of this subpopulation.

  4. Binding of the heterogeneous ribonucleoprotein K (hnRNP K to the Epstein-Barr virus nuclear antigen 2 (EBNA2 enhances viral LMP2A expression.

    Directory of Open Access Journals (Sweden)

    Henrik Gross

    Full Text Available The Epstein-Barr Virus (EBV -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively. EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2 Type 1. The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3 which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K. Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.

  5. An indirect antibody assay using haptenated antigen and 125I-labelled anti-hapten antibody

    International Nuclear Information System (INIS)

    Aalberse, R.C.; Amsterdam Univ.

    1978-01-01

    Hapten (trinitrophenyl) was coupled to antigen (ovalbumin). The haptenated antigen was bound by anti-ovalbumin antibody and binding was quantitated with 125 I-labelled anti-hapten antibodies. Thus, with a single radioactive reagent, antibodies against a variety of antigens can be detected while the problems inherent in a labelled antiglobulin binding test are avoided. In the ovalbumin system, the haptenated antigen binding test proved to be approximately 20 times as sensitive as the iodinated ovalbumin binding test

  6. Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule.

    Science.gov (United States)

    Atassi, M Zouhair; Jankovic, Joseph; Steward, Lance E; Aoki, K Roger; Dolimbek, Behzod Z

    2012-01-01

    We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs. Copyright © 2011. Published by Elsevier GmbH.

  7. Solution Structure, Membrane Interactions, and Protein Binding Partners of the Tetraspanin Sm-TSP-2, a Vaccine Antigen from the Human Blood Fluke Schistosoma mansoni*

    Science.gov (United States)

    Jia, Xinying; Schulte, Leigh; Loukas, Alex; Pickering, Darren; Pearson, Mark; Mobli, Mehdi; Jones, Alun; Rosengren, Karl J.; Daly, Norelle L.; Gobert, Geoffrey N.; Jones, Malcolm K.; Craik, David J.; Mulvenna, Jason

    2014-01-01

    The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument. PMID:24429291

  8. Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni.

    Science.gov (United States)

    Jia, Xinying; Schulte, Leigh; Loukas, Alex; Pickering, Darren; Pearson, Mark; Mobli, Mehdi; Jones, Alun; Rosengren, Karl J; Daly, Norelle L; Gobert, Geoffrey N; Jones, Malcolm K; Craik, David J; Mulvenna, Jason

    2014-03-07

    The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.

  9. Elevated insulin and reduced insulin like growth factor binding protein-3/prostate specific antigen ratio with increase in prostate size in Benign Prostatic Hyperplasia.

    Science.gov (United States)

    Sreenivasulu, Karli; Nandeesha, Hanumanthappa; Dorairajan, Lalgudi Narayanan; Rajappa, Medha; Vinayagam, Vickneshwaran

    2017-06-01

    Insulin and insulin like growth factor-1 (IGF-1) have growth promoting effects, while insulin like growth factor binding protein-3 (IGFBP-3) has growth inhibitory effects. The present study was designed to assess the concentrations of insulin, IGF-1, IGFBP-3 and their association with prostate size in patients with BPH. Ninety 90 BPH cases and 90 controls were enrolled in the study. Insulin, IGF-1, IGFBP-3, PSA, testosterone and estradiol were estimated in both the groups. Insulin, IGF-1 and estradiol were increased and IGFBP-3/PSA was decreased in BPH cases when compared with controls. Insulin (r=0.64, p=0.001) and IGF-1 (r=0.22, p=0.03) were positively correlated and IGFBP-3/PSA (r=-0.316, p=0.002) were negatively correlated with prostate size in BPH. Multivariate analysis showed that insulin (p=0.001) and IGFBP-3/PSA (p=0.004) predicts the prostate size in patients with BPH. Insulin was increased and IGFBP-3/PSA was reduced in BPH patients with increased prostate size. At a cutoff concentration of 527.52, IGFBP-3/PSA ratio was found to differentiate benign growth of prostate from normal prostate with 96% sensitivity and 96% specificity. Insulin is elevated and IGFBP-3/PSA is reduced with increase prostate size in BPH cases. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Enhanced effect of BCG vaccine against pulmonary Mycobacterium tuberculosis infection in mice with lung Th17 response to mycobacterial heparin-binding hemagglutinin adhesin antigen.

    Science.gov (United States)

    Fukui, Masayuki; Shinjo, Kikuko; Umemura, Masayuki; Shigeno, Satoko; Harakuni, Tetsuya; Arakawa, Takeshi; Matsuzaki, Goro

    2015-12-01

    Although the BCG vaccine can prevent tuberculosis (TB) in infants, its ability to prevent adult pulmonary TB is reportedly limited. Therefore, development of a novel effective vaccine against pulmonary TB has become an international research priority. We have previously reported that intranasal vaccination of mice with a mycobacterial heparin-binding hemagglutinin adhesin (HBHA) plus mucosal adjuvant cholera toxin (CT) enhances production of IFN-γ and anti-HBHA antibody and suppresses extrapulmonary bacterial dissemination after intranasal infection with BCG. In the present study, the effects of intranasal HBHA + CT vaccine on murine pulmonary Mycobacterium tuberculosis (Mtb) infection were examined. Intranasal HBHA + CT vaccination alone failed to reduce the bacterial burden in the infected lung. However, a combination vaccine consisting of s.c. BCG priming and an intranasal HBHA + CT booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14 compared with BCG vaccine alone. Further, it was found that intranasal HBHA + CT vaccine enhanced not only IFN-γ but also IL-17A production by HBHA-specific T cells in the lung after pulmonary Mtb infection. Therefore, this combination vaccine may be a good candidate for a new vaccine strategy against pulmonary TB. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

  11. The antigen-binding fragment of human gamma immunoglobulin prevents amyloid β-peptide folding into β-sheet to form oligomers

    Science.gov (United States)

    Valls-Comamala, Victòria; Guivernau, Biuse; Bonet, Jaume; Puig, Marta; Perálvarez-Marín, Alex; Palomer, Ernest; Fernàndez-Busquets, Xavier; Altafaj, Xavier; Tajes, Marta; Puig-Pijoan, Albert; Vicente, Rubén; Oliva, Baldomero; Muñoz, Francisco J.

    2017-01-01

    The amyloid beta-peptide (Aβ) plays a leading role in Alzheimer's disease (AD) physiopathology. Even though monomeric forms of Aβ are harmless to cells, Aβ can aggregate into β-sheet oligomers and fibrils, which are both neurotoxic. Therefore, one of the main therapeutic approaches to cure or delay AD onset and progression is targeting Aβ aggregation. In the present study, we show that a pool of human gamma immunoglobulins (IgG) protected cortical neurons from the challenge with Aβ oligomers, as assayed by MTT reduction, caspase-3 activation and cytoskeleton integrity. In addition, we report the inhibitory effect of IgG on Aβ aggregation, as shown by Thioflavin T assay, size exclusion chromatography and atomic force microscopy. Similar results were obtained with Palivizumab, a human anti-sincitial virus antibody. In order to dissect the important domains, we cleaved the pool of human IgG with papain to obtain Fab and Fc fragments. Using these cleaved fragments, we functionally identified Fab as the immunoglobulin fragment inhibiting Aβ aggregation, a result that was further confirmed by an in silico structural model. Interestingly, bioinformatic tools show a highly conserved structure able to bind amyloid in the Fab region. Overall, our data strongly support the inhibitory effect of human IgG on Aβ aggregation and its neuroprotective role. PMID:28467807

  12. Surface co-expression of two different PfEMP1 antigens on single Plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1

    DEFF Research Database (Denmark)

    Joergensen, Louise; Bengtsson, Dominique C; Bengtsson, Anja

    2010-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var...

  13. The solvent at antigen-binding site regulated C3d-CR2 interactions through the C-terminal tail of C3d at different ion strengths: insights from molecular dynamics simulation.

    Science.gov (United States)

    Zhang, Yan; Guo, Jingjing; Li, Lanlan; Liu, Xuewei; Yao, Xiaojun; Liu, Huanxiang

    2016-10-01

    The interactions of complement receptor 2 (CR2) and the degradation fragment C3d of complement component C3 play important links between the innate and adaptive immune systems. Due to the importance of C3d-CR2 interaction in the design of vaccines and inhibitors, a number of studies have been performed to investigate C3d-CR2 interaction. Many studies have indicated C3d-CR2 interactions are ionic strength-dependent. To investigate the molecular mechanism of C3d-CR2 interaction and the origin of effects of ionic strength, molecular dynamics simulations for C3d-CR2 complex together with the energetic and structural analysis were performed. Our results revealed the increased interactions between charged protein and ions weaken C3d-CR2 association, as ionic strengths increase. Moreover, ion strengths have similar effects on antigen-binding site and CR2 binding site. Meanwhile, Ala17 and Gln20 will transform between the activated and non-activated states mediated by His133 and Glu135 at different ion strengths. Our results reveal the origins of the effects of ionic strengths on C3d-CR2 interactions are due to the changes of water, ion occupancies and distributions. This study uncovers the origin of the effect of ionic strength on C3d-CR2 interaction and deepens the understanding of the molecular mechanism of their interaction, which is valuable for the design of vaccines and small molecule inhibitors. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. A Role of Sp1 Binding Motifs in Basal and Large T-Antigen-Induced Promoter Activities of Human Polyomavirus HPyV9 and Its Variant UF-1

    Directory of Open Access Journals (Sweden)

    Ugo Moens

    2017-11-01

    Full Text Available Human polyomavirus 9 (HPyV9 was originally detected in the serum of a renal transplant patient. Seroepidemiological studies showed that ~20–50% of the human population have antibodies against this virus. HPyV9 has not yet been associated with any disease and little is known about the route of infection, transmission, host cell tropism, and genomic variability in circulating strains. Recently, the HPyV9 variant UF-1 with an eight base-pair deletion, a thirteen base-pair insertion and with point mutations, creating three putative Sp1 binding sites in the late promoter was isolated from an AIDS patient. Transient transfection studies with a luciferase reporter plasmid driven by HPyV9 or UF1 promoter demonstrated that UF1 early and late promoters were stronger than HPyV9 promoters in most cell lines, and that the UF1 late promoter was more potently activated by HPyV9 large T-antigen (LTAg. Mutation of two Sp1 motifs strongly reduced trans-activation of the late UF1 promoter by HPyV9 LTAg in HeLa cells. In conclusion, the mutations in the UF1 late promoter seem to strengthen its activity and its response to stimulation by HPyV9 LTAg in certain cells. It remains to be investigated whether these promoter changes have an influence on virus replication and affect the possible pathogenic properties of the virus.

  15. Immunity to tumour antigens.

    Science.gov (United States)

    Li, Geng; Ali, Selman A; McArdle, Stephanie E B; Mian, Shahid; Ahmad, Murrium; Miles, Amanda; Rees, Robert C

    2005-01-01

    During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.

  16. Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

    Science.gov (United States)

    Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

    2015-07-01

    Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.

  17. HLA-DPβ1 Asp84-Lys69 antigen-binding signature predicts event-free survival in childhood B-cell precursor acute lymphoblastic leukaemia: results from the MRC UKALL XI childhood ALL trial.

    Science.gov (United States)

    Taylor, G M; Wade, R; Hussain, A; Thompson, P; Hann, I; Gibson, B; Eden, T; Richards, S

    2012-07-01

    We previously reported that children in the UKALL XI ALL trial with HLA-DP 1 and -DP 3 supertypes had significantly worse event-free survival (EFS) than children with other DP supertypes. As DP 1 and DP 3 share two of four key antigen-binding amino-acid polymorphisms (aspartic acid84-lysine69), we asked whether Asp84-Lys69 or Asp84 alone were independent prognostic indicators in childhood acute lymphoblastic leukemia (ALL). We analysed EFS in 798 UKALL XI patients, stratified by Asp84-Lys69 vs non-Asp84-Lys69, for a median follow-up of 12.5 years. Asp84-Lys69 was associated with a significantly worse EFS than non-Asp84-Lys69 (5-year EFS: Asp84-Lys69: 58.8% (95% CI (confidence of interval): 52.7-64.9%); non-Asp84-Lys69: 67.3% (63.4-71.2%); 2P=0.007). Post-relapse EFS was 10% less in Asp84-Lys69 than non-Asp84-Lys69 patients. EFS was significantly worse (P=0.03) and post-relapse EFS marginally worse (P=0.06) in patients with Asp84 compared with Gly84. These results suggest that Asp84-Lys69 predicted adverse EFS in the context of UKALL XI because of Asp84, and may have influenced post-relapse EFS. We speculate that this may be due to the recruitment of Asp84-Lys69-restricted regulatory T cells in the context of this regimen, leading to the re-emergence of residual disease. However, functional and molecular studies of the prognostic value of this and other HLA molecular signatures in other childhood ALL trials are needed.

  18. Biologic significance of receptor-binding cancer antigen expressed on SiSo cells (RCAS1) as a pivotal regulator of tumor growth through angiogenesis in human uterine cancer.

    Science.gov (United States)

    Sonoda, Kenzo; Miyamoto, Shingo; Yamazaki, Ayano; Kobayashi, Hiroaki; Nakashima, Manabu; Mekada, Eisuke; Wake, Norio

    2007-11-01

    The expression of receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is related significantly to the overall survival of patients with various cancers. RCAS1 reportedly induces apoptotic cell death in peripheral lymphocytes, which may contribute to the escape of tumor cells from immune surveillance. RCAS1 expression also has been related to tumor invasiveness and size in uterine cervical cancer. To clarify whether RCAS1 exacerbates tumor progression, the authors investigated the association between RCAS1 expression and tumor growth potential. The authors constructed small interfering ribonucleic acid (RNA) (siRNA) to target RCAS1. After transfection of siRNA and the RCAS1-encoding gene, growth of tumor cells was assessed in vitro and in vivo. The correlation between RCAS1 expression and angiogenesis was investigated in the transfected cells and in inoculated tumors from nude mice. In addition, the same association was investigated immunohistochemically with tissue samples from patients with uterine cervical cancer. Knockdown of RCAS1 expression by siRNA significantly suppressed the in vivo growth of SiSo and HOUA tumor cells (P cell growth was not affected significantly. Enhanced RCAS1 expression significantly promoted in vivo growth, but not in vitro growth, of tumors derived from COS-7 cells (P = .0039). Introduction of the RCAS1-encoding gene increased expression of vascular endothelial growth factor (VEGF). In uterine cervical cancer, RCAS1 expression was associated significantly with VEGF expression (P = .0407) and with microvessel density (P = .0108). RCAS1 may be a pivotal regulator of tumor growth through angiogenesis. Continued exploration of the biologic function of RCAS1 may allow the development of novel therapeutic strategies for uterine cancer.

  19. Lectin binding patterns and immunohistochemical antigen detection ...

    African Journals Online (AJOL)

    Ibrahim Eldaghayes

    2018-02-09

    Feb 9, 2018 ... placenta and lungs of Brucella abortus-bovine infected fetuses. María Andrea ... The present lectin histochemical study revealed a distinctive pattern of oligosaccharide .... tissue was used as a positive control and nonimmune.

  20. Carcinoma-associated antigens

    International Nuclear Information System (INIS)

    Bartorelli, A.; Accinni, R.

    1981-01-01

    This invention relates to novel antigens associated with breast carcinoma, anti-sera specific to said antigens, 125 I-labeled forms of said antigens and methods of detecting said antigens in serum or plasma. The invention also relates to a diagnostic kit containing standardised antigens or antisera or marked forms thereof for the detection of said antigens in human blood, serum or plasma. (author)

  1. Conservation of myeloid surface antigens on primate granulocytes.

    Science.gov (United States)

    Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

    1983-02-01

    Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

  2. Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases

    International Nuclear Information System (INIS)

    Falo, L.D. Jr.; Haber, S.I.; Herrmann, S.; Benacerraf, B.; Rock, K.L.

    1987-01-01

    To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) the authors analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A 2 and phospholipase C. In addition it is observed with three distinct antigens - ovalbumin, bovine insulin, and poly(LGlu 56 LLys 35 LPhe 9 )[(GluLysPhe)/sub n/]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to control in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or processing independent antigens. In parallel studies 125 I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125 I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. These studies demonstrate that phospholipase effectively removes processed cell surface antigen

  3. Leukemia-associated antigens in man.

    Science.gov (United States)

    Brown, G; Capellaro, D; Greaves, M

    1975-12-01

    Rabbit antisera raised against acute lymphoblastic leukemia (ALL) cells were used to distinguish ALL from other leukemias, to identify rare leukemia cells in the bone marrow of patients in remission, and to define human leukemia-associated antigens. Antibody binding was studied with the use of immunofluorescence reagents and the analytic capacity of the Fluorescence Activated Cell Sorter-1 (FACS-1). The results indicated that most non-T-cell ALL have three leukemia-associated antigens on their surface which are absent from normal lymphoid cells: 1) an antigen shared with myelocytes, myeloblastic leukemia cells, and fetal liver (hematopoietic) cells; 2) an antigen shared with a subset of intermediate normoblasts in normal bone marrow and fetal liver; and 3) an antigen found thus far only on non-T-cell ALL and in some acute undifferentiated leukemias, which we therefore regard as a strong candidate for a leukemia-specific antigen. These antigens are absent from a subgroup of ALL patients in which the lymphoblasta express T-cell surface markers. Preliminary studies on the bone marrow samples of patients in remission indicated that rare leukemia cells were present in some samples. The implications of these findings with respect to the heterogeneity and cell origin(s) of ALL, its diagnosis, and its potential monitoring during treatment were discussed.

  4. Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen

    Directory of Open Access Journals (Sweden)

    Anita Verma

    2018-02-01

    Full Text Available Toxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA, the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses. In contrast, a receptor binding-deficient mutant of PA, which does not bind receptors and only inefficiently enters antigen-presenting cells by macropinocytosis, elicited very poor antibody responses. A chimeric protein consisting of the receptor binding-deficient PA mutant tethered to the binding subunit of cholera toxin, which efficiently enters cells using the cholera toxin receptor rather than the PA receptor, elicited an anti-PA IgG antibody response similar to that elicited by wild-type PA; however, the chimeric protein elicited a poor toxin neutralizing antibody response. Taken together, our results demonstrate that the antigen capture pathway can dictate the magnitudes of the total IgG and toxin neutralizing antibody responses to PA as well as the ratio of the two responses.

  5. The interaction of beta 2-microglobulin (beta 2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse beta 2m

    DEFF Research Database (Denmark)

    Pedersen, L O; Stryhn, A; Holter, T L

    1995-01-01

    of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in beta 2m binding. We propose that mouse beta 2m interacts with the minor peptide binding (i.e. the "empty") fraction with a lower affinity than human beta 2m does, whereas mouse and human beta 2m interact......The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and beta 2-microglobulin...... (beta 2m) have been used to examine the assembly of the trimolecular MHC class I/beta 2m/peptide complex. Recombinant human beta 2m and mouse beta 2ma have been generated to compare the binding of the two beta 2m to mouse class I. It is frequently assumed that human beta 2m binds to mouse class I heavy...

  6. Bayesian nonparametric clustering in phylogenetics: modeling antigenic evolution in influenza.

    Science.gov (United States)

    Cybis, Gabriela B; Sinsheimer, Janet S; Bedford, Trevor; Rambaut, Andrew; Lemey, Philippe; Suchard, Marc A

    2018-01-30

    Influenza is responsible for up to 500,000 deaths every year, and antigenic variability represents much of its epidemiological burden. To visualize antigenic differences across many viral strains, antigenic cartography methods use multidimensional scaling on binding assay data to map influenza antigenicity onto a low-dimensional space. Analysis of such assay data ideally leads to natural clustering of influenza strains of similar antigenicity that correlate with sequence evolution. To understand the dynamics of these antigenic groups, we present a framework that jointly models genetic and antigenic evolution by combining multidimensional scaling of binding assay data, Bayesian phylogenetic machinery and nonparametric clustering methods. We propose a phylogenetic Chinese restaurant process that extends the current process to incorporate the phylogenetic dependency structure between strains in the modeling of antigenic clusters. With this method, we are able to use the genetic information to better understand the evolution of antigenicity throughout epidemics, as shown in applications of this model to H1N1 influenza. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  7. Protein A-containing staphylococcus aureus as an immunoglobulin-binding reagent: 1) in radioimmunoassays - 'staf-RIA' - recently also for antibiotics and microbial antigens/antibodies, and 2) in a non-radioactive surface immunoassay - 'Staph-ace ay' read by the naked eye - primarily for antibodies to antigens adsorbed to transparent surfaces

    International Nuclear Information System (INIS)

    Jonsson, S.

    1977-01-01

    This paper is intended to summarize recent developments for the use of protein A-containing staphylococci as an immunoglobulin-binding reagent in various types of radioimmunoassay and some related areas, particularly the staphylococcal surface immunoassay. The paper also presents a new process for the large scale preparation of a freeze-dried preparation of the immunoglobulin-binding, killed staphylococci, which thereby gain a much improved suspension stability. (orig.) [de

  8. Facts on the fragmentation of antigens in presenting cells, on the association of antigen fragments with MHC molecules in cell-free systems, and speculation on the cell biology of antigen processing

    DEFF Research Database (Denmark)

    Werdelin, O; Mouritsen, S; Petersen, B L

    1988-01-01

    The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic...... fragmentation. Some of the antigen fragments bind to MHC class II molecules and are transported to the surface of the antigen-presenting cell where the actual presentation to T lymphocytes occurs. The nature of the processed antigen, how and where it is derived and subsequently becomes associated with MHC...... molecules are the questions discussed in this review. To us, the entire concept of processing has appeal not only because it explains some hitherto well-established, but poorly understood, phenomena such as the fact that T lymphocytes focus their attention entirely upon antigens on other cells. It has...

  9. Carcinoembryonic antigen (CEA)

    International Nuclear Information System (INIS)

    Ephraim, K.H.; Cox, P.H.; Hamer, C.J.A. v.d.; Berends, W.; Delhez, H.

    1977-01-01

    The carcinoembryonic antigen (CEA) is a complex of antigen determinants and also the carrier of these determinants. Chemically it is a glycoprotein. Its occurrence in blood serum or urine is correlated with malignant disease. Several radioimmunoassays (RIA) have been developed, one by Hoffmann-Laroche and one by the Rotterdam Radiotherapeutic Institute. Both methods and the Hoffmann assay kit are tested. Specifications are given for isolation of the antigen, preparation of the antiserum, and the execution of the RIA. Biochemical and clinical aspects are discussed

  10. Antigen injection (image)

    Science.gov (United States)

    Leprosy is caused by the organism Mycobacterium leprae . The leprosy test involves injection of an antigen just under ... if your body has a current or recent leprosy infection. The injection site is labeled and examined ...

  11. Radioimmunoassay for hepatitis B core antigen

    International Nuclear Information System (INIS)

    Sagnelli, E.; Pereira, C.; Triolo, G.; Vernace, S.; Paronetto, F.

    1982-01-01

    Serum hepatitis B core antigen (HBcAg) is an important marker of hepatitis B virus replication. We describe an easy, sensitive radioimmunoassay for determination of HBcAg in detergent-treated serum pellets containing Dane particles. Components of a commercial kit for anticore determination are used, and HBcAG is measured by competitive inhibition of binding of 125 I-labeled antibodies to HBcAg with HBcAg-coated beads. We assayed for HBcAG in the sera of 49 patients with hepatitis B surface antigen (HBsAg)-positive chronic hepatitis, 50 patients with HBsAg-negative chronic hepatitis, and 30 healthy volunteers. HBcAg was detected in 41% of patients with HBsAg-positive chronic hepatitis but not in patients with HBsAg-negative chronic hepatitis. Hepatitis Be antigen (an antigen closely associated with the core of Dane particles) determined in the same sera by radioimmunoassay, was not detected in 50% of HBcAg-positive sera

  12. Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

    Science.gov (United States)

    Akl, Ahmed; Lepetit, Maud; Crochette, Romain; Giral, Magali; Lepourry, Julie; Pallier, Annaick; Castagnet, Stéphanie; Dugast, Emilie; Guillot-Gueguen, Cécile; Jacq-Foucher, Marylène; Saulquin, Xavier; Cesbron, Anne; Laplaud, David; Nicot, Arnaud; Brouard, Sophie; Soulillou, Jean-Paul

    2013-01-01

    In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27−IgD+ (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells. PMID:24386360

  13. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna

    2011-06-28

    The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

  14. Synthetic antigens, radiolabelled derivatives thereof, and methods of analysis using such derivatives

    International Nuclear Information System (INIS)

    Eisenhardt, W.A. Jr.; Hedaya, E.; Theodoropulos, S.

    1981-01-01

    This patent claim on behalf of Union Carbide Corporation, relates to a method of carrying out a competitive binding radioassay of a compound of interest in a clinical sample, using isocyanates labelled with radioiodine as synthetic antigens. (U.K.)

  15. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

    DEFF Research Database (Denmark)

    Jamnani, Fatemeh Rahimi; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali

    2014-01-01

    Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination...

  16. Humoral and cell-mediated immune response against human retinal antigens in relation to ocular onchocerciasis

    NARCIS (Netherlands)

    van der Lelij, A.; Rothova, A.; Stilma, J. S.; Vetter, J. C.; Hoekzema, R.; Kijlstra, A.

    1990-01-01

    Autoimmune mechanisms are thought to be involved in the pathogenesis of the chorioretinal changes in ocular onchocerciasis. The humoral autoimmune response was determined by measuring serum levels of autoantibodies, directed against human S-antigen and interphotoreceptor retinoid binding protein

  17. Antigen smuggling in tuberculosis.

    Science.gov (United States)

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Antigen detection systems

    Science.gov (United States)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

  19. Isocyanate test antigens

    International Nuclear Information System (INIS)

    Karol, M.H.; Alarie, Y.C.

    1980-01-01

    A test antigen for detecting antibodies to a diisocyanate comprises the reaction product of a protein and a monoisocyanate derived from the same radical as the diisocyanate. The diisocyanates most usually encountered and therefore calling for antibody detection are those of toluene, hexamethylene, methylene, isophorone and naphthylene. The preferred protein is human serum albumin. (author)

  20. Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

    NARCIS (Netherlands)

    Dolk, E.; Vliet, C. van; Perez, J.M.J.; Vriend, G.; Darbon, H.; Ferrat, G.; Cambillau, C.; Frenken, L.G.J.; Verrips, T.

    2005-01-01

    In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains

  1. The role of CD4 in antigen-independent activation of isolated single T lymphocytes

    DEFF Research Database (Denmark)

    Kelso, A; Owens, T

    1988-01-01

    The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An ass...

  2. Distribution of 125I-monoclonal antibodies to antigen Ly 2.1 of T-lymphocytes in mice under the influence of immunomodulators

    International Nuclear Information System (INIS)

    Mirolyubova, Sh.Yu.; Fadeev, N.P.; Serzhanina, V.A.; Klimovich, V.B.; Makarenko, M.V.; Korsakova, L.N.

    1991-01-01

    A study was made of the distribution of 125 I (a chloramine method of labelling) monoclonal antibodies to the surface antigen Ly 2.1 T-lymphocytes during action of immunomodulators (tactivin, hydrocortisone, tactivin administered after hydrocortisone) on ACR mice. These antibodies were shown to retain antigen binding capacity, permitting monitoring of the redistribution of the antigen in the body exposed to immunomodulators

  3. Deteksi Antigen pada Kriptokokosis

    Directory of Open Access Journals (Sweden)

    Robiatul Adawiyah

    2014-12-01

    Full Text Available AbstrakKriptokokosis merupakan infeksi sistemik yang disebabkan Cryptococcus sp. Predileksi jamur tersebut adalah susunan saraf pusat dan selaput otak. Terdapat 5 spesies Cryptococcus sp. yang menyebabkan penyakit pada manusia; yang paling banyak adalah Cr. neoformans dan Cr. gattii. Diagnosis kriptokokosis ditegakkan berdasarkan gejala klinis, pemeriksaan laboratoris serta radiologis. Pemeriksaan laboratoris dilakukan dengan identifikasi morfologi, serologi danPCR. Pemeriksaan secara morfologi dengan tinta India positif  bila jumlah sel jamur 10  sel/ml spesimen. Kultur dilakukan di media sabouraud dextrose agar (SDA dan niger sheed agar (NSA, jamur tumbuh setelah 5-7 hari. Deteksi antigen dan antibodi dilakukan pada cairan tubuh dan tidak membutuhkan waktu lama. Deteksi antibodi Cr.neoformans memiliki kelemahan yaitu tidak menunjukkan hasil positif pada infeksi akut, IgA masih positif setelah 1-2 tahun fase penyembuhan, IgG dapat persisten, pada individu imunokompromis menunjukkan hasil yang sangat kompleks dan dalam menentukan diagnosis sering tidak konsisten. Polisakarida adalah komponen paling berperan dalam virulensi Cr. neoformans. Komponen polisakarida terutama glucuronoxylomannan merupakan petanda penting dalam diagnosis kriptokokosis secara serologis. Deteksi antigen Cr. neoformans memiliki kelebihan yaitu menunjukkan hasil positif pada infeksi akut/kronis, sensitivitas dan spesifisitas tinggi, dapat mendeteksi polisakarida hingga 10 ng/ml sehingga dengan kadarantigen yang minimal tetap dapat mendiagnosis kriptokokosis.Kata kunci: Cr. neoformans, glucuronoxylomannan, antigenAbstractCryptococcosis is systemic infection that caused by Cryptococcus sp. Predilection of this fungi is the central nervous system and brain membrane. There are 5 species of Cryptococcus sp. that cause cryptococcosis in human; but the majority are caused by Cr. neoformans and Cr. gattii. The diagnosis of cryptococcosis is made based on clinical symptoms

  4. A radioimmunoassay to screen for antibodies to native conformational antigens and analyse ligand-induced structural states of antigenic proteins

    International Nuclear Information System (INIS)

    Bernotat-Danielowski, S.; Koepsell, H.

    1988-01-01

    A radioimmunoassay is described in which antigenic protein was immobilized by incubating nitrocellulose filters of defined diameter with antigen-containing solutions. Antigenic sites which are sensitive to protein denaturation by drying could be detected with the assay. The assay was also used to screen hybridoma supernatants for antibodies directed against Na + cotransport proteins from renal brush-border membranes. Monoclonal antibodies were selected which showed different binding charactertics depending on whether or not substrates of Na + cotransporters were present. One of the antibodies, which showed different antibody binding after addition of D-glucose or L-lactate, bound to a polypeptide component of the renal N + -D-glucose cotransporter and was able to inhibit Na + gradient-dependent. To investigate the effects of D-glucose and L-lactate on the binding of this antibody concentration dependence was measured. High and low affinity binding sites for D-glucose and L-lactate were characterized thereby demonstrating that the radioimmunoassay permits investigations of the properties of high and low affinity substrate binding sites. (author). refs.; 6 figs.; 2 tabs

  5. Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

    International Nuclear Information System (INIS)

    Abbas, A.K.; Haber, S.; Rock, K.L.

    1985-01-01

    Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes

  6. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-12-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  7. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    International Nuclear Information System (INIS)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-01-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae

  8. Carcino-Embryonic Antigen

    International Nuclear Information System (INIS)

    Akute, O.

    1999-02-01

    Tumour marker analysis has increased our understanding of the presence of tumours in the body. Carcino-embryonic antigen, CEA, is one of the best studied tumour markers and has proved an ideal diagnostic adjuvant. It has helped in quantifying the amount of disease present in a patient and thence to make accurate prognosis on the various diagnosed ailments. At UCH, it is observed that there is an increase in cancer related ailments and therefore the need for early diagnosis is more compelling in our environment to mitigate future cost of managing advanced manifestation

  9. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

    Directory of Open Access Journals (Sweden)

    Martin Kreutz

    Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

  10. Novel adenovirus encoded virus-like particles displaying the placental malaria associated VAR2CSA antigen

    DEFF Research Database (Denmark)

    Andersson, Anne-Marie C; dos Santos Marques Resende, Mafalda; Salanti, Ali

    2017-01-01

    The malaria parasite Plasmodium falciparum presents antigens on the infected erythrocyte surface that bind human receptors expressed on the vascular endothelium. The VAR2CSA mediated binding to a distinct chondroitin sulphate A (CSA) is a crucial step in the pathophysiology of placental malaria a...

  11. The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

    KAUST Repository

    Chailyan, Anna; Marcatili, Paolo; Tramontano, Anna

    2011-01-01

    of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface

  12. Structural requirements for the interaction between class II MHC molecules and peptide antigens

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Appella, E

    1990-01-01

    of binding, it is possible to define certain structural features of peptides that are associated with the capacity to bind to a particular MHC specificity (IA(d) or IE(d)); 3) IA(d) and IE(d) molecules recognize different and independent structures on the antigen molecule; 4) only about 10% of the single...... IA(d) and IE(d) molecules and their peptide ligands, we found that some structural characteristics apply to both antigen-MHC interactions. In particular, we found: 1) each MHC molecule is capable of binding many unrelated peptides through the same peptide-binding site; 2) despite this permissiveness...... amino acid substitutions tested on two IA(d)- and IE(d)-binding peptides had significant effect on their MHC-binding capacities, while over 80% of these substitutions significantly impaired T cell recognition of the Ia-peptide complex; 5) based on the segregation between residues that are crucial for T...

  13. Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

    Science.gov (United States)

    Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

    2018-02-01

    Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

  14. An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

    Directory of Open Access Journals (Sweden)

    Marcela V Maus

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

  15. Antigen antibody interactions

    CERN Document Server

    DeLisi, Charles

    1976-01-01

    1. 1 Organization of the Immune System One of the most important survival mechanisms of vertebrates is their ability to recognize and respond to the onslaught of pathogenic microbes to which they are conti- ously exposed. The collection of host cells and molecules involved in this recognition­ 12 response function constitutes its immune system. In man, it comprises about 10 cells 20 (lymphocytes) and 10 molecules (immunoglobulins). Its ontogenic development is c- strained by the requirement that it be capable of responding to an almost limitless variety of molecular configurations on foreign substances, while simultaneously remaining inert to those on self components. It has thus evolved to discriminate, with exquisite precision, between molecular patterns. The foreign substances which induce a response, called antigens, are typically large molecules such as proteins and polysaccharides. The portions of these with which immunoglobulins interact are called epitopes or determinants. A typical protein epitope m...

  16. The O-antigen structure of bacterium Comamonas aquatica CJG.

    Science.gov (United States)

    Wang, Xiqian; Kondakova, Anna N; Zhu, Yutong; Knirel, Yuriy A; Han, Aidong

    2017-11-01

    Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1 H and 13 C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

  17. Radioimmunoassays of hidden viral antigens

    International Nuclear Information System (INIS)

    Neurath, A.R.; Strick, N.; Baker, L.; Krugman, S.

    1982-01-01

    Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure

  18. Development of an epitope panel for consistent identification of antigen-specific T-cells in humans

    DEFF Research Database (Denmark)

    Fløe, Andreas; Løppke, Caroline; Hilberg, Ole

    2017-01-01

    Objective We aimed to establish a panel of MHC-peptide multimers suitable as a positive control in detection of HLA A*0201 restricted antigen specific T-cells (ASTC) by flow cytometry. Materials and methods MHC Dextramers were loaded with HLA A*0201 binding peptides from viral antigens and melano...

  19. Splenic B cells and antigen-specific B cells process anti-Ig in a similar manner

    International Nuclear Information System (INIS)

    Myers, C.D.; Vitetta, E.S.

    1989-01-01

    B lymphocytes can process and present antigen to T cells. However, the fate of native antigen after its binding to specific B cells, i.e., the intracellular events involved in the processing and recycling of the antigenic fragments to the cell surface for antigen presentation, are not well understood. In the present study, we demonstrate that murine B cells degrade anti-Ig molecules bound to their surface and release acid soluble fragments into the supernatant. We also demonstrate that the kinetics of this process are identical for anti-mu, anti-delta, and anti-light chain antibodies, indicating that both surface IgM and surface IgD are equally effective in binding antigen and directing its processing. We also describe the effects of azide, chloroquine, and irradiation on this process. To extend these studies to the processing of specifically bound antigen, we demonstrate that highly purified trinitrophenyl antigen-binding cells degrade anti-Ig molecules with the same kinetics as unpurified splenic B cells. Thus, this purified population provides a suitable model system for the analysis of antigen degradation by antigen-specific cells

  20. Modeling antigen-antibody nanoparticle bioconjugates and their polymorphs

    Science.gov (United States)

    Desgranges, Caroline; Delhommelle, Jerome

    2018-03-01

    The integration of nanomaterials with biomolecules has recently led to the development of new ways of designing biosensors, and through their assembly, to new hybrid structures for novel and exciting applications. In this work, we develop a coarse-grained model for nanoparticles grafted with antibody molecules and their binding with antigens. In particular, we isolate two possible states for antigen-antibody pairs during the binding process, termed as recognition and anchoring states. Using molecular simulation, we calculate the thermodynamic and structural features of three possible crystal structures or polymorphs, the body-centered cubic, simple cubic, and face-centered cubic phases, and of the melt. This leads us to determine the domain of stability of the three solid phases. In particular, the role played by the switching process between anchoring and recognition states during melting is identified, shedding light on the complex microscopic mechanisms in these systems.

  1. Doxorubicin-anti-carcinoembryonic antigen immunoconjugate activity in vitro.

    Science.gov (United States)

    Richardson, V J; Ford, C H; Tsaltas, G; Gallant, M E

    1989-04-01

    An in vitro model consisting of a series of 11 human cancer cell lines with varying density of expression of membrane carcinoembryonic antigen (CEA) has been used to evaluate conjugates of doxorubicin (Adriamycin) covalently linked by a carbodiimide method to goat polyclonal antibodies and mouse monoclonal antibodies to CEA. Conjugates were produced which retained both antigen binding and drug cytotoxicity. IC50 values were determined for free drug, free drug mixed with unconjugated antibodies and for the immunoconjugates. Cell lines that were very sensitive to free drug (IC50 less than 100 ng/ml) were also found to be highly sensitive to conjugated drug and similarly cell lines resistant to drug (IC50 greater than 1,000 ng/ml) were also resistant to conjugated drug. Although there was no correlation between CEA expression and conjugates efficacy, competitive inhibition studies using autologous antibody to block conjugate binding to cells indicated immunoconjugates specificity for the CEA target.

  2. Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy

    Science.gov (United States)

    Lin, Margaret; Krawitz, Denise; Callahan, Matthew D.; Deperalta, Galahad; Wecksler, Aaron T.

    2018-05-01

    We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. [Figure not available: see fulltext.

  3. Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy

    Science.gov (United States)

    Lin, Margaret; Krawitz, Denise; Callahan, Matthew D.; Deperalta, Galahad; Wecksler, Aaron T.

    2018-03-01

    We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. [Figure not available: see fulltext.

  4. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism

    OpenAIRE

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-01-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leuko...

  5. Patterns of predicted T-cell epitopes associated with antigenic drift in influenza H3N2 hemagglutinin.

    Directory of Open Access Journals (Sweden)

    E Jane Homan

    Full Text Available Antigenic drift allowing escape from neutralizing antibodies is an important feature of transmission and survival of influenza viruses in host populations. Antigenic drift has been studied in particular detail for influenza A H3N2 and well defined antigenic clusters of this virus documented. We examine how host immunogenetics contributes to determination of the antibody spectrum, and hence the immune pressure bringing about antigenic drift. Using uTOPE™ bioinformatics analysis of predicted MHC binding, based on amino acid physical property principal components, we examined the binding affinity of all 9-mer and 15-mer peptides within the hemagglutinin 1 (HA1 of 447 H3N2 virus isolates to 35 MHC-I and 14 MHC-II alleles. We provide a comprehensive map of predicted MHC-I and MHC-II binding affinity for a broad array of HLA alleles for the H3N2 influenza HA1 protein. Each HLA allele exhibited a characteristic predicted binding pattern. Cluster analysis for each HLA allele shows that patterns based on predicted MHC binding mirror those described based on antibody binding. A single amino acid mutation or position displacement can result in a marked difference in MHC binding and hence potential T-helper function. We assessed the impact of individual amino acid changes in HA1 sequences between 10 virus isolates from 1968-2002, representative of antigenic clusters, to understand the changes in MHC binding over time. Gain and loss of predicted high affinity MHC-II binding sites with cluster transitions were documented. Predicted high affinity MHC-II binding sites were adjacent to antibody binding sites. We conclude that host MHC diversity may have a major determinant role in the antigenic drift of influenza A H3N2.

  6. COLONOSCOPY AND CARCINOEMBRYONIC ANTIGEN VARIATIONS

    Directory of Open Access Journals (Sweden)

    Rita G SOUSA

    2014-03-01

    Full Text Available Context Colonoscopy is essential for synchronous and metachronous cancer detection. Carcinoembryonic antigen is a colorectal cancer tumor marker, important as a follow-up tool in patients with previous colorectal cancer. False-positive carcinoembryonic antigen elevation results in multiples exams and in patient anxiety. In literature, there is reference to transient carcinoembryonic antigen increase with colonoscopy. Objective To evaluate the influence of bowel preparation and colonoscopy in carcinoembryonic antigen blood levels. Methods We prospectively studied subjects that underwent routine colonoscopy in our institution. Blood samples were collected (1 before bowel cleaning, (2 before colonoscopy and (3 immediately after colonoscopy. Blood carcinoembryonic antigen levels were determined by “Sandwich” immunoassay. The statistical methods used were the paired t-test and ANOVA. Results Thirty-seven patients (22M/15F were included; age range 28-84 (mean 56 years. Mean carcinoembryonic antigen values were 1.9, 2 and 1.8 for (1, (2 and (3, respectively. An increase in value (2 compared with (1 was observed in 20/37 patients (P = 0.018, mainly in younger patients and in patients requiring more endoluminal interventions. In 29/37 patients, the CEA value decreased from (2 to (3 (P = 1.3x10-7. Conclusions A trend for carcinoembryonic antigen increase after bowel cleaning was observed, especially in younger patients and in patients with more endoluminal interventions, but without clinical meaning.

  7. Isolation and characterization of antigen-Ia complexes involved in T cell recognition

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1986-01-01

    Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes, it is he......Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes...... with glutaraldehyde revealed that the ovalbumin peptide was cross-linked solely to the alpha chain of I-Ad. Planar membranes containing I-Ad-OVA complexes stimulated a T cell response with 2 X 10(4) less antigen than required when uncomplexed antigen was used, thus demonstrating the biologic importance...

  8. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

    Directory of Open Access Journals (Sweden)

    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  9. Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge

    Science.gov (United States)

    Blanc, Pascal; Moro-Sibilot, Ludovic; Barthly, Lucas; Jagot, Ferdinand; This, Sébastien; de Bernard, Simon; Buffat, Laurent; Dussurgey, Sébastien; Colisson, Renaud; Hobeika, Elias; Fest, Thierry; Taillardet, Morgan; Thaunat, Olivier; Sicard, Antoine; Mondière, Paul; Genestier, Laurent; Nutt, Stephen L.; Defrance, Thierry

    2016-01-01

    Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge. PMID:27924814

  10. Shedding of leukemia-associated P24 antigen by lymphoblastoid cell lines.

    Science.gov (United States)

    Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

    1987-12-01

    We report the development of a unique enzyme-linked immunosorbent assay (ELISA) which makes possible the detection of leukemia-associated P24 antigen, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4 simultaneously. Using the RCA1/SJ-9A4-ELISA, P24 antigen, as few as 50 X 10(3) cells from a common acute lymphoblastic leukemia (C-ALL) cell line could be detected. The presence of D-galactose gave complete and specific inhibition of P24 antigen binding to RCA1. Matched concentrations of D-glucose and D-sucrose had no effect on binding. The release of the P24 antigen into the culture medium by a C-ALL cell line maintained at 37 degrees C could be detected; however, no P24 antigen was present in the culture medium when the cells were maintained at 4 degrees C. Sequential analysis of the culture medium for soluble P24 antigen revealed that release of the P24 antigen associated with cell growth. Molecular sieve chromatography of concentrated culture medium indicated that shed P24 antigen was eluted in the macromolecule fraction. P24 antigen was detected in the cerebrospinal fluid (CSF) of four patients with P24 positive ALL at the time of relapse of the central nervous system (CNS) and was undetectable while in complete remission. The CSF from three patients with P24 negative ALL and three patients with aseptic meningitis had no detectable activity.

  11. Oncogenic cancer/testis antigens

    DEFF Research Database (Denmark)

    Gjerstorff, Morten F; Andersen, Mads H; Ditzel, Henrik J

    2015-01-01

    Recent developments have set the stage for immunotherapy as a supplement to conventional cancer treatment. Consequently, a significant effort is required to further improve efficacy and specificity, particularly the identification of optimal therapeutic targets for clinical testing. Cancer....../testis antigens are immunogenic, highly cancer-specific, and frequently expressed in various types of cancer, which make them promising candidate targets for cancer immunotherapy, including cancer vaccination and adoptive T-cell transfer with chimeric T-cell receptors. Our current understanding of tumor...... immunology and immune escape suggests that targeting oncogenic antigens may be beneficial, meaning that identification of cancer/testis antigens with oncogenic properties is of high priority. Recent work from our lab and others provide evidence that many cancer/testis antigens, in fact, have oncogenic...

  12. Natural selection promotes antigenic evolvability.

    Science.gov (United States)

    Graves, Christopher J; Ros, Vera I D; Stevenson, Brian; Sniegowski, Paul D; Brisson, Dustin

    2013-01-01

    The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.

  13. Natural selection promotes antigenic evolvability.

    Directory of Open Access Journals (Sweden)

    Christopher J Graves

    Full Text Available The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish

  14. Structural and antigenic variation among diverse clade 2 H5N1 viruses.

    Directory of Open Access Journals (Sweden)

    David A Shore

    Full Text Available Antigenic variation among circulating H5N1 highly pathogenic avian influenza A viruses mandates the continuous production of strain-specific pre-pandemic vaccine candidates and represents a significant challenge for pandemic preparedness. Here we assessed the structural, antigenic and receptor-binding properties of three H5N1 HPAI virus hemagglutinins, which were recently selected by the WHO as vaccine candidates [A/Egypt/N03072/2010 (Egypt10, clade 2.2.1, A/Hubei/1/2010 (Hubei10, clade 2.3.2.1 and A/Anhui/1/2005 (Anhui05, clade 2.3.4]. These analyses revealed that antigenic diversity among these three isolates was restricted to changes in the size and charge of amino acid side chains at a handful of positions, spatially equivalent to the antigenic sites identified in H1 subtype viruses circulating among humans. All three of the H5N1 viruses analyzed in this study were responsible for fatal human infections, with the most recently-isolated strains, Hubei10 and Egypt10, containing multiple residues in the receptor-binding site of the HA, which were suspected to enhance mammalian transmission. However, glycan-binding analyses demonstrated a lack of binding to human α2-6-linked sialic acid receptor analogs for all three HAs, reinforcing the notion that receptor-binding specificity contributes only partially to transmissibility and pathogenesis of HPAI viruses and suggesting that changes in host specificity must be interpreted in the context of the host and environmental factors, as well as the virus as a whole. Together, our data reveal structural linkages with phylogenetic and antigenic analyses of recently emerged H5N1 virus clades and should assist in interpreting the significance of future changes in antigenic and receptor-binding properties.

  15. Proteins involved in invasion of human red blood cells by malaria parasites

    Directory of Open Access Journals (Sweden)

    Ewa Jaśkiewicz

    2010-11-01

    Full Text Available Malaria is a disease caused by parasites of Plasmodium species. It is responsible for around 1-2 million deaths annually, mainly children under the age of 5. It occurs mainly in tropical and subtropical areas.Malaria is caused by five Plasmodium species:[i] P. falciparum, P. malariae, P. vivax, P. knowlesi[/i] and [i]P. ovale[/i]. Mosquitoes spread the disease by biting humans. The malaria parasite has two stages of development: the human stage and the mosquito stage. The first stage occurs in the human body and is divided into two phases: the liver phase and the blood phase.The invasion of erythrocytes by [i]Plasmodium[/i] merozoites is a multistep process of specific protein interactions between the parasite and red blood cell. The first step is the reversible merozoite attachment to the erythrocyte followed by its apical reorientation, then formation of an irreversible “tight” junction and finally entry into the red cell in a parasitophorous vacuole.The blood phase is supported by a number of proteins produced by the parasite. The merozoite surface GPI-anchored proteins (MSP-1, 2, 4, 5, 8 and 10 assist in the process of recognition of susceptible erythrocytes, apical membrane antigen (AMA-1 may be directly responsible for apical reorientation of the merozoite and apical proteins which function in tight junction formation. These ligands are members of two families: Duffy binding-like (DBL and reticulocyte binding-like (RBL proteins. In [i]Plasmodium[/i] [i]falciparum[/i] the DBL family includes: EBA-175, EBA-140 (BAEBL, EBA-181 (JESEBL, EBA-165 (PEBL and EBL-1 ligands.To date, no effective antimalarial vaccine has been developed, but there are several studies for this purpose. Therefore, it is crucial to understand the molecular basis of host cells invasion by parasites. Major efforts are focused on developing a multiantigenic and multiepitope vaccine preventing all steps of [i]Plasmodium[/i] invasion.

  16. Anvendelse af prostataspecifikt antigen. En oversigt

    DEFF Research Database (Denmark)

    Brasso, K; Skaarup, P; Roosen, Jens Ulrik

    1998-01-01

    Since it was first introduced, measurement of prostate specific antigen has gained increasing interest, and prostate specific antigen is regarded as being the best tumour marker available. The antigen lacks cancer specificity, limiting the usefulness in early diagnosis, The use of prostate specific...... antigen in early diagnosis, staging, and in monitoring patients with prostate cancer is reviewed....

  17. Chemoselective ligation and antigen vectorization.

    Science.gov (United States)

    Gras-Masse, H

    2001-01-01

    The interest in cocktail-lipopeptide vaccines has now been confirmed by phase I clinical trials: highly diversified B-, T-helper or cytotoxic T-cell epitopes can be combined with a lipophilic vector for the induction of B- and T-cell responses of predetermined specificity. With the goal of producing an improved vaccine that should ideally induce a multispecific response in non-selected populations, increasing the diversity of the immunizing mixture represents one of the most obvious strategies.The selective delivery of antigens to professional antigen-presenting cells represents another promising approach for the improvement of vaccine efficacy. In this context, the mannose-receptor represents an attractive entry point for the targeting to dendritic cells of antigens linked to clustered glycosides or glycomimetics. In all cases, highly complex but fully characterized molecules must be produced. To develop a modular and flexible strategy which could be generally applicable to a large set of peptide antigens, we elected to explore the potentialities of chemoselective ligation methods. The hydrazone bond was found particularly reliable and fully compatible with sulphide ligation. Hydrazone/thioether orthogonal ligation systems could be developed to account for the nature of the antigens and the solubility of the vector systems. Copyright 2001 The International Association for Biologicals.

  18. Antigen-antibody reactions of UV-irradiated phage DNA

    International Nuclear Information System (INIS)

    Fink, A.

    1976-01-01

    The observation of others could be confirmed that UV-irradiated DNA is a better immunogen than unirradiated DNA. The author's immune sera contained a high amount of antibodies with a specific action against photoproducts in the DNA. The thymine dimer was identified as relevant photoproduct and thus as antigenic determinant. In comparison, the amount of unspecific antibodies reacting with denaturated DNA was low and varied between sera. Thymin-dimer antibodies showed a high specificity without cross-reaction with other pyrimidine dimers such as anti CC and anti CT; they belong to the class of IgG molecules. UV-irradiated dinucleotide dTpT is sufficient to induce the formation of antibodies reacting with the cis-syn thymine dimers in UV-irradiated DNA. Antibody binding is proportional to the UV doses applied to the DNA. When using completely denaturated DNA, there is a linear increase changing into a plateau at higher doses. The extent of antigen-antibody binding is strongly dependent on the degree of denaturation of the DNA. With increasing denaturation, the antibody binding of the DNA increases. The antigen-antibody reaction can thus be used to estimate the degree of denaturation of the DNA. There were no signs of an influence of the degree of denaturation of the DNA on the quantum yield of thymine dimers. The different amounts of antibodies is therefore due to the masking of thymine dimers in native DNA. When irradiating intact phage particles, there was no sign of an influence of the phages' protein covers on the antibody binding capacity of DNA compared with DNA irradiated in vitro. (orig.) [de

  19. A new application of scanning electrochemical microscopy for the label-free interrogation of antibody-antigen interactions

    Energy Technology Data Exchange (ETDEWEB)

    Holmes, Joanne L.; Davis, Frank; Collyer, Stuart D. [Cranfield Health, Cranfield University, Cranfield, MK43 0AL (United Kingdom); Higson, Seamus P.J., E-mail: s.p.j.higson@cranfield.ac.uk [Cranfield Health, Cranfield University, Cranfield, MK43 0AL (United Kingdom)

    2011-03-18

    Within this work we present a 'proof of principle' study for the use of scanning electrochemical microscopy (SECM) to detect and image biomolecular interactions in a label-free assay as a potential alternative to current fluorescence techniques. Screen-printed carbon electrodes were used as the substrate for the deposition of a dotted array, where the dots consist of biotinylated polyethyleneimine. These were then further derivatised, first with neutravidin and then with a biotinylated antibody to the protein neuron specific enolase (NSE). SECM using a ferrocene carboxylic acid mediator showed clear differences between the array and the surrounding unmodified carbon. Imaging of the arrays before and following exposure to various concentrations of the antigen showed clear evidence for specific binding of the NSE antigen to the antibody derivatised dots. Non-specific binding was quantified. Control experiments with other proteins showed only non-specific binding across the whole of the substrate, thereby confirming that specific binding does occur between the antibody and antigen at the surface of the dots. Binding of the antigen was accompanied by a measured increase in current response, which may be explained in terms of protein electrostatic interaction and hydrophobic interactions to the mediator, thereby increasing the localised mediator flux. A calibration curve was obtained between 500 fg mL{sup -1} to 200 pg mL{sup -1} NSE which demonstrated a logarithmic relationship between the current change upon binding and antigen concentration without the need for any labelling of the substrate.

  20. Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses.

    Science.gov (United States)

    Dowdall, S M J; Proudman, C J; Love, S; Klei, T R; Matthews, J B

    2003-12-01

    Cyathostomins are important equine gastrointestinal parasites. Mass emergence of mucosal stage larvae causes a potentially fatal colitis. Mucosal stages are undetectable non-invasively. An assay that would estimate mucosal larval stage infection would greatly assist in treatment, control and prognosis. Previously, we identified two putative diagnostic antigens (20 and 25 kDa) in somatic larval preparations. Here, we describe their purification and antigen-specific IgG(T) responses to them. Western blots confirmed the purity of the antigens and showed that epitopes in the 20 kDa complex were specific to larval cyathostomins. No cross-reactive antigens appeared to be present in Parascaris equorum or Strongyloides westeri species. Low levels of cross-reactivity were observed in Strongylus edentatus and Strongylus vulgaris species. Use of purified antigens greatly reduced background binding in equine sera. These results indicate that both antigen complexes may be of use in a diagnostic assay.

  1. A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40

    DEFF Research Database (Denmark)

    Götz, C; Koenig, M G; Issinger, O G

    1995-01-01

    by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed......The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we...... show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation...

  2. Interleukin 1-induced down-regulation of antibody binding to CD4 molecules on human lymphocytes

    DEFF Research Database (Denmark)

    Tvede, N; Christensen, L D; Ødum, Niels

    1988-01-01

    Interleukin 1 (IL-1) is involved in the early activation of T lymphocytes. The CD4 antigen, described as a phenotypic marker of helper T cells, is also important in early T-cell activation by its ability to bind to MHC class II molecules on antigen-presenting cells, and to transmit positive (and...

  3. THE SEARCH OF OPTIMAL COMBINATION OF ANTIGENS FOR SEROLOGICAL DIAGNOSTICS OF TUBERCULOSIS

    Directory of Open Access Journals (Sweden)

    E. V. Vasilyeva

    2013-01-01

    Full Text Available Abstract. The four chimeric recombinant antigens CBD-CFP10, CBD-ESAT6, ESAT6-CFP10 and CBD-P38 contained aminoacid sequences of full-size proteins ESAT6, CFP10 and matured protein P38 of M. tuberculosis, joined with aminoacid sequences of cellulose bind domain of endogluconase A (CBD from Cellumonas fimi have been obtained by gene engineering methods. Recombinant proteins were purified by affine chromatography in column with Ni-NTA-sepharose 6В-CL and as PPDN-3 were used for detection of their antigenic activity in indirect ELISA for TB serological diagnostics. The sera from patients with lung tuberculosis (n = 321, from persons who had professional contacts with TB patients (n = 42, from healthy blood donors (n = 366 and from patients with lung diseases of non-TB etiology were tested. It was detected that there was positive correlation between antibodies level for all studied antigens compared by pair. It has been demonstrated that although antigens were different by antigenic and immunobiological characteristics they add each other in the content of antigenic diagnostics compositions. Thus, all these antigens can be used in the test kits for serological diagnostics of TB. Using of these antigens will allow to detect persons infected by TB and patients with active tuberculosis. 

  4. Effect of antigen shedding on targeted delivery of immunotoxins in solid tumors from a mathematical model.

    Directory of Open Access Journals (Sweden)

    Youngshang Pak

    Full Text Available Most cancer-specific antigens used as targets of antibody-drug conjugates and immunotoxins are shed from the cell surface (Zhang & Pastan (2008 Clin. Cancer Res. 14: 7981-7986, although at widely varying rates and by different mechanisms (Dello Sbarba & Rovida (2002 Biol. Chem. 383: 69-83. Why many cancer-specific antigens are shed and how the shedding affects delivery efficiency of antibody-based protein drugs are poorly understood questions at present. Before a detailed numerical study, it was assumed that antigen shedding would reduce the efficacy of antibody-drug conjugates and immunotoxins. However, our previous study using a comprehensive mathematical model showed that antigen shedding can significantly improve the efficacy of the mesothelin-binding immunotoxin, SS1P (anti-mesothelin-Fv-PE38, and suggested that receptor shedding can be a general mechanism for enhancing the effect of inter-cellular signaling molecules. Here, we improved this model and applied it to both SS1P and another recombinant immunotoxin, LMB-2, which targets CD25. We show that the effect of antigen shedding is influenced by a number of factors including the number of antigen molecules on the cell surface and the endocytosis rate. The high shedding rate of mesothelin is beneficial for SS1P, for which the antigen is large in number and endocytosed rapidly. On the other hand, the slow shedding of CD25 is beneficial for LMB-2, for which the antigen is small in number and endocytosed slowly.

  5. Specific binding-adsorbent assay method and test means

    International Nuclear Information System (INIS)

    1981-01-01

    A description is given of an improved specific binding assay method and test means employing a nonspecific adsorbent for the substance to be determined, particularly hepatitis B surface (HBsub(s)) antigen, in its free state or additionally in the form of its immune complex. The invention is illustrated by 1) the radioimmunoadsorbent assay for HBsub(s) antigen, 2) the radioimmunoadsorbent assay for HBsub(s) antigen in the form of immune complex with antibody, 3) a study of adsorption characteristics of various anion exchange materials for HBsub(s) antigen, 4) the use of hydrophobic adsorbents in a radioimmunoadsorbent assay for HBsub(s) antigen and 5) the radioimmunoadsorbent assay for antibody to HBsub(s) antigen. The advantages of the present method for detecting HBsub(s) antigen compared to previous methods include the manufacturing advantages of eliminating the need for insolubilised anti-HBsub(s) and the advantages of a single incubation step, fewer manipulations, storability of adsorbent materials, increased sensitivity and versatility of detecting HBsub(s) antigen in the form of its immune complex if desired. (U.K.)

  6. [Examination about utility of a Streptococcus pneumoniae capsular antigen swiftness search kit urine in a pneumonia patient].

    Science.gov (United States)

    Hashikita, Giichi; Yamaguti, Toshiyuki; Tachi, Yoshimi; Kishi, Etsuko; Kawamura, Toru; Takahashi, Shun; Arai, Yukie; Koyama, Sachie; Huruhata, Toshihumi; Itabashi, Akira; Oka, Yoko; Yamazaki, Tsutomu; Maesaki, Sigefumi

    2005-01-01

    We investigated the usefullness of Binax NOW urine antigen test, an immunochromatographic assay that binds any soluble Streptococcus pneumoniae antigen (C polysaccharide) for the diagnosis of penumoniae form September 2003 to March 2005. We used 372 samples form the patinets with pneumoniae diagnosed for blood or sputum cultuter or gram-stained sputum smear. Out of 24 culture positive specimens, Binax NOW urine antigen test, showed positive in 18 (75%) specimens. The sensitivity of sputum and blood culture was 71.7% and 83.3%, respectively. Binax NOW urine antigen test was seemed false positives in 55 samples, false negatives in 6 samples. The specificity of Binax NOW urine antigen test was evaluated 84.1%. Overall agreement among tests was 83.6%. When compared to culture, false negative urine antigen may be the result of colonizing S. pneumoniae in sputum or pneumonia caused by an agent other than S. pneumoniae. CRP values for cases were both urine antigen and culture were positive ranged from 40 mg/dl to 10 mg/dl while urine antigen and culture negative cases were predominantly less than 10 mg/dl. Positive blood and pleural fluid culture cases were consistently associated with strongly positive urine antigen tests. Non-agreement between urine antigen, culture, and microscopy may be the result of specimen quality, labile nature of S. pneumoniae and antimicrobial therapy.

  7. Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

    Energy Technology Data Exchange (ETDEWEB)

    Archbold, Julia K.; Macdonald, Whitney A.; Gras, Stephanie; Ely, Lauren K.; Miles, John J.; Bell, Melissa J.; Brennan, Rebekah M.; Beddoe, Travis; Wilce, Matthew C.J.; Clements, Craig S.; Purcell, Anthony W.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie; (Monash); (Queensland Inst. of Med. Rsrch.); (Melbourne)

    2009-07-10

    Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

  8. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    Holers, V.M.; Kotzin, B.L.

    1985-01-01

    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

  9. Natural selection promotes antigenic evolvability

    NARCIS (Netherlands)

    Graves, C.J.; Ros, V.I.D.; Stevenson, B.; Sniegowski, P.D.; Brisson, D.

    2013-01-01

    The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide

  10. Chimeric Antigen Receptors to CD276 for Treating Cancer | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    This licensing opportunity from the National Cancer Institute concerns the development of CARs comprising an antigen-binding fragment derived from the MGA271 antibody. The resulting CARs can be used in adoptive cell therapy treatment for neuroblastoma and other tumors that express CD276.

  11. Interaction forces between salivary proteins and Streptococcus mutans with and without antigen I/II

    NARCIS (Netherlands)

    Xu, C.P.; Belt-Gritter, van de B.; Dijkstra, R.J.B.; Norde, W.; Mei, van der H.C.; Busscher, H.J.

    2007-01-01

    The antigen I/II family of surface proteins is expressed by oral streptococci, including Streptococcus mutans, and mediates specific binding to, among others, salivary films. The aim of this study was to investigate the interaction forces between salivary proteins and S. mutans with (LT11) and

  12. Concepts and applications for influenza antigenic cartography

    Science.gov (United States)

    Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2011-01-01

    Influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. A robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. However, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incompleteness, high noises, and low reactors. To overcome these challenges, we developed a computational method, temporal Matrix Completion-Multidimensional Scaling (MC-MDS), by adapting the low rank MC concept from the movie recommendation system in Netflix and the MDS method from geographic cartography construction. The application on H3N2 and 2009 pandemic H1N1 influenza A viruses demonstrates that temporal MC-MDS is effective and efficient in constructing influenza antigenic cartography. The web sever is available at http://sysbio.cvm.msstate.edu/AntigenMap. PMID:21761589

  13. Microbial F-type lectin domains with affinity for blood group antigens.

    Science.gov (United States)

    Mahajan, Sonal; Khairnar, Aasawari; Bishnoi, Ritika; Ramya, T N C

    2017-09-23

    F-type lectins are fucose binding lectins with characteristic fucose binding and calcium binding motifs. Although they occur with a selective distribution in viruses, prokaryotes and eukaryotes, most biochemical studies have focused on vertebrate F-type lectins. Recently, using sensitive bioinformatics search techniques on the non-redundant database, we had identified many microbial F-type lectin domains with diverse domain organizations. We report here the biochemical characterization of F-type lectin domains from Cyanobium sp. PCC 7001, Myxococcus hansupus and Leucothrix mucor. We demonstrate that while all these three microbial F-type lectin domains bind to the blood group H antigen epitope on fucosylated glycans, there are fine differences in their glycan binding specificity. Cyanobium sp. PCC 7001 F-type lectin domain binds exclusively to extended H type-2 motif, Myxococcus hansupus F-type lectin domain binds to B, H type-1 and Lewis b motifs, and Leucothrix mucor F-type lectin domain binds to a wide range of fucosylated glycans, including A, B, H and Lewis antigens. We believe that these microbial lectins will be useful additions to the glycobiologist's toolbox for labeling, isolating and visualizing glycans. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Characterization of a human antigen specific helper factor

    International Nuclear Information System (INIS)

    Richardson, B.

    1986-01-01

    While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with 35 S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEM line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants

  15. A rapid one-step radiometric assay for hepatitis B surface antigen utilising monoclonal antibodies

    International Nuclear Information System (INIS)

    Goodall, A.H.; Meek, F.L.; Waters, J.A.; Miescher, G.C.; Janossy, G.; Thomas, H.C.

    1982-01-01

    A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available. (Auth.)

  16. Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites

    International Nuclear Information System (INIS)

    Doinel, C.; Ropars, C.; Salmon, C.

    1978-01-01

    Homogeneous cold agglutinins, purified and labelled with 125 I, have been used in a study of the effects of neuraminidase and proteolytic enzymes on the I and i reactivities of human adult erythrocytes. Measurements were made of antigen site numbers, equilibrium constants and thermodynamic parameters. There was enhanced reactivity after enzyme treatment as well as after the release of N-acetylneuraminic acid. Steric factors were shown to be of primary importance in the accessibility of the I and i antigenic determinant. After enzyme treatment, the antigenic structures became more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane. (author)

  17. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus

    International Nuclear Information System (INIS)

    Salfeld, J.; Pfaff, E.; Noah, M.; Schaller, H.

    1989-01-01

    The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis virus nucleocapsid

  18. Radioimmunoassay for antibodies against surface membrane antigens using adhering cells

    Energy Technology Data Exchange (ETDEWEB)

    Tax, A; Manson, L A [Wistar Inst. of Anatomy and Biology, Philadelphia, Pa. (USA)

    1976-07-01

    A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit /sup 125/I-antimouse-Fab. The bound radioactive material was eluted with glycine-HCl buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolic or noncytolic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface.

  19. Effects of Co60 gamma radiation on the immunogenic and antigenic properties of Bothrops jararacussu venom

    International Nuclear Information System (INIS)

    Spencer, Patrick J.; Nascimento, Nanci do; Rogero, Jose R.

    1997-01-01

    Ionizing radiation has been successfully employed to attenuate animals toxins and venoms for immunizing antisera producing animals. However, the radiation effects on antigenicity and immunogenecity have not yet been elucidated. In the present work, we investigated the effects of gamma rays on the antigenic and immunogenicity have not yet been elucidated. In the present work, we investigated the effects of gamma rays on the antigenic and immunogenic behaviour of Bothrops jararacussu venon. Venom samples (2mg/ml in 150 mM NaCl) were irradiated with 500, 1000 and 2000 Gy of 60 Co gamma rays. These samples were submitted to antigen capture ELISA on plates coated with commercial bothropic antiserum. Results suggest a loss of reactivity of the 1000 and 2000 Gy irradiated samples. Antibodies against native and 2000 Gy irradiated venoms were produced in rabbits. Both sera able to bind native venom with a slightly higher titer for anti-irradiated serum. These data suggest that radiation promoted structural modification on the antigen molecules. However since the antibodies produced against irradiated antivenom were able to recognize native venom, there must have been preservation of some antigenic determinants. It has already been demosntrated that irradiation of proteins leads to structural modifications and unfolding of the molecules. Our data suggest that irradiation led to conformational epitopes destruction with preservation of linear epitopes and that the response against irradiated venom may be attributed to these linear antigenic determinants. (author). 8 refs., 3 figs

  20. Shedding of CD9 antigen into cerebrospinal fluid by acute lymphoblastic leukemia cells.

    Science.gov (United States)

    Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

    1990-07-01

    The accurate identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of acute lymphoblastic leukemia (ALL). We demonstrated that soluble CD9 antigen was shed into CSF obtained from children with ALL, using enzyme-linked immunosorbent assay (ELISA), which used the activity of CD9 antigen to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4, simultaneously. Using RCA1/SJ-9A4 ELISA, CD9 antigen was detectable in CSF but not in plasma from 12 cases of CD9+ ALL in central nervous system (CNS) relapse. However, CD9 antigen was not released into CSF from 11 cases of CD9- ALL with CNS involvement, 136 cases of CD9+ ALL in complete remission (CR), 29 cases of CD9- ALL in CR, or 21 cases of aseptic meningitis. Interestingly, the levels of CD9 antigen were elevated in CSF from 7 of 10 CD9+ ALL patients without cytologically proven CNS involvement at diagnosis, with subsequent return to undetectable levels after initial induction chemotherapy was begun. In addition, sequential analysis of CSF from a 5-year-old boy with CD9+ ALL in CNS relapse showed that levels of CD9 antigen correlated well with the number of leukemic cells in CSF. Serial quantitative analysis of CD9 antigen in CSF could be useful to detect the proliferation of residual leukemic cells before the clinical manifestation.

  1. Distance between two binding sites of the same antibody molecule

    International Nuclear Information System (INIS)

    Cser, L.; Gladkikh, I.A.; Ostanevich, Y.M.; Franek, F.; Novotny, J.; Nezlin, R.S.

    1978-01-01

    Neutron small-angle scattering experiments are reported, aimed at determining the distance between the two binding sites of the same antibody molecule employing complexes of anti-Dnp antibody with an antigenically univalent, high molecular weight ligand. Although the distance values could be determined only with a large statistical error, the data allowed the conclusion that the geometrical parameters of the complexes formed with the early (i.e., precipitating) antibody are significantly different from those of the complexes formed with the late (i.e, non-precipitating) antibody. The data suggest that the precipitating antibody complexed with a high molecular weight antigen assumes an extended shape with an antigen to antigen distance of 35.8 +- 1.3 nm. (Auth.)

  2. Radioprotective activity of shigella antigens

    International Nuclear Information System (INIS)

    Klemparskaya, N.N.; Gorbunova, E.S.; Dobronravova, N.N.

    1981-01-01

    The possibility of using experimental microbe antigenous preparation out of Flexner and Zonne shigellas as a protector and a remedy in the case of gamma irradiation, is investigated. The experiments are carried out on mice of both sexes immunized before or after irradiation by two methods: subcutaneously and enerally. It is found that in most cases investigated, the introduction of the experimental preparation 3, 5, 7 and 10 days before irradiation increases the survivability of animals [ru

  3. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1987-01-01

    The capacity of purified I-Ad, I-Ed, I-Ak, and I-Ek to bind to protein derived peptides that have been previously reported to be T cell immunogens has been examined. For each of the 12 peptides studied strong binding to the relevant Ia restriction element was observed. All the peptides bound more...... than one Ia molecule; however, for 11 of 12 peptides, the dominant binding was to the restriction element, whereas in one instance the dominant binding was to a nonrestriction element. When the peptides were used to inhibit the presentation of antigen by prefixed accessory cells to T cells......, an excellent correlation was found between the capacity of a peptide to inhibit the binding of an antigen to purified Ia and the capacity of the peptide to inhibit accessory cell presentation of the antigen. Thus, the binding of peptide to purified Ia is immunologically relevant, and Ia seems to be the only...

  4. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each ...

  5. Chlorphenesin: an antigen-associated immunosuppressant.

    Science.gov (United States)

    Whang, H Y; Neter, E

    1970-07-01

    Chlorphenesin (3-p-chlorophenoxy-1,2-propanediol), when injected intravenously together with either of two common bacterial antigens, inhibits the antibody response of the rabbit. The antigens studied are those common to Enterobacteriaceae and to gram-positive bacteria. The immunosuppression is contingent upon incubation of chlorphenesin and antigen in vitro prior to administration, since separate injection of antigen and inhibitor or of mixtures without prior incubation yields undiminished antibody response. Chlorphenesin, as shown by hemagglutination-inhibition tests, does not alter the antigenic determinants, because antibody neutralization occurs in the presence or absence of the drug. The immunosuppressive effect is reversible, since precipitation of chlorphenesin at 4 C substantially restores immunogenicity. Animals immunized with antigen-drug mixtures, which fail to respond with significant antibody production, nonetheless are immunologically primed. It is concluded that chlorphenesin represents another example of antigen-associated immunosuppressants.

  6. Presentation of lipid antigens to T cells.

    Science.gov (United States)

    Mori, Lucia; De Libero, Gennaro

    2008-04-15

    T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

  8. Ultraviolet radiation (UVR) induces cell-surface Ro/SSA antigen expression by human keratinocytes in vitro: a possible mechanism for the UVR induction of cutaneous lupus lesions

    International Nuclear Information System (INIS)

    Jones, S.K.

    1992-01-01

    Antinuclear antibodies are useful markers of connective tissue disease. In this study, UVB but not UVA induced the expression of Ro/SSA antigen on keratinocyte surfaces in vitro. This expression was also found with the extractable nuclear antigens RnP and Sm, but not with single or double-stranded DNA. The expression was prevented by blocking protein synthesis, suggesting that it was an active process. The results suggest that UVB exposure may result in the expression of Ro/SSA antigen on the surfaces of basal keratinocytes in vivo. This antigen could then bind circulating antibody leading to the cutaneous lesions in neonatal and subacute cutaneous lupus erythematosus. (Author)

  9. Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia

    Science.gov (United States)

    Ghiotto, Fabio; Fais, Franco; Valetto, Angelo; Albesiano, Emilia; Hashimoto, Shiori; Dono, Mariella; Ikematsu, Hideyuki; Allen, Steven L.; Kolitz, Jonathan; Rai, Kanti R.; Nardini, Marco; Tramontano, Anna; Ferrarini, Manlio; Chiorazzi, Nicholas

    2004-01-01

    Studies of B cell antigen receptors (BCRs) expressed by leukemic lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that B lymphocytes with some level of BCR structural restriction become transformed. While analyzing rearranged VHDJH and VLJL genes of 25 non–IgM-producing B-CLL cases, we found five IgG+ cases that display strikingly similar BCRs (use of the same H- and L-chain V gene segments with unique, shared heavy chain third complementarity-determining region [HCDR3] and light chain third complementarity-determining region [LCDR3] motifs). These H- and L-chain characteristics were not identified in other B-CLL cases or in normal B lymphocytes whose sequences are available in the public databases. Three-dimensional modeling studies suggest that these BCRs could bind the same antigenic epitope. The structural features of the B-CLL BCRs resemble those of mAb’s reactive with carbohydrate determinants of bacterial capsules or viral coats and with certain autoantigens. These findings suggest that the B lymphocytes that gave rise to these IgG+ B-CLL cells were selected for this unique BCR structure. This selection could have occurred because the precursors of the B-CLL cells were chosen for their antigen-binding capabilities by antigen(s) of restricted nature and structure, or because the precursors derived from a B cell subpopulation with limited BCR heterogeneity, or both. PMID:15057307

  10. Immunogenetic mechanisms driving norovirus GII.4 antigenic variation.

    Directory of Open Access Journals (Sweden)

    Lisa C Lindesmith

    Full Text Available Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs. Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987-1997, contemporary (2004-2009, and broad (1987-2009. NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294-298 and 368-372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393-395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing

  11. A nonself sugar mimic of the HIV glycan shield shows enhanced antigenicity

    Energy Technology Data Exchange (ETDEWEB)

    Doores, Katie J.; Fulton, Zara; Hong, Vu; Patel, Mitul K.; Scanlan, Christopher N.; Wormald, Mark R.; Finn, M.G.; Burton, Dennis R.; Wilson, Ian A.; Davis, Benjamin G. (Scripps); (Oxford)

    2011-08-24

    Antibody 2G12 uniquely neutralizes a broad range of HIV-1 isolates by binding the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. Antigens that resemble these natural epitopes of 2G12 would be highly desirable components for an HIV-1 vaccine. However, host-produced (self)-carbohydrate motifs have been unsuccessful so far at eliciting 2G12-like antibodies that cross-react with gp120. Based on the surprising observation that 2G12 binds nonproteinaceous monosaccharide D-fructose with higher affinity than D-mannose, we show here that a designed set of nonself, synthetic monosaccharides are potent antigens. When introduced to the terminus of the D1 arm of protein glycans recognized by 2G12, their antigenicity is significantly enhanced. Logical variation of these unnatural sugars pinpointed key modifications, and the molecular basis of this increased antigenicity was elucidated using high-resolution crystallographic analyses. Virus-like particle protein conjugates containing such nonself glycans are bound more tightly by 2G12. As immunogens they elicit higher titers of antibodies than those immunogenic conjugates containing the self D1 glycan motif. These antibodies generated from nonself immunogens also cross-react with this self motif, which is found in the glycan shield, when it is presented in a range of different conjugates and glycans. However, these antibodies did not bind this glycan motif when present on gp120.

  12. Experimental radioimmunotherapy with I-131-antibody against a differentiation antigen

    International Nuclear Information System (INIS)

    Badger, C.C.; Krohn, K.A.; Bernstein, I.D.

    1985-01-01

    The authors have previously shown that I-131-labeled antibodies (Ab) against the Thyl.l antigen can care AKR/Cu (Thyl.2+) mice bearing the AKR/J (Thy 1l.1+) SL2 T-cell lymphoma. The authors have now extended these studies to therapy with I-131-anti-Thyl.1 of SL2 lymphoma in AKR/J mice where Ab reacts with both tumor and normal cells. A 25 μg bolus was rapidly cleared from serum by binding to spleen cells (75% with Tl/2 <60 min.) and only low concentrations of Ab(<2% ID/gm) were present in tumor after infusion. Therapy of AKR/J mice bearing established s.c. lymphoma nodules with 1500 μCi I-131-anti-Thyl.1 resulted in complete regression of the nodule in 6/6 animals although tumor eventually regrew and all animals died of metastatic lymphoma. In contrast, I-131-irrelevant Ab given to produce the same amount of whole body radiation (750 μCi) did not affect tumor growth. These studies suggest that radiolabeled-AB against differentiation antigens may be useful for therapy in spite of binding to normal cell populations

  13. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Shayla K Shorter

    Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  14. Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

    Directory of Open Access Journals (Sweden)

    Hinrich eAbken

    2013-11-01

    Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

  15. Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies

    DEFF Research Database (Denmark)

    Samuelsen, Simone V; Solov'yov, Ilia A.; Balboni, Imelda M.

    2016-01-01

    New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing...... molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best...... that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies....

  16. Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

    International Nuclear Information System (INIS)

    Chao, S.; Chao, L.; Chao, J.

    1989-01-01

    A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

  17. [Blood groups - minuses and pluses. Do the blood group antigens protect us from infectious diseases?].

    Science.gov (United States)

    Czerwiński, Marcin

    2015-06-25

    Human blood can be divided into groups, which is a method of blood classification based on the presence or absence of inherited erythrocyte surface antigens that can elicit immune response. According to the International Society of Blood Transfusion, there are 341 blood group antigens collected in 35 blood group systems. These antigens can be proteins, glycoproteins or glycosphingolipids, and function as transmembrane transporters, ion channels, adhesion molecules or receptors for other proteins. The majority of blood group antigens is present also on another types of cells. Due to their localization on the surface of cells, blood group antigens can act as receptors for various pathogens or their toxins, such as protozoa (malaria parasites), bacteria (Helicobacter pylori, Vibrio cholerae and Shigella dysenteriae) and viruses (Noroviruses, Parvoviruses, HIV). If the presence of group antigen (or its variant which arised due to mutation) is beneficial for the host (e.g. because pathogens are not able to bind to the cells), the blood group may become a selection trait, leading to its dissemination in the population exposed to that pathogen. There are thirteen blood group systems that can be related to pathogen resistance, and it seems that the particular influence was elicit by malaria parasites. It is generally thought that the high incidence of blood groups such as O in the Amazon region, Fy(a-b-) in Africa and Ge(-) in Papua-New Guinea is the result of selective pressure from malaria parasite. This review summarizes the data about relationship between blood groups and resistance to pathogens.

  18. Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

    International Nuclear Information System (INIS)

    Kennel, S.J.; Foote, L.J.; Lankford, P.K.; Johnson, M.; Mitchell, T.; Braslawsky, G.R.

    1983-01-01

    For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. The authors have studied the binding of 125 I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind =Ka[Ag total]/1 + Ka[Ag total]. Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10 8 M -1 are not likely to be useful for drug targeting or tumor imaging

  19. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    International Nuclear Information System (INIS)

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-01-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

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

    International Nuclear Information System (INIS)

    Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

    1986-01-01

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

  1. HA03 as an Iranian Candidate Concealed Antigen for Vaccination against Hyalomma anatolicum anatolicum: Comparative Structural and In silico Studies

    Directory of Open Access Journals (Sweden)

    Mohammadi, A.

    2013-12-01

    Full Text Available In the last decades researchers had focused on developing a vaccine against tick based on protective antigen. Recombinant vaccines based on concealed antigen from Boophilus microplus have been developed in Australia and Cuba by the name of TICKGARD and GAVAC (De La Fuente and Kocan, 2006. Further studies on this antigen have shown some extent of protection against other species (De Vos et al., 2001. In Iran most important species is Hyalomma anatolicum and limited information about its control are available. This paper reports structural and polymorphic analysis of HA03 as an Iranian candidate concealed antigen of H. a. anatolicum deposited in Gen-Bank .(Aghaeipour et al. GQ228820. The comparison between this antigen and other mid gut concealed antigen that their characteristics are available in GenBank showed there are high rate of similarity between them. The HA03 amino acid sequence had a homology of around 89%, 64%, 56% with HA98, BM86, BM95 respectively. Potential of MHC class I and II binding region indicated a considerable variation between BM86 antigen and its efficiency against Iranian H. a. anatolicum. In addition, predicted major of hydrophobisity and similarity in N-glycosylation besides large amount of cystein and seven EGF like regions presented in protein structure revealed that value of HA03 as a new protective antigen and the necessity of the development, BM86 homolog of H. a. anatolicum HA03 based recombinant vaccine.

  2. Characterization of Leishmania Soluble Exo-Antigen

    National Research Council Canada - National Science Library

    Cui, Liwang

    2003-01-01

    .... Vaccine development is the ultimate solution for this problem. Our previous research indicates that Leishmania parasites secrete, excrete, or shed antigens into the medium during in vitro culture...

  3. Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

    International Nuclear Information System (INIS)

    Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

    1987-01-01

    We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex

  4. Assessment of cancer and virus antigens for cross-reactivity in human tissues.

    Science.gov (United States)

    Jaravine, Victor; Raffegerst, Silke; Schendel, Dolores J; Frishman, Dmitrij

    2017-01-01

    Cross-reactivity (CR) or invocation of autoimmune side effects in various tissues has important safety implications in adoptive immunotherapy directed against selected antigens. The ability to predict CR (on-target and off-target toxicities) may help in the early selection of safer therapeutically relevant target antigens. We developed a methodology for the calculation of quantitative CR for any defined peptide epitope. Using this approach, we performed assessment of 4 groups of 283 currently known human MHC-class-I epitopes including differentiation antigens, overexpressed proteins, cancer-testis antigens and mutations displayed by tumor cells. In addition, 89 epitopes originating from viral sources were investigated. The natural occurrence of these epitopes in human tissues was assessed based on proteomics abundance data, while the probability of their presentation by MHC-class-I molecules was modelled by the method of Keşmir et al. which combines proteasomal cleavage, TAP affinity and MHC-binding predictions. The results of these analyses for many previously defined peptides are presented as CR indices and tissue profiles. The methodology thus allows for quantitative comparisons of epitopes and is suggested to be suited for the assessment of epitopes of candidate antigens in an early stage of development of adoptive immunotherapy. Our method is implemented as a Java program, with curated datasets stored in a MySQL database. It predicts all naturally possible self-antigens for a given sequence of a therapeutic antigen (or epitope) and after filtering for predicted immunogenicity outputs results as an index and profile of CR to the self-antigens in 22 human tissues. The program is implemented as part of the iCrossR webserver, which is publicly available at http://webclu.bio.wzw.tum.de/icrossr/ CONTACT: d.frishman@wzw.tum.deSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press

  5. Antigen delivery by α2-macroglobulin enhances the cytotoxic T lymphocyte response

    Science.gov (United States)

    Bowers, Edith V.; Horvath, Jeffrey J.; Bond, Jennifer E.; Cianciolo, George J.; Pizzo, Salvatore V.

    2009-01-01

    α2M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, α2M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that α2M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that α2M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA257–264 (SIINFEKL) demonstrated up to 25-fold greater IFN-γ and IL-2 secretion when treated in vitro with α2M*-OVA compared with soluble OVA. The frequency of IFN-γ-producing cells was increased ∼15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by α2M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α2M*-OVA. These α2M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that α2M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support α2M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing. PMID:19652028

  6. Influence of maternal gestational treatment with mycobacterial antigens on postnatal immunity in an experimental murine model.

    Directory of Open Access Journals (Sweden)

    Muhammad Jubayer Rahman

    Full Text Available BACKGROUND: It has been proposed that the immune system could be primed as early as during the fetal life and this might have an impact on postnatal vaccination. Therefore, we addressed in murine models whether gestational treatment with mycobacterial antigens could induce better immune responses in the postnatal life. METHODS/FINDINGS: BALB/c mice were treated subcutaneously (s.c. at the second week of gestation with antigen (Ag85A or heparin-binding hemagglutinin (HBHA in the absence of adjuvant. Following birth, offspring mice were immunized intranasally (i.n. with the same antigens formulated with the adjuvant cholera toxin (CT at week 1 and week 4. One week after the last immunization, we assessed antigen-specific recall interferon gamma (IFN-gamma responses by in vitro restimulation of lung-derived lymphocytes. Protection against infection was assessed by challenge with high dose Mycobacterium bovis Bacille Calmette-Guérin (BCG given i.n. We found that recall IFN-gamma responses were higher in the offspring born to the treated mother compared to the untreated-mother. More importantly, we observed that the offspring born to the treated mother controlled infection better than the offspring born to the untreated mother. Since the gestational treatment was done in absence of adjuvant, essentially there was no antibody production observed in the pregnant mice and therefore no influence of maternal antibodies was expected. We hypothesized that the effect of maternal treatment with antigen on the offspring occurred due to antigen transportation through placenta. To trace the antigens, we conjugated fluorescent nanocrystals with Ag85A (Qdot-ITK-Ag85A. After inoculation in the pregnant mice, Qdot-ITK-Ag85A conjugates were detected in the liver, spleen of pregnant females and in all the fetuses and placentas examined. CONCLUSION: The fetal immune system could be primed in utero by mycobacterial antigens transported through the placenta.

  7. Studies on the surface antigenicity and susceptibility to antibody-dependent killing of developing schistosomula using sera from chronically infected mice and mice vaccinated with irradiated cercariae

    International Nuclear Information System (INIS)

    Bickle, Q.D.; Ford, M.J.

    1982-01-01

    Changes in the surface antigenicity and susceptibility to in vitro killing during development of schistosomula of Schistosoma mansoni were studied using serum from chronically infected mice (CIS) and from mice vaccinated with highly irradiated (20 krad) cercariae (VS). Binding of these sera was quantitated by counting the number of P388D 1 cells (a transformed, macrophage-like cell of mouse origin, bearing Fc receptors for IgG) binding to the parasite surface. Compared with schistosomula derived in vitro by mechanical transformation (MS), schistosomula recovered 3 hr after skin penetration in vitro (SS) showed a significant loss in surface binding of CIS. Schistosomula recovered 3 hr after skin penetration in vivo (SRS) showed even less binding, and this trend continued such that parasites recovered from the lungs 5 days after infection (LS) showed only minimal binding, and 10-day-old worms from the portal system showed no significant binding. In contrast, VS, which bound significantly less well to MS than CIS, showed enhanced binding to SS, and in the face of their declining antigenicity with respect to CIS, 3- to 24-hr SRS maintained this raised level of antigenicity. Although there appeared to be a decline in binding of VS thereafter, LS remained antigenic, still binding as many cells as MS did despite the fact that they also expressed host antigens detected usng antisera raised against mouse RBC. In spite of this persistence of VS binding up to the lung stage, resistance to eosinophil-mediated killing in vitro had developed by 48 hr post-infection, and LS were totally resistant to both eosinophil- and C-mediated killing

  8. Total iron binding capacity

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003489.htm Total iron binding capacity To use the sharing features on this page, please enable JavaScript. Total iron binding capacity (TIBC) is a blood test to ...

  9. Experiments on the radioimmunological determination of the triiodothyronine binding capacity of human blood serum

    International Nuclear Information System (INIS)

    Traub, H.W.J.

    1980-01-01

    Bovine serum antibodies have been obtained from rabbits. Constant amounts of antibodies have been incubated with increasing amounts of antigens. The maximum value found for T 3 binding to the antigen-antibody complex was 66 μg RA/mg antigen. Human serum samples containing 0,05 ng 125 J-T 3 /μl were incubated with 600 μg RA, and the antigen was precipitated with 8,4 mg anti-RA. The mean value obtained for 30 enthyreotic test persons was a binding to RA of 7.9 +- 0,52% 125 J-T 3 . Comparative measurements of patient and standard serum samples shared only slight differences. This means that the measured data do not reflect the thyroid function. (orig./MG) [de

  10. Highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Avraham, H.; Golenser, J.; Gazitt, Y.; Spira, D.T.; Sulitzeanu, D. (Hebrew Univ., Jerusalem (Israel). Hadassah Medical School)

    1982-08-27

    A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10/sup 6/ RBC.

  11. A simplified radioimmunoassay for triiodothyronine (T3) using pre-incubated labelled antigen and antibody

    International Nuclear Information System (INIS)

    Pillai, M.R.A.; Nagvekar, U.H.; Desai, C.N.; Mani, R.S.

    1986-01-01

    The development of a simplified radioimmunoassay for triiodothyronine (T 3 ) using pre-incubated labelled T 3 and antibody is described. The assay is carried out by adding 50 μl of standard or sample to 0.4 ml of pre-incubated reagent dispensed in assay tubes. The reaction was allowed to proceed for about four hours and the antigen-antibody complex precipitated by the addition of 1 cm 3 of 22% polyethylene glycol solution. Due to the high dissociation constant of T 3 -antibody complex at 37 deg C, the labelled antigen-antibody complex dissociates and thereby the unlabelled antigen binds with the antibody. The sensitivity of this assay is comparable to an assay done by the equilibrium method using the same antibody. Sixty serum samples were analyzed using this method and were compared with the equilibrium assay. (author)

  12. Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Monica Casucci, Attilio Bondanza

    2011-01-01

    Full Text Available Chimeric antigen receptors (CARs are generated by fusing the antigen-binding motif of a monoclonal antibody (mAb with the signal transduction machinery of the T-cell receptor (TCR. The genetic modification of T lymphocytes with chimeric receptors specific for tumor-associated antigens (TAAs allows for the redirection towards tumor cells. Clinical experience with CAR-redirected T cells suggests that antitumor efficacy associates with some degree of toxicity, especially when TAA expression is shared with healthy tissues. This situation closely resembles the case of allogeneic hematopoietic stem cell transplantation (HSCT, wherein allorecognition causes both the graft-versus-leukemia (GVL effect and graft-versus-host disease (GVHD. Suicide gene therapy, i.e. the genetic induction of a conditional suicide phenotype into donor T cells, enables dissociating the GVL effect from GVHD. Applying suicide gene modification to CAR-redirected T cells may therefore greatly increase their safety profile and facilitate their clinical development.

  13. Isolation and characterization of two antigenic glycoproteins from the pollen of Prosopis juliflora.

    Science.gov (United States)

    Thakur, I S

    1991-03-01

    Two antigenically active glycoprotein fractions were isolated from crude extract of the pollen of Prosopis juliflora using DEAE-cellulose ion exchange chromatography. The glycoproteins gave single band on polyacrylamide gel electrophoresis. The molecular weight of these two glycoprotein was 20,000 and 10,000 as determined by gel filtration on Sephadex G-75. With the help of crossed immunoelectrophoresis and gel diffusion crude extract exhibited twelve and three precipitating antigens suggesting its heterogeneous nature; and the purified glycoprotein fractions however formed single precipitin band on gel diffusion test and immunoelectrophoresis. As tested by ELISA the polyclonal antisera raised in rabbit showed strong binding affinity with glycoprotein of MW 20,000. These result indicates that the two glycoprotein fractions are not antigenically identical.

  14. A highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies

    International Nuclear Information System (INIS)

    Avraham, H.; Golenser, J.; Gazitt, Y.; Spira, D.T.; Sulitzeanu, D.

    1982-01-01

    A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10 6 RBC. (Auth.)

  15. Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

    Science.gov (United States)

    Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

    2015-09-01

    The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  16. IDENTIFICATION OF ACROSOME AS THE MAIN ANTIGEN OF THE SPERM CELLS PROVOKING AUTOANTIBODIES IN VASECTOMIZED IRANIAN MEN

    Directory of Open Access Journals (Sweden)

    M R Nowroozi

    2008-12-01

    Full Text Available "nVasectomy is one of the extensively used methods of contraception in family planning programs. Antisperm antibodies (ASA develop after vasectomy which can result in auto-immune male infertility. The precise sperm antigens involved in the autoimmune response are still poorly defined, therefore we determined the circulating ASA and identified relevant sperm antigens based on localization of binding sites of ASA to sperm cell antigens, using a rapid, inexpensive and clinically relevant assay in vasectomized men. Results showed that 2.5% of men had ASA at the time of vasectomy, whereas 53.5% of the study population subsequently developed ASA. The numbers of men with circulating ASA increased significantly for the first three months after vasectomy. These antibodies were distinguishable into three groups based on their bindings to different sites of sperm cell antigens including against acrosome and tail in 67.56% and 10.8%, respectively; 21.6% of subjects had antibody to the other parts of the sperm cell antigens. The results of this study are discussed in terms of an autoimmune response against sperm antigens and development of ASA.

  17. Virosomes for antigen and DNA delivery

    NARCIS (Netherlands)

    Daemen, T; de Mare, A; Bungener, L; de Jonge, J; Huckriede, A; Wilschut, J

    2005-01-01

    Specific targeting and delivery as well as the display of antigens on the surface of professional antigen-presenting cells (APCs) are key issues in the design and development of new-generation vaccines aimed at the induction of both humoral and cell-mediated immunity. Prophylactic vaccination

  18. Radioimmunoassay for a human prostate specific antigen

    International Nuclear Information System (INIS)

    Machida, T.; Miki, M.; Ohishi, Y.; Kido, A.; Morikawa, J.; Ogawa, Y.

    1983-01-01

    As a marker for prostatic cancer, a prostate-specific antigen was purified from human prostatic tissues. Double antibody radioimmunoassay utilizing immune reaction was developed on the basis of the purified prostatic antigen (PA). Measurement results have revealed that PA radioimmunoassay is much better than prostatic acid phosphatase (PAP) radioimmunoassay in the diagnosis of prostatic cancer

  19. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

  20. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    Directory of Open Access Journals (Sweden)

    Sean A Gray

    Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  1. Studies on antigenic cross-reactivity of Trichuris ovis with host mucosal antigens in goat

    Directory of Open Access Journals (Sweden)

    Gautam Patra

    2015-12-01

    Full Text Available Objective: To ascertain whether immunodominant antigens of Trichuris ovis might share and cross react with host molecule. Methods: Two crude protein preparations from anterior and posterior parts of Trichuris ovis were characterized along with host mucosal antigen by double immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting technique. Conventional scanning electron microscopy was performed as per standard procedure. Results: Sharp and distinct bands of three antigens have been found in double immunodiffusion using hyperimmune serum raised in rabbit indicating the presence of specific antibody against each antigen. All three antigens have shown major and minor bands with molecular weight ranging from 15 to 110 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Conclusions: The antigenic cross-reactivity was thought to result from shared antigens. The existence of paracloacal papillae found in the anterior part of the male was not a unique feature for species differentiation.

  2. Adoptive transfer of natural antibodies to non-immunized chickens affects subsequent antigen-specific humoral and cellular immune responses

    NARCIS (Netherlands)

    Lammers, A.; Klomp, M.E.V.; Nieuwland, M.G.B.; Savelkoul, H.F.J.; Parmentier, H.K.

    2004-01-01

    To determine a regulatory function of natural antibodies in the immune response of chickens, pooled plasma obtained from non-immunized (naive) 15 months old hens was subjected to keyhole limpet hemocyanin (KLH) antigen-affinity chromatography. Purified KLH-binding antibodies were adoptively

  3. Target-specific activation of mast cells by immunoglobulin E reactive with a renal cell carcinoma-associated antigen

    NARCIS (Netherlands)

    Luiten, R. M.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1996-01-01

    Immunoglobulin E (IgE) that specifically binds to antigens present on carcinoma cells may represent a useful tool to combat carcinomas. Induction of an inflammatory response at the tumor site by tumor-specific IgE may result in reduced tumor growth and tumor regression. Local mast cells may be

  4. The value of serum Hepatitis B surface antigen quantification in ...

    African Journals Online (AJOL)

    The value of serum Hepatitis B surface antigen quantification in determining viralactivity in chronic Hepatitis B virus infection. ... ofCHB andalso higher in hepatitis e antigen positive patients compared to hepatitis e antigen negative patients.

  5. Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stanfield, R.L.; Dooley, H.; Verdino, P.; Flajnik, M.F.; Wilson, I.A.; /Scripps Res. Inst. /Maryland U.

    2007-07-13

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  6. Characterization of the Apa antigen from M. avium subsp. paratuberculosis: a conserved Mycobacterium antigen that elicits a strong humoral response in cattle.

    Science.gov (United States)

    Gioffré, A; Echeverría-Valencia, G; Arese, A; Morsella, C; Garbaccio, S; Delgado, F; Zumárraga, M; Paolicchi, F; Cataldi, A; Romano, M I

    2009-12-15

    Johne's disease or paratuberculosis is widespread in almost all countries and remains difficult to eradicate. Nowadays, diagnosis of Mycobacterium avium subsp. paratuberculosis (MPTB) infection is one of the main concerns. In this work, we evaluated the expression, biochemical properties and antigenicity of the Apa antigen, encoded by the gene annotated as MAP1569, in the MPTB genome. We confirmed its expression in MPTB and its glycosylation by the ConA binding assay. Although the MPTB-Apa is not an immunodominant antigen, MPTB-infected cattle showed a strong humoral response to recombinant Apa by Western blot and ELISA. Milk was also a suitable sample to be tested by ELISA. We comparatively analysed the humoral cross-reactivity to the Apa from MPTB (MPTB-Apa) and the orthologue from Mycobacterium tuberculosis (MT-Apa, identical to that from Mycobacterium bovis) in both infected and control cows. Response of M. bovis- and MPTB-infected animals against MT-Apa was similar (P=0.6985) but the response of the M. bovis-infected ones to MPTB-Apa was differential, being significantly diminished (PApa stimulation in the IFNgamma release assay, we found no significant differences when compared infected herds with non-infected ones (P=0.34). This antigen, in contrast to bovine Purified Protein Derivative (PPDb), was strongly represented in avian PPD (PPDa), as shown by the recognition of BALB/c mice hyperimmune sera against MPTB-Apa by Dot-blot immunoassay. We therefore demonstrated the antigenicity of Apa in MPTB-infected animals and a differential response to the recombinant antigen when compared to M. bovis-infected animals. These traits herein described, added to the usefulness of milk samples to detect IgG anti-Apa, could be important for routine screening in dairy cattle, considering a multiantigenic approach to overcome the lack of immunodominance.

  7. Radiation and chemical effects on viral transformation and tumor antigen expression. Annual progress report, August 1, 1978--May 1, 1979

    International Nuclear Information System (INIS)

    Coggin, J.H. Jr.

    1979-01-01

    Studies aimed at the biological, biochemical, and immunologic characterization of fetal antigens (EA) in hamsters and mice and locating and determining the distribution of fetal antigens in tumor tissues and in developing fetuses have been underway for several months. Progress has been made in isolating embryonic or fetal antigens from fetuses and from tumor cells. We have developed and reported a reliable lymphocyte transformation assay (LTA) which meets our needs in routinely assaying cell free tumor associated antigen (TAA) preparations from fetal and tumor cells. The assay correlated with transplantation resistance assays and has appropriate specificity. We have also developed the staph-A protein binding assay utilizing anti-serum derived against embryonic antigens present on SV40 tumor cells. In other studies, we have reported increases and perturbations in thymocytes during viral and chemical oncogenesis in hamsters, have developed a simple technique for preserving functional lymphocytes sensitized against TAA by freezing for use in our model system work, have reported the cross-reactivity of tranplantation resistance antigen on a spectrum of chemically induced tumors previously believed to only contain individually specific TSTAs and have recently reported the cross-reactivity of papovavirus induced transplantation resistance antigen in sarcoma cells induced by different viruses. We have concluded our studies of glycosyltransferases in the membranes of developing fetuses and noted no differences in their levels with advancing days of gestation using whold embryo cell populations

  8. 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.

  9. Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

    Science.gov (United States)

    Engelhard, V H; Powers, G A; Moore, L C; Holterman, M J; Correa-Freire, M C

    1984-01-01

    HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

  10. Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

    Directory of Open Access Journals (Sweden)

    Stefanie Hausammann

    Full Text Available Inhibitory antibodies directed against coagulation factor VIII (FVIII can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

  11. Calcium-binding proteins from human platelets

    International Nuclear Information System (INIS)

    Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

    1983-01-01

    Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

  12. The interfacial character of antibody paratopes: analysis of antibody-antigen structures.

    Science.gov (United States)

    Nguyen, Minh N; Pradhan, Mohan R; Verma, Chandra; Zhong, Pingyu

    2017-10-01

    In this study, computational methods are applied to investigate the general properties of antigen engaging residues of a paratope from a non-redundant dataset of 403 antibody-antigen complexes to dissect the contribution of hydrogen bonds, hydrophobic, van der Waals contacts and ionic interactions, as well as role of water molecules in the antigen-antibody interface. Consistent with previous reports using smaller datasets, we found that Tyr, Trp, Ser, Asn, Asp, Thr, Arg, Gly, His contribute substantially to the interactions between antibody and antigen. Furthermore, antibody-antigen interactions can be mediated by interfacial waters. However, there is no reported comprehensive analysis for a large number of structured waters that engage in higher ordered structures at the antibody-antigen interface. From our dataset, we have found the presence of interfacial waters in 242 complexes. We present evidence that suggests a compelling role of these interfacial waters in interactions of antibodies with a range of antigens differing in shape complementarity. Finally, we carry out 296 835 pairwise 3D structure comparisons of 771 structures of contact residues of antibodies with their interfacial water molecules from our dataset using CLICK method. A heuristic clustering algorithm is used to obtain unique structural similarities, and found to separate into 368 different clusters. These clusters are used to identify structural motifs of contact residues of antibodies for epitope binding. This clustering database of contact residues is freely accessible at http://mspc.bii.a-star.edu.sg/minhn/pclick.html. minhn@bii.a-star.edu.sg, chandra@bii.a-star.edu.sg or zhong_pingyu@immunol.a-star.edu.sg. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  13. Posttransplant chimeric antigen receptor therapy.

    Science.gov (United States)

    Smith, Melody; Zakrzewski, Johannes; James, Scott; Sadelain, Michel

    2018-03-08

    Therapeutic T-cell engineering is emerging as a powerful approach to treat refractory hematological malignancies. Its most successful embodiment to date is based on the use of second-generation chimeric antigen receptors (CARs) targeting CD19, a cell surface molecule found in most B-cell leukemias and lymphomas. Remarkable complete remissions have been obtained with autologous T cells expressing CD19 CARs in patients with relapsed, chemo-refractory B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Allogeneic CAR T cells may also be harnessed to treat relapse after allogeneic hematopoietic stem cell transplantation. However, the use of donor T cells poses unique challenges owing to potential alloreactivity. We review different approaches to mitigate the risk of causing or aggravating graft-versus-host disease (GVHD), including CAR therapies based on donor leukocyte infusion, virus-specific T cells, T-cell receptor-deficient T cells, lymphoid progenitor cells, and regulatory T cells. Advances in CAR design, T-cell selection and gene editing are poised to enable the safe use of allogeneic CAR T cells without incurring GVHD. © 2018 by The American Society of Hematology.

  14. Predictive value of prostate-specific antigen for prostate cancer

    DEFF Research Database (Denmark)

    Shepherd, Leah; Borges, Alvaro Humberto; Ravn, Lene

    2014-01-01

    INTRODUCTION: Although prostate cancer (PCa) incidence is lower in HIV+ men than in HIV- men, the usefulness of prostate-specific antigen (PSA) screening in this population is not well defined and may have higher false negative rates than in HIV- men. We aimed to describe the kinetics and predict......INTRODUCTION: Although prostate cancer (PCa) incidence is lower in HIV+ men than in HIV- men, the usefulness of prostate-specific antigen (PSA) screening in this population is not well defined and may have higher false negative rates than in HIV- men. We aimed to describe the kinetics...... and predictive value of PSA in HIV+ men. METHODS: Men with PCa (n=21) and up to two matched controls (n=40) with prospectively stored plasma samples before PCa (or matched date in controls) were selected. Cases and controls were matched on date of first and last sample, age, region of residence and CD4 count...... at first sample date. Total PSA (tPSA), free PSA (fPSA), testosterone and sex hormone binding globulin (SHBG) were measured. Conditional logistic regression models investigated associations between markers and PCa. Sensitivity and specificity of using tPSA >4 µg/L to predict PCa was calculated. Mixed...

  15. A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

    Science.gov (United States)

    Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

    2012-10-12

    The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

  16. MRl of prostate cancer antigen expression for diagnosis and immunotherapy.

    Directory of Open Access Journals (Sweden)

    Jing Ren

    Full Text Available BACKGROUND: Tumor antigen (TA-targeted monoclonal antibody (mAb immunotherapy can be effective for the treatment of a broad range of cancer etiologies; however, these approaches have demonstrated variable clinical efficacy for the treatment of patients with prostate cancer (PCa. An obstacle currently impeding translational progress has been the inability to quantify the mAb dose that reaches the tumor site and binds to the targeted TAs. The coupling of mAb to nanoparticle-based magnetic resonance imaging (MRI probes should permit in vivo measurement of patient-specific biodistributions; these measurements could facilitate future development of novel dosimetry paradigms wherein mAb dose is titrated to optimize outcomes for individual patients. METHODS: The prostate stem cell antigen (PSCA is broadly expressed on the surface of prostate cancer (PCa cells. Anti-human PSCA monoclonal antibodies (mAb 7F5 were bound to Au/Fe(3O(4 (GoldMag nanoparticles (mAb 7F5@GoldMag to serve as PSCA-specific theragnostic MRI probe permitting visualization of mAb biodistribution in vivo. First, the antibody immobilization efficiency of the GoldMag particles and the efficacy for PSCA-specific binding was assessed. Next, PC-3 (prostate cancer with PSCA over-expression and SMMC-7721 (hepatoma cells without PSCA expression tumor-bearing mice were injected with mAb 7F5@GoldMag for MRI. MRI probe biodistributions were assessed at increasing time intervals post-infusion; therapy response was evaluated with serial tumor volume measurements. RESULTS: Targeted binding of the mAb 7F5@GoldMag probes to PC-3 cells was verified using optical images and MRI; selective binding was not observed for SMMC-7721 tumors. The immunotherapeutic efficacy of the mAb 7F5@GoldMag in PC-3 tumor-bearing mice was verified with significant inhibition of tumor growth compared to untreated control animals. CONCLUSION: Our promising results suggest the feasibility of using mAb 7F5@GoldMag probes as a

  17. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    International Nuclear Information System (INIS)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-01-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with 111 In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity

  18. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    Energy Technology Data Exchange (ETDEWEB)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-05-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with /sup 111/In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity.

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

    Science.gov (United States)

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

    2015-01-01

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

  20. Feature Binding in Zebrafish

    Directory of Open Access Journals (Sweden)

    P Neri

    2012-07-01

    Full Text Available Binding operations are primarily ascribed to cortex or similarly complex avian structures. My experiments show that the zebrafish, a lower vertebrate lacking cortex, supports visual feature binding of form and motion for the purpose of social behavior. These results challenge the notion that feature binding may require highly evolved neural structures and demonstrate that the nervous system of lower vertebrates can afford unexpectedly complex computations.

  1. Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

    DEFF Research Database (Denmark)

    Deufel, T; Grove, A; Kofod, Hans

    1985-01-01

    Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera...... chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N-terminal sequences of each polypeptide....

  2. Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

    International Nuclear Information System (INIS)

    Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

    1987-01-01

    Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of 125 I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of 125 I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen

  3. Antibody binding constants from Farr test and other radioimmunoassays. A theoretical and experimental analysis

    International Nuclear Information System (INIS)

    Engel, J.; Schalch, W.

    1980-01-01

    For the reaction of monovalently reacting antibody (116-700pIEF) with its antigen (streptococcal group A-variant polysaccharide), an apparent binding constant Ksub(a) was derived by the ammonium sulfate precipitation technique (Farr assay) which was 40 times larger than the true binding constant K = 10 6 M -1 determined by fluorescence titration and equilibrium dialysis. For monovalently reacting antibodies the time needed for re-equilibration of the binding reaction is short as compared to the time of ammonium sulfate incubation. A thermodynamic analysis was therefore performed for the case of complete equilibration of all components in solution and in the ammonium sulfate precipitate. It was found that in this limiting case Ksub(a)/K is equal to the ratio of the solubilities of the antibody and the antibody complex corrected by the activity coefficients of the components in the precipitate. For other antibody-antigen reactions in which the antibody reacts with both binding sites to the same antigen molecule, re-equilibration of the binding reaction in solution is much slower. For such systems a disturbance of the binding reaction by the precipitation is less likely and correct binding constants may be obtained by the Farr technique or other radioimmunoassays involving precipitation. (author)

  4. Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum.

    Science.gov (United States)

    Oyebola, Kolapo M; Idowu, Emmanuel T; Olukosi, Yetunde A; Awolola, Taiwo S; Amambua-Ngwa, Alfred

    2017-06-29

    The burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions. We performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima's D) and integrated haplotype score (iHS) to identify genes under selection. Fourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima's D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively. We have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known

  5. Crystal structure of a TAPBPR–MHC I complex reveals the mechanism of peptide editing in antigen presentation

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jiansheng; Natarajan, Kannan; Boyd, Lisa F.; Morozov, Giora I.; Mage, Michael G.; Margulies, David H. (NIH); (Hebrew)

    2017-10-12

    Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

  6. Tumor Associated Antigenic Peptides in Prostate Cancer

    National Research Council Canada - National Science Library

    Tiwari, Raj

    1999-01-01

    .... We proposed to identify these novel antigens in an experimental rat model using purified preparations of the heat shock protein gp96 and a library of synthetic distinct antibodies that were available...

  7. Prostate-Specific Antigen (PSA) Test

    Science.gov (United States)

    ... Cancer Prostate Cancer Screening Research Prostate-Specific Antigen (PSA) Test On This Page What is the PSA ... parts of the body before being detected. The PSA test may give false-positive or false-negative ...

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

    Science.gov (United States)

    Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

    2004-01-01

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

  9. Allosensibilisation to erythrocyte antigens (literature review

    Directory of Open Access Journals (Sweden)

    N. V. Mineeva

    2015-01-01

    Full Text Available In this article literature review of the causes of allosensibilisation to erythrocyte antigens are presented. It is shown that the ability to produce antierythrocyte antibodies is affected by many factors, principal of whom it is difficult to identify. For the allosensibilisation development requires genetically determined differences in erythrocyte antigens phenotypes of donor and recipient, mother and fetus, which can lead to immune response and antibodies production. The biochemical nature of erythrocyte antigens, antigen dose (the amount of transfused doses, the number of antigens determinants on donor and fetus erythrocytes, the number of pregnancies are important. Individual patient characteristics: age, gender, diseases, the use of immunosuppressive therapy and the presence of inflammatory processes, are also relevant. Note that antibody to one erythrocyte antigens have clinical value, and to the other – have no. The actual data about frequency of clinically significant antibodies contribute to the development of post-transfusion hemolytic complications prophylaxis as well as the improvement of laboratory diagnosis of hemolytic disease of the newborn in the presence of maternal antierythrocyte antibodies.

  10. Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A.

    Science.gov (United States)

    Cole, C N; Tornow, J; Clark, R; Tjian, R

    1986-01-01

    The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional

  11. Melanin-binding radiopharmaceuticals

    International Nuclear Information System (INIS)

    Packer, S.; Fairchild, R.G.; Watts, K.P.; Greenberg, D.; Hannon, S.J.

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed

  12. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  13. Genetic distribution of noncapsular meningococcal group B vaccine antigens in Neisseria lactamica.

    Science.gov (United States)

    Lucidarme, Jay; Gilchrist, Stefanie; Newbold, Lynne S; Gray, Stephen J; Kaczmarski, Edward B; Richardson, Lynne; Bennett, Julia S; Maiden, Martin C J; Findlow, Jamie; Borrow, Ray

    2013-09-01

    The poor immunogenicity of the meningococcal serogroup B (MenB) capsule has led to the development of vaccines targeting subcapsular antigens, in particular the immunodominant and diverse outer membrane porin, PorA. These vaccines are largely strain specific; however, they offer limited protection against the diverse MenB-associated diseases observed in many industrialized nations. To broaden the scope of its protection, the multicomponent vaccine (4CMenB) incorporates a PorA-containing outer membrane vesicle (OMV) alongside relatively conserved recombinant protein components, including factor H-binding protein (fHbp), Neisseria adhesin A (NadA), and neisserial heparin-binding antigen (NHBA). The expression of PorA is unique to meningococci (Neisseria meningitidis); however, many subcapsular antigens are shared with nonpathogenic members of the genus Neisseria that also inhabit the nasopharynx. These organisms may elicit cross-protective immunity against meningococci and/or occupy a niche that might otherwise accommodate pathogens. The potential for 4CMenB responses to impact such species (and vice versa) was investigated by determining the genetic distribution of the primary 4CMenB antigens among diverse members of the common childhood commensal, Neisseria lactamica. All the isolates possessed nhba but were devoid of fhbp and nadA. The nhba alleles were mainly distinct from but closely related to those observed among a representative panel of invasive MenB isolates from the same broad geographic region. We made similar findings for the immunogenic typing antigen, FetA, which constitutes a major part of the 4CMenB OMV. Thus, 4CMenB vaccine responses may impact or be impacted by nasopharyngeal carriage of commensal neisseriae. This highlights an area for further research and surveillance should the vaccine be routinely implemented.

  14. Purification of antibodies to bacterial antigens by an immunoadsorbent and a method to quantify their reaction with insoluble bacterial targets

    International Nuclear Information System (INIS)

    Mathews, H.L.; Minden, P.

    1979-01-01

    A combination of procedures was employed to develop a radioimmunoassay which quantified the binding of antibodies to antigens of either intact Propionibacterium acnes or to antigens of insoluble extracts derived from the bacteria. Reactive antibody populations were purified by use of bacterial immunoadsorbents which were prepared by coupling P. acnes to diethylaminoethyl cellulose. Binding of antibodies was detected with [ 125 I]staphylococcal protein A ([ 125 I]SpA) and optimal conditions for the assay defined by varying the amounts of antibodies, bacterial antigenic targets and [ 125 I]SpA. In antibody excess, 100% of available [ 125 I]SpA was bound by the target-antibody complexes. However, when antibody concentration was limiting, a linear relationship was demonstrated between per cent specific binding of[ 125 I]SpA and antibodies bound to bacterial targets. These results were achieved only with immunoadsorbent-purified antibody populations and not with hyperimmune sera or IgG. The radioimmunoassay detected subtle antigenic differences and similarities between P. acnes, P. acnes extracts and a variety of unrelated microorganisms. (Auth.)

  15. Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

    Science.gov (United States)

    Hasanzadeh, Leila; Ghaznavi-Rad, Ehsanollah; Soufian, Safieh; Farjadi, Vahideh; Abtahi, Hamid

    2013-01-01

    Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis . PMID:23997913

  16. Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

    Directory of Open Access Journals (Sweden)

    Leila Hasanzadeh

    2013-07-01

    Full Text Available Objective(s: Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity.   Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3 pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

  17. Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies

    OpenAIRE

    Payne, Ruth O.; Silk, Sarah E.; Elias, Sean C.; Milne, Kathryn H.; Rawlinson, Thomas A.; Llewellyn, David; Shakri, A. Rushdi; Jin, Jing; Labb?, Genevi?ve M.; Edwards, Nick J.; Poulton, Ian D.; Roberts, Rachel; Farid, Ryan; J?rgensen, Thomas; Alanine, Daniel G.W.

    2017-01-01

    BACKGROUND: Plasmodium vivax is the most widespread human malaria geographically; however, no effective vaccine exists. Red blood cell invasion by the P. vivax merozoite depends on an interaction between the Duffy antigen receptor for chemokines (DARC) and region II of the parasite's Duffy-binding protein (PvDBP_RII). Naturally acquired binding-inhibitory antibodies against this interaction associate with clinical immunity, but it is unknown whether these responses can be induced by human vac...

  18. Antigen Cross-Presentation of Immune Complexes

    Science.gov (United States)

    Platzer, Barbara; Stout, Madeleine; Fiebiger, Edda

    2014-01-01

    The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of interest to physicians, as well as basic scientists, that aim to establish efficient cell-based cancer immune therapy. A prerequisite for exploiting this pathway for therapeutic purposes is a better understanding of the mechanisms that underlie the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses when initiated by DCs via cross-presentation. The ability of humans DC to perform cross-presentation is of utmost interest, as this cell type is a main target for cell-based immunotherapy in humans. The outcome of a cross-presentation event is guided by the nature of the antigen, the form of antigen uptake, and the subpopulation of DCs that performs presentation. Generally, CD8α+ DCs are considered to be the most potent cross-presenting DCs. This paradigm, however, only applies to soluble antigens. During adaptive immune responses, immune complexes form when antibodies interact with their specific epitopes on soluble antigens. Immunoglobulin G (IgG) immune complexes target Fc-gamma receptors on DCs to shuttle exogenous antigens efficiently into the cross-presentation pathway. This receptor-mediated cross-presentation pathway is a well-described route for the induction of strong CD8+ T cell responses. IgG-mediated cross-presentation is intriguing because it permits the CD8− DCs, which are commonly considered to be weak cross-presenters, to efficiently cross-present. Engaging multiple DC subtypes for cross-presentation might be a superior strategy to boost CTL responses in vivo. We here summarize our current understanding of how DCs use IgG-complexed antigens for the efficient induction of CTL responses. Because of its importance for human cell therapy, we also review the recent advances in the characterization of cross-presentation properties of human DC subsets. PMID:24744762

  19. Treatment of Schistosoma mansoni with miltefosine in vitro enhances serological recognition of defined worm surface antigens.

    Directory of Open Access Journals (Sweden)

    Marwa H El-Faham

    2017-08-01

    Full Text Available Miltefosine, an anti-cancer drug that has been successfully repositioned for treatment of Leishmania infections, has recently also shown promising effects against Schistosoma spp targeting all life cycle stages of the parasite. The current study examined the effect of treating Schistosoma mansoni adult worms with miltefosine on exposure of worm surface antigens in vitro.In an indirect immunofluorescence assay, rabbit anti-S.mansoni adult worm homogenate and anti-S. mansoni infection antisera gave strong immunofluorescence of the S. mansoni adult worm surface after treatment with miltefosine, the latter antiserum having previously been shown to synergistically enhance the schistosomicidal activity of praziquantel. Rabbit antibodies that recognised surface antigens exposed on miltefosine-treated worms were recovered by elution off the worm surface in low pH buffer and were used in a western immunoblotting assay to identify antigenic targets in a homogenate extract of adult worms (SmWH. Four proteins reacting with the antibodies in immunoblots were purified and proteomic analysis (MS/MS combined with specific immunoblotting indicated they were the S. mansoni proteins: fructose-1,6 bisphosphate aldolase (SmFBPA, Sm22.6, alkaline phosphatase and malate dehydrogenase. These antibodies were also found to bind to the surface of 3-hour schistosomula and induce immune agglutination of the parasites, suggesting they may have a role in immune protection.This study reveals a novel mode of action of miltefosine as an anti-schistosome agent. The immune-dependent hypothesis we investigated has previously been lent credence with praziquantel (PZQ, whereby treatment unmasks parasite surface antigens not normally exposed to the host during infection. Antigens involved in this molecular mechanism could have potential as intervention targets and antibodies against these antigens may act to increase the drug's anti-parasite efficacy and be involved in the development

  20. Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

    International Nuclear Information System (INIS)

    Mueller, H.; Fehr, J.

    1986-01-01

    The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

  1. Studies on antigenic cross-reactivity of Trichuris ovis with host mucosal antigens in goat

    OpenAIRE

    Gautam Patra; Seikh Sahanawaz Alam; Sonjoy Kumar Borthakur; Hridayesh Prasad

    2015-01-01

    Objective: To ascertain whether immunodominant antigens of Trichuris ovis might share and cross react with host molecule. Methods: Two crude protein preparations from anterior and posterior parts of Trichuris ovis were characterized along with host mucosal antigen by double immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting technique. Conventional scanning electron microscopy was performed as per standard procedure. Results: Sharp...

  2. Forecasting of interaction between bee propolis and protective antigenic domain in anthrax using the software and bioinformatics web servers

    Directory of Open Access Journals (Sweden)

    Elmira Mohammadi

    2017-01-01

    Full Text Available Background: Protective antigen of anthrax toxin, after touching the cell receptors, plays an important role in the pathogenesis of toxin. The purpose of this study was to investigate the interaction of anthrax toxin protective antigen and four great combination propolis included caffeic acid, benzyl caffeate, cinnamic acid and kaempferol using the softwares and bioinformatics web servers. Methods: Three-dimensional structure of protective antigen (receptor obtains from Protein Data Bank (PDB. Four of the main components from propolis were selected          as ligand and their 3D-structures were obtained from ChemSpider and ZINC     compound database. The interaction of each ligand and receptor was assessed                   by SwissDock server (http://www.swissdock.ch/ and BSP-SLIM server (http://zhanglab.ccmb.med.umich.edu/BSP-SLIM. Docking results appears with Fullfitness numbers (in kcal/mol. Identification of amino acids involved in ligand and receptor interaction, was performed using the Chimera software; UCSF Chimera program (http://www.cgl.ucsf.edu/. Results: The results of interaction between propolis components and protective antigen by BSP-SLIM server showed that the most interaction was related with benzyl caffeate, caffeic acid, kaempferol and cinnamic acid, respectively. Results for the desired ligand Interaction with protective antigen genes using SwissDock server showed that the caffeic acid had ΔG equals -9.10 kcal/mol and FullFitness equal to -993.16 kcal/mol respectively. The analysis of interaction between ligands with amino-acids of protective antigen indicated that the interaction of Caffeic acid whit Glutamic acid 117 had energy -15.5429 kcal/mol. Conclusion: Finding strong and safe inhibitors for anthrax toxin is very useful method for inhibiting its toxicity to cell. In this study the binding ability of four flavonoids to protective antigen was studied. Glutamic acid 117 is very effective

  3. Carcinoembryonic Antigen Level in Liver Disease

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Kyoo Ok; Kim, Ki Whang; Park, Chang Yun [Yonsei University College of Medicine, Seoul (Korea, Republic of)

    1978-09-15

    Carcinoembryonic antigen was initially known as tumor specific antigen and had a potential diagnostic value in the detection of digestive tract malignancies. However, subsequent studies showed CEA and CEA-like antigen present in benign disease, particularly in liver. We had collected sera from 58 patients who had liver scan and later were diagnosed clinically and histologically as liver disease. We estimated CEA values and correlations were made with liver function tests in liver cirrhosis cases. The results: 1) The raised plasma carcinoembryonic antigen level were found in 13 (68.4%) of 19 patients cirrhosis, 5 (27.8%) of 18 patients in hepatoma, 5 (71%) of 7 patients in chronic active hepatitis, all 3 patients in liver abscesses, 2 (66.7%) of 3 patients in liver abscesses, 2 (66.7%) of 3 patients in obstructive biliary disease and none in each one patient of traumatic liver hematoma, subphrenic abscess and clonorchiasis. 2) There is no linear correlation between carcinoembryonic antigen level and liver function tests including serum bilirubin, alkaline phosphatase, SGOT and prothrombin time in liver patients.

  4. Human Tumor Antigens Yesterday, Today, and Tomorrow.

    Science.gov (United States)

    Finn, Olivera J

    2017-05-01

    The question of whether human tumors express antigens that can be recognized by the immune system has been answered with a resounding YES. Most were identified through spontaneous antitumor humoral and cellular immune responses found in cancer patients and include peptides, glycopeptides, phosphopeptides, viral peptides, and peptides resulting from common mutations in oncogenes and tumor-suppressor genes, or common gene fusion events. Many have been extensively tested as candidates for anticancer vaccines. More recently, attention has been focused on the potentially large number of unique tumor antigens, mutated neoantigens, that are the predicted products of the numerous mutations revealed by exome sequencing of primary tumors. Only a few have been confirmed as targets of spontaneous immunity and immunosurveillance, and even fewer have been tested in preclinical and clinical settings. The field has been divided for a long time on the relative importance of shared versus mutated antigens in tumor surveillance and as candidates for vaccines. This question will eventually need to be answered in a head to head comparison in well-designed clinical trials. One advantage that shared antigens have over mutated antigens is their potential to be used in vaccines for primary cancer prevention. Cancer Immunol Res; 5(5); 347-54. ©2017 AACR . ©2017 American Association for Cancer Research.

  5. Carcinoembryonic Antigen Level in Liver Disease

    International Nuclear Information System (INIS)

    Choi, Kyoo Ok; Kim, Ki Whang; Park, Chang Yun

    1978-01-01

    Carcinoembryonic antigen was initially known as tumor specific antigen and had a potential diagnostic value in the detection of digestive tract malignancies. However, subsequent studies showed CEA and CEA-like antigen present in benign disease, particularly in liver. We had collected sera from 58 patients who had liver scan and later were diagnosed clinically and histologically as liver disease. We estimated CEA values and correlations were made with liver function tests in liver cirrhosis cases. The results: 1) The raised plasma carcinoembryonic antigen level were found in 13 (68.4%) of 19 patients cirrhosis, 5 (27.8%) of 18 patients in hepatoma, 5 (71%) of 7 patients in chronic active hepatitis, all 3 patients in liver abscesses, 2 (66.7%) of 3 patients in liver abscesses, 2 (66.7%) of 3 patients in obstructive biliary disease and none in each one patient of traumatic liver hematoma, subphrenic abscess and clonorchiasis. 2) There is no linear correlation between carcinoembryonic antigen level and liver function tests including serum bilirubin, alkaline phosphatase, SGOT and prothrombin time in liver patients.

  6. Original antigenic sin responses to influenza viruses.

    Science.gov (United States)

    Kim, Jin Hyang; Skountzou, Ioanna; Compans, Richard; Jacob, Joshy

    2009-09-01

    Most immune responses follow Burnet's rule in that Ag recruits specific lymphocytes from a large repertoire and induces them to proliferate and differentiate into effector cells. However, the phenomenon of "original antigenic sin" stands out as a paradox to Burnet's rule of B cell engagement. Humans, upon infection with a novel influenza strain, produce Abs against older viral strains at the expense of responses to novel, protective antigenic determinants. This exacerbates the severity of the current infection. This blind spot of the immune system and the redirection of responses to the "original Ag" rather than to novel epitopes were described fifty years ago. Recent reports have questioned the existence of this phenomenon. Hence, we revisited this issue to determine the extent to which original antigenic sin is induced by variant influenza viruses. Using two related strains of influenza A virus, we show that original antigenic sin leads to a significant decrease in development of protective immunity and recall responses to the second virus. In addition, we show that sequential infection of mice with two live influenza virus strains leads to almost exclusive Ab responses to the first viral strain, suggesting that original antigenic sin could be a potential strategy by which variant influenza viruses subvert the immune system.

  7. Molecular basis of immunogenicity to botulinum neurotoxins and uses of the defined antigenic regions.

    Science.gov (United States)

    Atassi, M Z

    2015-12-01

    Intensive research in this laboratory over the last 19 years has aimed at understanding the molecular bases for immune recognition of botulinum neurotoxin, types A and B and the role of anti-toxin immune responses in defense against the toxin. Using 92 synthetic 19-residue peptides that overlapped by 5 residues and comprised an entire toxin (A or B) we determined the peptides' ability to bind anti-toxin Abs of human, mouse, horse and chicken. We also localized the epitopes recognized by Abs of cervical dystonia patients who developed immunoresistance to correlate toxin during treatment with BoNT/A or BoNT/B. For BoNT/A, patients' blocking Abs bound to 13 regions (5 on L and 8 on H subunit) on the surface and the response to each region was under separate MHC control. The responses were defined by the structure of the antigen and by the MHC of the host. The antigenic regions coincided or overlapped with synaptosomes (SNPS) binding regions. Antibody binding blocked the toxin's ability to bind to neuronal cells. In fact selected synthetic peptides were able to inhibit the toxin's action in vivo. A combination of three synthetic strong antigenic peptides detected blocking Abs in 88% of immunoresistant patients' sera. Administration of selected epitopes, pre-linked at their N(α) group to monomethoxyployethylene glycol, into mice with ongoing blocking anti-toxin Abs, reduced blocking Ab levels in the recipients. This may be suitable for clinical applications. Defined epitopes should also be valuable in synthetic vaccines design. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Recognition of oxidized albumin and thyroid antigens by psoriasis autoantibodies. A possible role of reactive-oxygen-species induced epitopes in chronic plaque psoriasis

    Directory of Open Access Journals (Sweden)

    Hani A. Al-Shobaili

    2015-12-01

    Full Text Available Objectives: To investigate the role of reactive-oxygen-species (ROS induced epitopes on human-serum-albumin (HSA and thyroid antigens in psoriasis autoimmunity. Methods: This study was performed in the College of Medicine, Qassim University, Buraidah, Saudi Arabia between May 2014 and February 2015. The study was designed to explore the role of ROS-induced epitopes in psoriasis autoimmunity. Singlet-oxygen (or ROS-induced epitopes on protein (ROS-epitopes-albumin was characterized by in-vitro and in-vivo. Thyroid antigens were prepared from rabbit thyroid, and thyroglobulin was isolated from thyroid extract. Immunocross-reactions of protein-A purified anti-ROS-epitopes-HSA-immunoglobulin G (IgGs with thyroid antigen, thyroglobulin, and their oxidized forms were determined. Binding characteristics of autoantibodies in chronic plaque psoriasis patients (n=26 against ROS-epitopes-HSA and also with native and oxidized thyroid antigens were screened, and the results were compared with age-matched controls (n=22. Results: The anti-ROS-epitopes-HSA-IgGs showed cross-reactions with thyroid antigen, thyroglobulin and with their oxidized forms. High degree of specific binding by psoriasis IgGs to ROS-epitopes-HSA, ROS-thyroid antigen and ROS-thyroglobulin was observed. Immunoglobulin G from normal-human-controls showed negligible binding with all tested antigens. Moreover, sera from psoriasis patients had higher levels of carbonyl contents compared with control sera. Conclusion: Structural alterations in albumin, thyroid antigens by ROS, generate unique neo-epitopes that might be one of the factors for the induction of autoantibodies in psoriasis.

  9. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

    Science.gov (United States)

    Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

    2018-01-01

    Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

  10. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

    Directory of Open Access Journals (Sweden)

    Isabel Correa

    2018-03-01

    Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

  11. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

    Science.gov (United States)

    Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

    2018-01-01

    Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

  12. A negative feedback modulator of antigen processing evolved from a frameshift in the cowpox virus genome.

    Directory of Open Access Journals (Sweden)

    Jiacheng Lin

    2014-12-01

    Full Text Available Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.

  13. Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

    International Nuclear Information System (INIS)

    Dominguez, J.M.; Lanoix, J.; Paiement, J.

    1991-01-01

    We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha- 32 P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha- 32 P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations

  14. Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability

    Science.gov (United States)

    Schmidt, Michael M.; Thurber, Greg M.

    2010-01-01

    Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids. PMID:18408925

  15. A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

    Science.gov (United States)

    Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

    2010-10-01

    Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

  16. Rationalization and Design of the Complementarity Determining Region Sequences in an Antibody-Antigen Recognition Interface

    Science.gov (United States)

    Chen, Ing-Chien; Lee, Yu-Ching; Chen, Jun-Bo; Tsai, Keng-Chang; Chen, Ching-Tai; Chang, Jeng-Yih; Yang, Ei-Wen; Hsu, Po-Chiang; Jian, Jhih-Wei; Hsu, Hung-Ju; Chang, Hung-Ju; Hsu, Wen-Lian; Huang, Kai-Fa; Ma, Alex Che; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are critical determinants in biological systems. Engineered proteins binding to specific areas on protein surfaces could lead to therapeutics or diagnostics for treating diseases in humans. But designing epitope-specific protein-protein interactions with computational atomistic interaction free energy remains a difficult challenge. Here we show that, with the antibody-VEGF (vascular endothelial growth factor) interaction as a model system, the experimentally observed amino acid preferences in the antibody-antigen interface can be rationalized with 3-dimensional distributions of interacting atoms derived from the database of protein structures. Machine learning models established on the rationalization can be generalized to design amino acid preferences in antibody-antigen interfaces, for which the experimental validations are tractable with current high throughput synthetic antibody display technologies. Leave-one-out cross validation on the benchmark system yielded the accuracy, precision, recall (sensitivity) and specificity of the overall binary predictions to be 0.69, 0.45, 0.63, and 0.71 respectively, and the overall Matthews correlation coefficient of the 20 amino acid types in the 24 interface CDR positions was 0.312. The structure-based computational antibody design methodology was further tested with other antibodies binding to VEGF. The results indicate that the methodology could provide alternatives to the current antibody technologies based on animal immune systems in engineering therapeutic and diagnostic antibodies against predetermined antigen epitopes. PMID:22457753

  17. A solid phase radio immunoassay on hydrophobic membrane filters: detection of antibodies to gonocal surface antigens

    International Nuclear Information System (INIS)

    Lambden, P.R.; Watt, P.J.

    1978-01-01

    A solid phase radioimmunoassay (SPRIA) has been developed for detection of IgG antibodies to gonococcal outer membrane components. Gonococcal antigens was immobilised on a solid support by covalent coupling to CNBr-activated Sepharose in the presence of the detergent Triton X-100. Binding of specific antibody to the Sepharose-antigen complex was detected using radiolabelled Protein A as the antiglobulin. Protein A was labelled by radioacetylation with tritiated acetic anhydride, yielding a product of high specific activity and high stability. No detectable loss of activity was observed over a ten month period. The entire assay was performed on Mitex teflon hydrophobic membrane filters which held the Sepharose beads and aqueous supernatant as a discrete drop of liquid. The supernatants and incubation were easily and rapidly removed from the beads by suction on a specially-designed manifold system. This procedure removed the need for repeated and time-consuming centrifugations. Titres were obtained graphically from double log plots of cpm bound versus antiserum dilution by extrapolation of the straight line to a point corresponding to twice the control level of radioactivity binding. The assay proved to be a very reliable and simple procedure for the detection of IgG antibodies to gonococcal surface antigens. (Auth.)

  18. Application of fluorescent monocytes for probing immune complexes on antigen microarrays.

    Directory of Open Access Journals (Sweden)

    Zoltán Szittner

    Full Text Available Microarrayed antigens are used for identifying serum antibodies with given specificities and for generating binding profiles. Antibodies bind to these arrayed antigens forming immune complexes and are conventionally identified by secondary labelled antibodies.In the body immune complexes are identified by bone marrow derived phagocytic cells, such as monocytes. In our work we were looking into the possibility of replacing secondary antibodies with monocytoid cells for the generation of antibody profiles. Using the human monocytoid cell line U937, which expresses cell surface receptors for immune complex components, we show that cell adhesion is completely dependent on the interaction of IgG heavy chains and Fcγ receptors, and this recognition is susceptible to differences between heavy chain structures and their glycosylation. We also report data on a possible application of this system in autoimmune diagnostics.Compared to secondary antibodies, fluorescent monocytesas biosensors are superior in reflecting biological functions of microarray-bound antibodies and represent an easy and robust alternative for profiling interactions between serum proteins and antigens.

  19. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... Links Patient Resources For Health Professionals Subscribe Search Sex Hormone Binding Globulin (SHBG) Send Us Your Feedback ... As Testosterone-estrogen Binding Globulin TeBG Formal Name Sex Hormone Binding Globulin This article was last reviewed ...

  20. Optimization of the Small Glycan Presentation for Binding a Tumor-Associated Antibody

    DEFF Research Database (Denmark)

    Kveton, Filip; Blšáková, Anna; Hushegyi, Andras

    2017-01-01

    on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective...... bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various...... techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding...

  1. Structure and binding properties of a cameloid nanobody raised against KDM5B

    DEFF Research Database (Denmark)

    Wiuf, Anders; Kristensen, Line Hyltoft; Kristensen, Ole

    2015-01-01

    The histone demethylase KDM5B is considered to be a promising target for anticancer therapy. Single-chain antibodies from llama (nanobodies) have been raised to aid in crystallization and structure determination of this enzyme. The antigen-binding properties of 15 of these nanobodies have been...

  2. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L

    1991-01-01

    with the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential...

  3. Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins

    Science.gov (United States)

    Schatten, G.; Schatten, H.; Simerly, C.; Maul, G. G.; Chaly, N.

    1985-01-01

    Nuclear structural changes during fertilization and embryogenesis in mice and sea urchins are traced using four antibodies. The oocytes from virgin female mice, morulae and blastocytes from mated females, and gametes from the sea urchin Lytechnius variegatis are studied using mouse monoclonal antibodies to nuclear lamin A/C, monoclonal antibody to P1, human autoimmune antibodies to lamin A/C, and to lamin B. The mouse fertilization data reveal no lamins on the oocyte; however, lamins are present on the pronuclei, and chromosomes are found on the oocytes and pronuclei. It is detected that on the sea urchin sperm the lamins are reduced to acrosomal and centriolar fossae and peripheral antigens are around the sperm nucleus. The mouse sperm bind lamin antibodies regionally and do not contain antigens. Lamins and antigens are observed on both pronuclei and chromosomes during sea urchin fertilization. Mouse embryogenesis reveals that lamin A/C is not recognized at morula and blastocyst stages; however, lamin B stains are retained. In sea urchin embryogenesis lamin recognition is lost at the blastrula, gastrula, and plutei stages. It is noted that nuclear lamins lost during spermatogenesis are restored at fertilization and peripheral antigens are associated with the surface of chromosomes during meiosis and mitosis and with the periphery of the pronuclei and nuclei during interphase.

  4. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

    International Nuclear Information System (INIS)

    Brodzik, R.; Bandurska, K.; Deka, D.; Golovkin, M.; Koprowski, H.

    2005-01-01

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP

  5. Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

    Science.gov (United States)

    Akiba, Hiroki; Tsumoto, Kouhei

    2015-07-01

    Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. Antigenic typing Polish isolates of canine parvovirus

    Energy Technology Data Exchange (ETDEWEB)

    Mizak, B. [National Veterinary Research Institute, Pulawy (Poland); Plucienniczak, A. [Polish Academy ofd Sciences. Microbiology and Virology Center, Lodz (Poland)

    1995-12-31

    Polish strains of canine parvovirus isolated between 1982 and 1993 were examined to determine the extent to which the virus has evolved antigenically and genetically over eleven years. Two CPV isolates obtained in Warsaw in 1982 and Pulawy in 1993, were examined using monoclonal antibody typing, restriction analysis and sequencing VP-2 protein gene. Five other isolates from Warsaw and Pulawy were tested with the panel of monoclonal antibodies specific to CPV-2, CPV-2a and common for canine parvovirus, feline panleukopenia virus and milk enteritis virus. Results of the studies demonstrated that all isolates tested represented CPV-2a antigenic type. Rapid antigenic strain replacement recorded by Parrish and Senda in the U.S.A and Japan was not confirmed in Poland. (author). 30 refs, 2 tabs.

  7. Harnessing Dendritic Cells for Tumor Antigen Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Nierkens, Stefan [Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, Nijmegen 6525 GA (Netherlands); Janssen, Edith M., E-mail: edith.janssen@cchmc.org [Division of Molecular Immunology, Cincinnati Children' s Hospital Research Foundation, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229 (United States)

    2011-04-26

    Dendritic cells (DC) are professional antigen presenting cells that are crucial for the induction of anti-tumor T cell responses. As a consequence, research has focused on the harnessing of DCs for therapeutic interventions. Although current strategies employing ex vivo-generated and tumor-antigen loaded DCs have been proven feasible, there are still many obstacles to overcome in order to improve clinical trial successes and offset the cost and complexity of customized cell therapy. This review focuses on one of these obstacles and a pivotal step for the priming of tumor-specific CD8{sup +} and CD4{sup +} T cells; the in vitro loading of DCs with tumor antigens.

  8. Antigenic typing Polish isolates of canine parvovirus

    International Nuclear Information System (INIS)

    Mizak, B.; Plucienniczak, A.

    1995-01-01

    Polish strains of canine parvovirus isolated between 1982 and 1993 were examined to determine the extent to which the virus has evolved antigenically and genetically over eleven years. Two CPV isolates obtained in Warsaw in 1982 and Pulawy in 1993, were examined using monoclonal antibody typing, restriction analysis and sequencing VP-2 protein gene. Five other isolates from Warsaw and Pulawy were tested with the panel of monoclonal antibodies specific to CPV-2, CPV-2a and common for canine parvovirus, feline panleukopenia virus and milk enteritis virus. Results of the studies demonstrated that all isolates tested represented CPV-2a antigenic type. Rapid antigenic strain replacement recorded by Parrish and Senda in the U.S.A and Japan was not confirmed in Poland. (author). 30 refs, 2 tabs

  9. Original antigenic sin: A comprehensive review.

    Science.gov (United States)

    Vatti, Anup; Monsalve, Diana M; Pacheco, Yovana; Chang, Christopher; Anaya, Juan-Manuel; Gershwin, M Eric

    2017-09-01

    The concept of "original antigenic sin" was first proposed by Thomas Francis, Jr. in 1960. This phenomenon has the potential to rewrite what we understand about how the immune system responds to infections and its mechanistic implications on how vaccines should be designed. Antigenic sin has been demonstrated to occur in several infectious diseases in both animals and humans, including human influenza infection and dengue fever. The basis of "original antigenic sin" requires immunological memory, and our immune system ability to autocorrect. In the context of viral infections, it is expected that if we are exposed to a native strain of a pathogen, we should be able to mount a secondary immune response on subsequent exposure to the same pathogen. "Original antigenic sin" will not contradict this well-established immunological process, as long as the subsequent infectious antigen is identical to the original one. But "original antigenic sin" implies that when the epitope varies slightly, then the immune system relies on memory of the earlier infection, rather than mount another primary or secondary response to the new epitope which would allow faster and stronger responses. The result is that the immunological response may be inadequate against the new strain, because the immune system does not adapt and instead relies on its memory to mount a response. In the case of vaccines, if we only immunize to a single strain or epitope, and if that strain/epitope changes over time, then the immune system is unable to mount an accurate secondary response. In addition, depending of the first viral exposure the secondary immune response can result in an antibody-dependent enhancement of the disease or at the opposite, it could induce anergy. Both of them triggering loss of pathogen control and inducing aberrant clinical consequences. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi.

    Science.gov (United States)

    Choong, Yee Siew; Lim, Theam Soon; Chew, Ai Lan; Aziah, Ismail; Ismail, Asma

    2011-04-01

    The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ∼50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ∼50 kDa channel protein is a good specific target for typhoid detection test. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Influence of clinical and laboratory variables on faecal antigen ELISA results in dogs with canine parvovirus infection.

    Science.gov (United States)

    Proksch, A L; Unterer, S; Speck, S; Truyen, U; Hartmann, K

    2015-06-01

    False negative faecal canine parvovirus (CPV) antigen ELISA results in dogs with CPV infection are common, but the factors that lead to these false negative results are still unknown. The aim of this study was to investigate whether dogs with a false negative faecal CPV antigen ELISA result have milder clinical signs and laboratory changes, a lower faecal virus load, higher faecal and serum CPV antibody titres and a faster recovery than dogs with a positive result. Eighty dogs with CPV infection, confirmed by the presence of clinical signs and a positive faecal CPV polymerase chain reaction (PCR), were assigned to two groups according to their faecal antigen ELISA result. Time until presentation, severity of symptoms, laboratory parameters, faecal virus load, faecal and serum antibody titres, and CPV sequencing data were compared between both groups. In 38/80 dogs that were hospitalised until recovery, the time to recovery, mortality, and the course of the disease were compared between dogs with positive and negative faecal antigen ELISA results. Of the 80 dogs included, 41 (51.3%) had a false negative faecal antigen ELISA result. ELISA-negative dogs had a significantly shorter time until presentation, lower frequency of defaecation, lower faecal virus load, and higher serum antibody concentrations than ELISA-positive dogs. Laboratory changes, CPV shedding, and outcomes were not associated with faecal antigen ELISA results. In conclusion, low faecal CPV load and antibodies binding to CPV antigen in faeces are likely to be important reasons for false negative faecal antigen ELISA results. Dogs with clinical signs of CPV infection should be retested by faecal PCR. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Effect of the pre-erythrocytic candidate malaria vaccine RTS,S/AS01E on blood stage immunity in young children

    DEFF Research Database (Denmark)

    Bejon, Philip; Cook, Jackie; Bergmann-Leitner, Elke

    2011-01-01

    (See the article by Greenhouse et al, on pages 19-26.) Background. RTS,S/AS01(E) is the lead candidate malaria vaccine and confers pre-erythrocytic immunity. Vaccination may therefore impact acquired immunity to blood-stage malaria parasites after natural infection. Methods. We measured, by enzyme......, MSP-1(42), and MSP-3 antibody concentrations and no significant change in GIA. Increasing anti-merozoite antibody concentrations and GIA were prospectively associated with increased risk of clinical malaria. Conclusions. Vaccination with RTS,S/AS01E reduces exposure to blood-stage parasites and, thus......-linked immunosorbent assay, antibodies to 4 Plasmodium falciparum merozoite antigens (AMA-1, MSP-1(42), EBA-175, and MSP-3) and by growth inhibitory activity (GIA) using 2 parasite clones (FV0 and 3D7) at 4 times on 860 children who were randomized to receive with RTS,S/AS01(E) or a control vaccine. Results. Antibody...

  13. Plasmodium falciparum field isolates from South America use an atypical red blood cell invasion pathway associated with invasion ligand polymorphisms

    DEFF Research Database (Denmark)

    Lopez-Perez, Mary; Villasis, Elizabeth; Machado, Ricardo L D

    2012-01-01

    Studies of Plasmodium falciparum invasion pathways in field isolates have been limited. Red blood cell (RBC) invasion is a complex process involving two invasion protein families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins, which are polymorphic and not fully...... characterized in field isolates. To determine the various P. falciparum invasion pathways used by parasite isolates from South America, we studied the invasion phenotypes in three regions: Colombia, Peru and Brazil. Additionally, polymorphisms in three members of the EBL (EBA-181, EBA-175 and EBL-1) and five...... pathways and the ligand polymorphisms differed substantially among the Colombian and Brazilian isolates while the Peruvian isolates represent an amalgam of those present in the Colombian and Brazilian field isolates. The NrTrCr invasion profile was associated with the presence of the PfRh2a pepC variant...

  14. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    Science.gov (United States)

    Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

    2012-01-01

    Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

  15. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    Directory of Open Access Journals (Sweden)

    Ewoud Bernardus Compeer

    2012-03-01

    Full Text Available The cross-presentation of endocytosed antigen as peptide/class I MHC complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells (APC capable of antigen cross-presentation, description of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC, there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlight DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, recycling and maturation including the sorting of membrane proteins, dynamic remodeling of endosomal structures and cell-surface directed endosomal trafficking. We will conclude with description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

  16. Efficacy demonstration of tetanus vaccines by double antigen ELISA.

    Science.gov (United States)

    Rosskopf, U; Noeske, K; Werner, E

    2005-09-01

    This paper describes a double antigen ELISA (DAE) for rapid, specific and reliable assessment of the antitetanus immune status of horses and sheep. Compared with the indirect ELISA, the double antigen ELISA has the advantage of species-independent testing of sera. Thanks to its test design, it is more specific since the detected antibodies are forced to bind tetanus toxoid twice. In addition, it is very sensitive to tetanus antibodies, enabling the detection of low antibody titres, in range which is relevant for the assessment of the protective status (tetanus toxin neutralising antibodies). The detection limit of the DAE for tetanus antibodies is in the order of 10(-4) EU/ml. A comparison of in vitro results of individual sera with in vivo titres showed that horse sera with titres of 0.04 and 0.05 EU/ml in the DAE showed titres of > 0.05 IU and 0.034 IU/ml respectively during in vivo testing thus indicating good agreement. For tested sheep sera which were rated > 0.05 IU/ml in vivo, the corresponding titre in the DAE was 0.24 EU/ml. Clear tetanus antitoxin establishment of protective ELISA limits requires further comparative examination of sera with low titres (tetanus vaccines ad us. vet. As a consequence, the toxin neutralisation test (still being the standard method of choice for quantifying tetanus toxin neutralising antitoxin titres) could be replaced, since it requires too great a number of animals per test and involves considerable suffering for the animals. The test described here reduces the use of mice and guinea pigs within vaccine efficacy testing. In addition, it involves less exposure of the laboratory personnel to toxin.

  17. Identification of antigenic proteins of setaria cervi by immunoblotting technique

    International Nuclear Information System (INIS)

    Kaushal, N.A.; Kaushal, D.C.; Ghatak, S.

    1987-01-01

    Identification and characterization of antigenic proteins of Setaria cervi (bovine filarial parasite) adults and microfilariae was done by immunoblotting technique using hyperimmune rabbit sera against S. cervi and Brugia malayi. The antigens recognized by these sera were detected by using 125 I protein-A followed by autoradiography. Fifteen different antigens were observed to be common between adult and microfilarial stages of the parasite. Some stage specific antigens were also identified. Many antigens of S. cervi adults and microfilariae were also recognized by rabbit anti-B.malayi serum showing the existence of common antigenic determinants between the bovine and human filarial parasites

  18. Blood group antigen studies using CdTe quantum dots and flow cytometry

    Directory of Open Access Journals (Sweden)

    Cabral Filho PE

    2015-07-01

    , this methodology with high sensibility and specificity can be applied to study a variety of RBC antigens, and, as a quantitative tool, can help in achieving a better comprehension of the antigen expression patterns on RBC membranes. Keywords: erythrocytes, nanoparticles, ABO antigens, carbohydrates, covalent binding

  19. The antibody response of pregnant Cameroonian women to VAR2CSA ID1-ID2a, a small recombinant protein containing the CSA-binding site

    DEFF Research Database (Denmark)

    Babakhanyan, Anna; Leke, Rose G F; Salanti, Ali

    2014-01-01

    In pregnant women, Plasmodium falciparum-infected erythrocytes expressing the VAR2CSA antigen bind to chondroitin sulfate A in the placenta causing placental malaria. The binding site of VAR2CSA is present in the ID1-ID2a region. This study sought to determine if pregnant Cameroonian women natura...

  20. Detection of autoimmune antibodies in localized scleroderma by synthetic oligonucleotide antigens

    DEFF Research Database (Denmark)

    Samuelsen, Simone; Jørgensen, Christian Damsgaard; Mellins, Elizabeth D

    2018-01-01

    In this study, we developed a series of synthetic oligonucleotides that allowed us to investigate the details on the antigen recognition by autoimmune antibodies in localized scleroderma subjects. Besides dramatically improved analytical specificity of the assay, our data suggests a potential...... linking for antibodies to DNA to the biological status of disease state in localized scleroderma. Moreover, introducing chemical modifications into short synthetic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules completely changed the binding titers of corresponding antibodies...... and their clinical relevance. The strongest observed effect was registered for the localized scleroderma skin damage index (LoSDI) on the IgG antibodies to TC dinucleotide-rich double-stranded antigen (p

  1. Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

    Directory of Open Access Journals (Sweden)

    Marc Cartellieri

    2010-01-01

    Full Text Available CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs. First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

  2. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  3. Small organic compounds enhance antigen loading of class II major histocompatibility complex proteins by targeting the polymorphic P1 pocket

    DEFF Research Database (Denmark)

    Höpner, Sabine; Dickhaut, Katharina; Hofstätter, Maria

    2006-01-01

    the peptide loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position beta86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently......, transient occupation of this pocket by the organic compound stabilizes the peptide-receptive conformation permitting rapid antigen loading. This interaction appeared restricted to the larger Gly(beta86) pocket and allowed striking enhancements of T cell responses for antigens presented by these "adamantyl......-susceptible" MHC molecules. As catalysts of antigen loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce...

  4. Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries.

    Science.gov (United States)

    Nuttall, S D; Krishnan, U V; Hattarki, M; De Gori, R; Irving, R A; Hudson, P J

    2001-08-01

    The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

  5. Discovery of native autoantigens via antigen surrogate technology: application to type 1 diabetes.

    Science.gov (United States)

    Doran, Todd M; Simanski, Scott; Kodadek, Thomas

    2015-02-20

    A fundamental goal in understanding the mechanisms of autoimmune disease is the characterization of autoantigens that are targeted by autoreactive antibodies and T cells. Unfortunately, the identification of autoantigens is a difficult problem. We have begun to explore a novel route to the discovery of autoantibody/autoantigen pairs that involves comparative screening of combinatorial libraries of unnatural, synthetic molecules for compounds that bind antibodies present at much higher levels in the serum of individuals with a given autoimmune disease than in the serum of control individuals. We have shown that this approach can yield "antigen surrogates" capable of capturing disease-specific autoantibodies from serum. In this report, we demonstrate that the synthetic antigen surrogates can be used to affinity purify the autoantibodies from serum and that these antibodies can then be used to identify their cognate autoantigen in an appropriate tissue lysate. Specifically, we report the discovery of a peptoid able to bind autoantibodies present in about one-third of nonobese diabetic (NOD) mice. The peptoid-binding autoantibodies were highly enriched through peptoid affinity chromatography and employed to probe mouse pancreatic and brain lysates. This resulted in identification of murine GAD65 as the native autoantigen. GAD65 is a known humoral autoantigen in human type 1 diabetes mellitus (T1DM), but its existence in mice had been controversial. This study demonstrates the potential of this chemical approach for the unbiased identification of autoantigen/autoantibody complexes.

  6. [Radiocompetitive method of H antigen determination].

    Science.gov (United States)

    Semenova, G B; Sokolov, Ia A; Liashenko, V A

    1978-06-01

    The authors describe a radiocompetitive method of H-d-monomere determination with the sensitivity of 2 ng/ml in double antibodies modification; this method was used for comparing the immunological affinity of the affiliated H-antigens. A difference between the immunological affinity to the antibodies in a monomere, polymere and the flagellum was shown.

  7. Immune responses to red blood cell antigens

    NARCIS (Netherlands)

    Stegmann, T.C.

    2016-01-01

    The research described in this thesis is aimed towards elucidation of the mechanism of action of anti-D. Anti-D is administered prophylactivly to prevent alloimmunization against the immunogenic D-antigen to D⁻ pregnant women carrying a D⁺ fetus. The plasma of women who became immunized during

  8. Antigen dynamics of follicular dendritic cells

    NARCIS (Netherlands)

    Heesters, B.A.

    2015-01-01

    Stromal-derived follicular dendritic cells (FDCs) are a major depot for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate and high-affinity antibody production takes place. Historically, FDCs have been characterized as ‘accessory’

  9. Evaluation of an Antigen-Antibody

    African Journals Online (AJOL)

    GB

    replication would lead to the production of various antigens. Today with BMT history of over 30 years, infection ... Study design: The study involved both retrospective and prospective laboratory-based analysis of ..... core protein of a molecular mass 19 x 103 Da, one picogram (pg) of virus core corresponds to 1.3 x. 105 HCV ...

  10. Lea blood group antigen on human platelets

    International Nuclear Information System (INIS)

    Dunstan, R.A.; Simpson, M.B.; Rosse, W.F.

    1985-01-01

    One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125 I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125 I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125 I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined

  11. Antigenic characterisation of lyssaviruses in South Africa

    Directory of Open Access Journals (Sweden)

    Ernest Ngoepe

    2014-09-01

    Full Text Available There are at least six Lyssavirus species that have been isolated in Africa, which include classical rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus and Ikoma lyssavirus. In this retrospective study, an analysis of the antigenic reactivity patterns of lyssaviruses in South Africa against a panel of 15 anti-nucleoprotein monoclonal antibodies was undertaken. A total of 624 brain specimens, collected between 2005 and 2009, confirmed as containing lyssavirus antigen by direct fluorescent antibody test, were subjected to antigenic differentiation. The lyssaviruses were differentiated into two species, namely rabies virus (99.5% and Mokola virus (0.5%. Furthermore, rabies virus was further delineated into two common rabies biotypes in South Africa: canid and mongoose. Initially, it was found that the canid rabies biotype had two reactivity patterns; differential staining was observed with just one monoclonal antibody. This difference was likely to have been an artefact related to sample quality, as passage in cell culture restored staining. Mongoose rabies viruses were more heterogeneous, with seven antigenic reactivity patterns detected. Although Mokola viruses were identified in this study, prevalence and reservoir host species are yet to be established. These data demonstrate the usefulness of monoclonal antibody typing panels in lyssavirus surveillance with reference to emergence of new species or spread of rabies biotypes to new geographic zones.

  12. Antigenic and genetic variability of human metapneumoviruses

    NARCIS (Netherlands)

    S. Herfst (Sander); L. Sprong; P.A. Cane; E. Forleo-Neto; A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); R.L. de Swart (Rik); B.G. van den Hoogen (Bernadette)

    2004-01-01

    textabstractHuman metapneumovirus (HMPV) is a member of the subfamily Pneumovirinae within the family Paramyxo- viridae. Other members of this subfamily, respiratory syncytial virus and avian pneumovirus, can be divided into subgroups on the basis of genetic or antigenic differences or both. For

  13. Understanding original antigenic sin in influenza with a dynamical system.

    Science.gov (United States)

    Pan, Keyao

    2011-01-01

    Original antigenic sin is the phenomenon in which prior exposure to an antigen leads to a subsequent suboptimal immune response to a related antigen. Immune memory normally allows for an improved and rapid response to antigens previously seen and is the mechanism by which vaccination works. I here develop a dynamical system model of the mechanism of original antigenic sin in influenza, clarifying and explaining the detailed spin-glass treatment of original antigenic sin. The dynamical system describes the viral load, the quantities of healthy and infected epithelial cells, the concentrations of naïve and memory antibodies, and the affinities of naïve and memory antibodies. I give explicit correspondences between the microscopic variables of the spin-glass model and those of the present dynamical system model. The dynamical system model reproduces the phenomenon of original antigenic sin and describes how a competition between different types of B cells compromises the overall effect of immune response. I illustrate the competition between the naïve and the memory antibodies as a function of the antigenic distance between the initial and subsequent antigens. The suboptimal immune response caused by original antigenic sin is observed when the host is exposed to an antigen which has intermediate antigenic distance to a second antigen previously recognized by the host's immune system.

  14. Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform.

    Directory of Open Access Journals (Sweden)

    Sara A Bumgardner

    Full Text Available Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785, L. acidophilus (strain NCFM, or NCFM-derived mutants-NCK2025 and NCK2031-elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2, the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2 receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL-1β in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1 Gag or membrane proximal external region (MPER, we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.

  15. Manipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin

    Directory of Open Access Journals (Sweden)

    Sara eLawrence

    2012-11-01

    Full Text Available The cholesterol-dependent cytolysins (CDCs attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modelling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases.

  16. Increasing vaccine potency through exosome antigen targeting.

    Science.gov (United States)

    Hartman, Zachary C; Wei, Junping; Glass, Oliver K; Guo, Hongtao; Lei, Gangjun; Yang, Xiao-Yi; Osada, Takuya; Hobeika, Amy; Delcayre, Alain; Le Pecq, Jean-Bernard; Morse, Michael A; Clay, Timothy M; Lyerly, Herbert K

    2011-11-21

    While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develop efficient TAA "cancer vaccines" using classical vaccine approaches have been largely ineffective. Recently, a process to specifically target proteins to exosomes has been established which takes advantage of the ability of the factor V like C1C2 domain of lactadherin to specifically address proteins to exosomes. Using this approach, we hypothesized that TAAs could be targeted to exosomes to potentially increase their immunogenicity, as exosomes have been demonstrated to traffic to antigen presenting cells (APC). To investigate this possibility, we created adenoviral vectors expressing the extracellular domain (ECD) of two non-mutated TAAs often found in tumors of cancer patients, carcinoembryonic antigen (CEA) and HER2, and coupled them to the C1C2 domain of lactadherin. We found that these C1C2 fusion proteins had enhanced expression in exosomes in vitro. We saw significant improvement in antigen specific immune responses to each of these antigens in naïve and tolerant transgenic animal models and could further demonstrate significantly enhanced therapeutic anti-tumor effects in a human HER2+ transgenic animal model. These findings demonstrate that the mode of secretion and trafficking can influence the immunogenicity of different human TAAs, and may explain the lack of immunogenicity of non-mutated TAAs found in cancer patients. They suggest that exosomal targeting could enhance future anti-tumor vaccination protocols. This targeting exosome process could also be adapted for the development of more potent vaccines in some viral and parasitic diseases where the classical vaccine approach has demonstrated limitations. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Computational and Experimental Validation of B and T-Cell Epitopes of the In Vivo Immune Response to a Novel Malarial Antigen

    Science.gov (United States)

    2013-08-16

    KRATOS software package to determine which peptides were positive for binding with the immune antibodies. Immunizations Recombinant PfCelTOS with...Improved technique for the preparation of water-in-oil emulsions containing protein antigens. BioTechniques 20: 797–800. 34. Greenbaum JA, Andersen PH

  18. Energetic changes caused by antigenic module insertion in a virus-like particle revealed by experiment and molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Lin Zhang

    Full Text Available The success of recombinant virus-like particles (VLPs for human papillomavirus and hepatitis B demonstrates the potential of VLPs as safe and efficacious vaccines. With new modular designs emerging, the effects of antigen module insertion on the self-assembly and structural integrity of VLPs should be clarified so as to better enabling improved design. Previous work has revealed insights into the molecular energetics of a VLP subunit, capsomere, comparing energetics within various solution conditions known to drive or inhibit self-assembly. In the present study, molecular dynamics (MD simulations coupled with the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA method were performed to examine the molecular interactions and energetics in a modular capsomere of a murine polyomavirus (MPV VLP designed to protect against influenza. Insertion of an influenza antigenic module is found to lower the binding energy within the capsomere, and a more active state is observed in Assembly Buffer as compared with that in Stabilization Buffer, which has been experimentally validated through measurements using differential scanning calorimetry. Further in-depth analysis based on free-energy decomposition indicates that destabilized binding can be attributed to electrostatic interaction induced by the chosen antigen module. These results provide molecular insights into the conformational stability of capsomeres and their abilities to be exploited for antigen presentation, and are expected to be beneficial for the biomolecular engineering of VLP vaccines.

  19. Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma

    International Nuclear Information System (INIS)

    Kato, H.; Torigoe, T.

    1977-01-01

    A heterologous antiserum for human cervical squamous cell carcinoma was prepared and specificity determined by Ouchterlony immunodiffusion and immunofluorescence studies. With this antiserum, a tumor antigen was purified from human cervical squamous cell carcinoma tissue. The specificities of the antigen and the antiserum were then re-examined by a radioimmunoassay method using 125 I-labeled purified antigen. Although normal cervical tissue extract showed a moderate cross-reactivity in the radioimmunoassay, the circulating antigen activity could not be detected in normal women or in several patients with other carcinomas, whereas 27 of 35 patients with cervical squamous cell carcinoma showed detectable serum antigen activity. All patients with advanced stages of cervical squamous cell carcinoma showed detectable antigen levels. These results indicate that there is a quantitative abnormality, at least, of this tumor antigen in patients with cervical squamous cell carcinoma and that the radioimmunoassay for the antigen is a potentially useful tool in clinical care

  20. Evaluating the use of dedicated swab for rapid antigen detection ...

    African Journals Online (AJOL)

    Evaluating the use of dedicated swab for rapid antigen detection testing in group a ... African Journal of Clinical and Experimental Microbiology ... Several generations of rapid antigen detection tests (RADTs) have been developed to facilitate ...

  1. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    DEFF Research Database (Denmark)

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt

    2011-01-01

    En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner.......En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner....

  2. Dengue virus-like particles mimic the antigenic properties of the infectious dengue virus envelope.

    Science.gov (United States)

    Metz, Stefan W; Thomas, Ashlie; White, Laura; Stoops, Mark; Corten, Markus; Hannemann, Holger; de Silva, Aravinda M

    2018-04-02

    The 4 dengue serotypes (DENV) are mosquito-borne pathogens that are associated with severe hemorrhagic disease. DENV particles have a lipid bilayer envelope that anchors two membrane glycoproteins prM and E. Two E-protein monomers form head-to-tail homodimers and three E-dimers align to form "rafts" that cover the viral surface. Some human antibodies that strongly neutralize DENV bind to quaternary structure epitopes displayed on E protein dimers or higher order structures forming the infectious virus. Expression of prM and E in cell culture leads to the formation of DENV virus-like particles (VLPs) which are smaller than wildtype virus particles and replication defective due to the absence of a viral genome. There is no data available that describes the antigenic landscape on the surface of flavivirus VLPs in comparison to the better studied infectious virion. A large panel of well characterized antibodies that recognize epitope of ranging complexity were used in biochemical analytics to obtain a comparative antigenic surface view of VLPs in respect to virus particles. DENV patient serum depletions were performed the show the potential of VLPs in serological diagnostics. VLPs were confirmed to be heterogeneous in size morphology and maturation state. Yet, we show that many highly conformational and quaternary structure-dependent antibody epitopes found on virus particles are efficiently displayed on DENV1-4 VLP surfaces as well. Additionally, DENV VLPs can efficiently be used as antigens to deplete DENV patient sera from serotype specific antibody populations. This study aids in further understanding epitopic landscape of DENV VLPs and presents a comparative antigenic surface view of VLPs in respect to virus particles. We propose the use VLPs as a safe and practical alternative to infectious virus as a vaccine and diagnostic antigen.

  3. Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

    Directory of Open Access Journals (Sweden)

    Schendel Dolores J

    2010-09-01

    Full Text Available Abstract Background Antigen-loaded dendritic cells (DC are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility. Methods In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC compared with conventional DC prepared in seven days (7d mDC, which represent the most common form of DC used for vaccines to date. Results Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivtRNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivtRNA. Conclusions This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivtRNA as sources of specific antigen.

  4. Cell membrane antigen-antibody complex dissociation by the widely used glycine-HCL method: an unreliable procedure for studying antibody internalization.

    Science.gov (United States)

    Tsaltas, G; Ford, C H

    1993-02-01

    Methods following the process of binding and internalization of antibodies to cell surface antigens have often employed low pH isoosmolar buffers in order to dissociate surface antigen-antibody complexes. One of the most widely used buffers is a 0.05 M glycine-HCL buffer pH 2.8. Since the efficacy of action of this buffer was critical to a series of internalization experiments employing monoclonal antibodies (Mabs) to carcinoembryonic antigen (CEA) expressing cancer cell lines in this laboratory, we tested its performance in a number of different assays. Our results indicate that this buffer only partially dissociates antigen-antibody bonds and therefore can introduce major inaccuracies in internalization experiments.

  5. Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection

    Directory of Open Access Journals (Sweden)

    Awobode Henrietta O

    2009-11-01

    Full Text Available Abstract Background MSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP119, inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP119 had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP119 would affect critical T-cell responses to epitopes in this antigen. Methods The cellular responses to wild-type MSP119 and a panel of modified MSP119 antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults. Results Interestingly, stimulation indices (SI for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP119. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu had the highest stimulation index (SI up to 360 and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins. Conclusion This study suggests that specific MSP119 variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.

  6. Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

    Science.gov (United States)

    Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

    2004-10-01

    Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

  7. Identification of antigenic Sarcoptes scabiei proteins for use in a diagnostic test and of non-antigenic proteins that may be immunomodulatory

    Science.gov (United States)

    Morgan, Marjorie S.; Rider, S. Dean; Arlian, Larry G.

    2017-01-01

    Background Scabies, caused by the mite, Sarcoptes scabiei, infects millions of humans, and many wild and domestic mammals. Scabies mites burrow in the lower stratum corneum of the epidermis of the skin and are the source of substances that are antigenic or modulate aspects of the protective response of the host. Ordinary scabies is a difficult disease to diagnose. Objective The goal of this project was to identify S. scabiei proteins that may be candidate antigens for use in a diagnostic test or may be used by the mite to modulate the host’s protective response. Methods An aqueous extract of S. scabiei was separated by 2-dimensional electrophoresis and proteins were identified by mass spectrometry. A parallel immunoblot was probed with serum from patients with ordinary scabies to identify IgM and/or IgG-binding antigens. The genes coding for 23 selected proteins were cloned into E. coli and the expressed recombinant proteins were screened with serum from patients with confirmed ordinary scabies. Results We identified 50 different proteins produced by S. scabiei, 34 of which were not previously identified, and determined that 66% were recognized by patient IgM and/or IgG. Fourteen proteins were screened for use in a diagnostic test but none possessed enough sensitivity and specificity to be useful. Six of the 9 proteins selected for the possibility that they may be immunomodulatory were not recognized by antibodies in patient serum. Conclusions Thirty-three proteins that bound IgM and/or IgG from the serum of patients with ordinary scabies were identified. None of the 14 tested were useful for inclusion in a diagnostic test. The identities of 16 proteins that are not recognized as antigens by infected patients were also determined. These could be among the molecules that are responsible for this mite’s ability to modulate its host’s innate and adaptive immune responses. PMID:28604804

  8. Identification of antigenic Sarcoptes scabiei proteins for use in a diagnostic test and of non-antigenic proteins that may be immunomodulatory.

    Directory of Open Access Journals (Sweden)

    Marjorie S Morgan

    2017-06-01

    Full Text Available Scabies, caused by the mite, Sarcoptes scabiei, infects millions of humans, and many wild and domestic mammals. Scabies mites burrow in the lower stratum corneum of the epidermis of the skin and are the source of substances that are antigenic or modulate aspects of the protective response of the host. Ordinary scabies is a difficult disease to diagnose.The goal of this project was to identify S. scabiei proteins that may be candidate antigens for use in a diagnostic test or may be used by the mite to modulate the host's protective response.An aqueous extract of S. scabiei was separated by 2-dimensional electrophoresis and proteins were identified by mass spectrometry. A parallel immunoblot was probed with serum from patients with ordinary scabies to identify IgM and/or IgG-binding antigens. The genes coding for 23 selected proteins were cloned into E. coli and the expressed recombinant proteins were screened with serum from patients with confirmed ordinary scabies.We identified 50 different proteins produced by S. scabiei, 34 of which were not previously identified, and determined that 66% were recognized by patient IgM and/or IgG. Fourteen proteins were screened for use in a diagnostic test but none possessed enough sensitivity and specificity to be useful. Six of the 9 proteins selected for the possibility that they may be immunomodulatory were not recognized by antibodies in patient serum.Thirty-three proteins that bound IgM and/or IgG from the serum of patients with ordinary scabies were identified. None of the 14 tested were useful for inclusion in a diagnostic test. The identities of 16 proteins that are not recognized as antigens by infected patients were also determined. These could be among the molecules that are responsible for this mite's ability to modulate its host's innate and adaptive immune responses.

  9. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

  10. A radioimmunoassay for antibodies against surface membrane antigens using adhering cells

    International Nuclear Information System (INIS)

    Tax, A.; Manson, L.A.

    1976-01-01

    A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit 125 I-antimouse-Fab. The bound radioactive material was eluted with glycine-HCl buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolic or noncytolic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface

  11. 21 CFR 660.40 - Hepatitis B Surface Antigen.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

  12. Potential radioimmunoassay system for detection of Hanganutziu-Deicher type heterophile antigen(s) and antibodies in tissues and fluids

    Energy Technology Data Exchange (ETDEWEB)

    Mukuria, J C; Naiki, Masaharu; Hashimoto, Masato; Nishiura, Katsumi; Okabe, Masahiro; Kato, Shiro

    1985-06-12

    A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The SVI-labeled H-D antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D antigen potency. Different H-D antigen-active molecules were compared for heterophile H-D antigen potency. Eight different lung cancer tissues were assayed for H-D antigen. The sera from the 8 lung cancer patients were also screened by ELISA and RIA in an attmept to correlate expression of H-D antigen on tissues with elevation of H-D antibodies.

  13. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    International Nuclear Information System (INIS)

    Kim, Sun Young; Song, Kyung-A; Kieff, Elliott; Kang, Myung-Soo

    2012-01-01

    Highlights: ► Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. ► A small molecule and a peptide as EBNA1 dimerization inhibitors identified. ► Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. ► Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)’s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459–607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-Jκ binding to the Jκ site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560–574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two

  14. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Young; Song, Kyung-A [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Kieff, Elliott [Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Kang, Myung-Soo, E-mail: mkang@skku.edu [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated

  15. Sequential memory: Binding dynamics

    Science.gov (United States)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

  16. Cellulose binding domain proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  17. Binding and Bulgarian

    NARCIS (Netherlands)

    Schürcks-Grozeva, Lilia Lubomirova

    2003-01-01

    In haar proefschrift analyseert Lilia Schürcks de anaforische verschijnselen in de Bulgaarse taal. Het gaat dan om wederkerende aspecten, uitgedrukt bij woorden als ‘zich’ en ‘elkaar’. De situatie in het Bulgaars blijkt moeilijk in te passen in de klassieke Binding Theory van Noam Chomsky. Bron: RUG

  18. NetMHCpan 4.0: Improved peptide-MHC class I interaction predictions integrating eluted ligand and peptide binding affinity data

    OpenAIRE

    Jurtz, Vanessa; Paul, Sinu; Andreatta, Massimo; Marcatili, Paolo; Peters, Bjoern; Nielsen, Morten

    2017-01-01

    Cytotoxic T cells are of central importance in the immune systems response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC (major histocompatibility complex) class I molecules. Peptide binding to MHC molecules is the single most selective step in the antigen presentation pathway. On the quest for T cell epitopes, the prediction of peptide binding to MHC molecules has therefore attracted large attention. In the past, predictors of peptide-...

  19. Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Yu, Ruijin [College of Science, Northwest A& F University, Yangling, Shaanxi 712100 (China); Lai, Weihua [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China)

    2017-06-15

    Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC{sub 50} value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

  20. Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

    International Nuclear Information System (INIS)

    Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin; Yu, Ruijin; Lai, Weihua; Xiong, Yonghua

    2017-01-01

    Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC_5_0 value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

  1. The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

    Directory of Open Access Journals (Sweden)

    Blaise Ndjamen

    2014-03-01

    Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

  2. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  3. Computational elucidation of potential antigenic CTL epitopes in Ebola virus.

    Science.gov (United States)

    Dikhit, Manas R; Kumar, Santosh; Vijaymahantesh; Sahoo, Bikash R; Mansuri, Rani; Amit, Ajay; Yousuf Ansari, Md; Sahoo, Ganesh C; Bimal, Sanjiva; Das, Pradeep

    2015-12-01

    Cell-mediated immunity is important for the control of Ebola virus infection. We hypothesized that those HLA A0201 and HLA B40 restricted epitopes derived from Ebola virus proteins, would mount a good antigenic response. Here we employed an immunoinformatics approach to identify specific 9mer amino acid which may be capable of inducing a robust cell-mediated immune response in humans. We identified a set of 28 epitopes that had no homologs in humans. Specifically, the epitopes derived from NP, RdRp, GP and VP40 share population coverage of 93.40%, 84.15%, 74.94% and 77.12%, respectively. Based on the other HLA binding specificity and population coverage, seven novel promiscuous epitopes were identified. These 7 promiscuous epitopes from NP, RdRp and GP were found to have world-wide population coverage of more than 95% indicating their potential significance as useful candidates for vaccine design. Epitope conservancy analysis also suggested that most of the peptides are highly conserved (100%) in other virulent Ebola strain (Mayinga-76, Kikwit-95 and Makona-G3816- 2014) and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Stereophysicochemical variability plots highlight conserved antigenic areas in Flaviviruses

    Directory of Open Access Journals (Sweden)

    Zhou Bin

    2005-04-01

    Full Text Available Abstract Background Flaviviruses, which include Dengue (DV and West Nile (WN, mutate in response to immune system pressure. Identifying escape mutants, variant progeny that replicate in the presence of neutralizing antibodies, is a common way to identify functionally important residues of viral proteins. However, the mutations typically occur at variable positions on the viral surface that are not essential for viral replication. Methods are needed to determine the true targets of the neutralizing antibodies. Results Stereophysicochemical variability plots (SVPs, 3-D images of protein structures colored according to variability, as determined by our PCPMer program, were used to visualize residues conserved in their physical chemical properties (PCPs near escape mutant positions. The analysis showed 1 that escape mutations in the flavivirus envelope protein are variable residues by our criteria and 2 two escape mutants found at the same position in many flaviviruses sit above clusters of conserved residues from different regions of the linear sequence. Conservation patterns in T-cell epitopes in the NS3- protease suggest a similar mechanism of immune system evasion. Conclusion The SVPs add another dimension to structurally defining the binding sites of neutralizing antibodies. They provide a useful aid for determining antigenically important regions and designing vaccines.

  5. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    International Nuclear Information System (INIS)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin

    2014-01-01

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

  6. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  7. HLA antigens in juvenile onset diabetes.

    Science.gov (United States)

    Kikuchi, T; Toyota, T; Ouchi, E

    1980-11-01

    To study association between juvenile onset diabetes (JOD) and major histocompatibility gene complex, 40 patients with childhood onset diabetes and 120 healthy subjects were typed for HLA. Bw54 was present in 33 percent of the patients with JOD, while it appeared in 8 percent of the controls. Expressed as a relative risk, the antigen Bw54 confers a susceptibility to the development of JOD which is 5.3 times that in the controls. JOD shows a little high degree of association with A9 (78%). However, the A9-antigen is common in the Japanese and appears in 58 percent. Though less striking, the decreased frequency of B12 was 3 percent of JOD, less than 15 percent of the controls (p less than 0.05). There was no association between Bw54 and JOD with family history of diabetes.

  8. Radionuclide-labelled antigens in serological epidemiology

    International Nuclear Information System (INIS)

    Felsenfeld, O.; Parrott, M.W.

    1977-01-01

    The feasibility of tests using radionuclide-labelled antigens in serological surveys was studied, with particular attention to the likely availability of facilities and personnel in the tropics and arctics, where measurements may be disturbed by climatic influences. The methodology required was to be simple, rapid and suitable for examining large numbers of sera, as for epidemological surveys. In the introduction, limitations of labelled antigen tests are discussed, the choice of radionuclide and measurement methods, test procedures and evaluation of results. Collection, preservation and shipment of speciments (serum, faeces, cerebrospinal fluid, sputum, etc.) are described. Experiments with bacteria and bacterial toxins (Enterobacteriaceae, vibrios, staphylococci, meningococci, etc.), with protozoa and metazoa (Entamoeba hystolytica, Schistosoma mansoni, Trypanosoma cruzi, Plasmodia and other parasites), with viruses (vaccinia, adeno-, polio-, and influenza viruses, etc.), and with fungi are discussed

  9. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    International Nuclear Information System (INIS)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-01-01

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

  10. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

  11. Novel Drosophila receptor that binds multiple growth factors

    International Nuclear Information System (INIS)

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-01-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10 -6 to 10 -8 M. The 100 kDa protein can be affinity-labeled with these 125 I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by 125 I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors

  12. A competitive-inhibiton radioimmunoassay for influenza virus envelope antigens

    International Nuclear Information System (INIS)

    Russ, G.; Styk, B.; Vareckova, E.; Polakova, K.

    1976-01-01

    A double-antibody competitive-inhibition radioimmunoassay for influenza virus envelope antigens is described. A viral antigen preparation from influenza A virus recombinant MRC11 [antigenically identical to A/Port Chalmers/1/73 (H3N2)] consisting of haemagglutinin and neuraminidase was labelled with radioiodine. Rabbit antisera were allowed to react with the labelled antigen and the resultant antigen-antibody complexes were precipitated with the appropriate antiglobulin. The competitive-inhibition radioimmunoassay very sensitively elucidated differences even among closely related influenza virus strains. Attempts have been made to eliminate neuraminidase from radioimmunoprecipitation to obtain a competitive-inhibition radioimmunoassay system for haemagglutinin alone. (author)

  13. Mannan-binding lectin activates C3 and the

    DEFF Research Database (Denmark)

    Selander, B.; Martensson, U.; Weintraub, A.

    2006-01-01

    Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL) or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera...... and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen-specific oligosaccharides derived from Salmonella typhimurium, S...

  14. Binding induced conformational changes of proteins correlate with their intrinsic fluctuations: a case study of antibodies

    Directory of Open Access Journals (Sweden)

    Keskin Ozlem

    2007-05-01

    Full Text Available Abstract Background How antibodies recognize and bind to antigens can not be totally explained by rigid shape and electrostatic complimentarity models. Alternatively, pre-existing equilibrium hypothesis states that the native state of an antibody is not defined by a single rigid conformation but instead with an ensemble of similar conformations that co-exist at equilibrium. Antigens bind to one of the preferred conformations making this conformation more abundant shifting the equilibrium. Results Here, two antibodies, a germline antibody of 36–65 Fab and a monoclonal antibody, SPE7 are studied in detail to elucidate the mechanism of antibody-antigen recognition and to understand how a single antibody recognizes different antigens. An elastic network model, Anisotropic Network Model (ANM is used in the calculations. Pre-existing equilibrium is not restricted to apply to antibodies. Intrinsic fluctuations of eight proteins, from different classes of proteins, such as enzymes, binding and transport proteins are investigated to test the suitability of the method. The intrinsic fluctuations are compared with the experimentally observed ligand induced conformational changes of these proteins. The results show that the intrinsic fluctuations obtained by theoretical methods correlate with structural changes observed when a ligand is bound to the protein. The decomposition of the total fluctuations serves to identify the different individual modes of motion, ranging from the most cooperative ones involving the overall structure, to the most localized ones. Conclusion Results suggest that the pre-equilibrium concept holds for antibodies and the promiscuity of antibodies can also be explained this hypothesis: a limited number of conformational states driven by intrinsic motions of an antibody might be adequate to bind to different antigens.

  15. Changes in antigenicity of porcine serum albumin in gamma-irradiated sausage extract by treatment with pepsin and trypsin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Koth-Bong-Woo-Ri; Song, Eu-Jin [Department of Food Science and Technology/Institute of Food Science, Pukyong National University, Busan 608-737 (Korea, Republic of); Lee, So-Young [Traditional Food Research Group, Korea Food Research Institute, Seongnam 463-746 (Korea, Republic of); Park, Jin-Gyu [Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongup 580-185 (Korea, Republic of); Lee, Ju-Woon [National Federation of Fisheries Cooperatives, Fisheries Economic Institute, Seoul 138-827 (Korea, Republic of); Byun, Myung-Woo [Department of Culinary Nutrition, Woosong University, Daejon 300-718 (Korea, Republic of); Ahn, Dong-Hyun, E-mail: dhahn@pknu.ac.kr [Department of Food Science and Technology/Institute of Food Science, Pukyong National University, Busan 608-737 (Korea, Republic of)

    2011-11-15

    Pork is known as an allergenic food with porcine serum albumin (PSA, 66 kDa) representing the major allergen. This study was conducted to investigate the change in antigenicity of PSA in gamma-irradiated sausage extract treated with pepsin and trypsin. Sausage products (A and B) were irradiated at 1, 3, 10, and 20 kGy. After irradiation, sausage proteins were extracted and digested with pepsin (1:200, 30 min) and trypsin (1:300, 5, 30, 60, 90, and 120 min). The binding ability of PSA in extracts of the irradiated sausages (A and B) decreased by over 3 kGy relative to the binding ability of PSA in extracts of intact sausages and showed no notable differences when the dose of radiation ranged from 3 to 20 kGy. After treatment with pepsin and trypsin, the binding ability of PSA in extracts of the irradiated sausages was decreased more relative to that of intact sausages and showed no significant differences when the period of trypsin treatment is increased or when the dose of irradiation is increased. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that there was no visible change in the intensity of the PSA band in extracts of the irradiated sausages. After pepsin and trypsin treatment, the intensity of PSA band faded with increasing doses of irradiation. In conclusion, antigenicity of PSA in pork sausages could be reduced by gamma irradiation. - Highlights: > Change in antigenicity of PSA in irradiated sausage extract (ISE) was examined. > Binding ability of PSA in ISE was decreased compared to intact extract. > Binding ability of PSA in ISE after enzyme treatments was also further decreased. > Intensity of PSA band in ISE after enzyme treatments became weak.

  16. Changes in antigenicity of porcine serum albumin in gamma-irradiated sausage extract by treatment with pepsin and trypsin

    International Nuclear Information System (INIS)

    Kim, Koth-Bong-Woo-Ri; Song, Eu-Jin; Lee, So-Young; Park, Jin-Gyu; Lee, Ju-Woon; Byun, Myung-Woo; Ahn, Dong-Hyun

    2011-01-01

    Pork is known as an allergenic food with porcine serum albumin (PSA, 66 kDa) representing the major allergen. This study was conducted to investigate the change in antigenicity of PSA in gamma-irradiated sausage extract treated with pepsin and trypsin. Sausage products (A and B) were irradiated at 1, 3, 10, and 20 kGy. After irradiation, sausage proteins were extracted and digested with pepsin (1:200, 30 min) and trypsin (1:300, 5, 30, 60, 90, and 120 min). The binding ability of PSA in extracts of the irradiated sausages (A and B) decreased by over 3 kGy relative to the binding ability of PSA in extracts of intact sausages and showed no notable differences when the dose of radiation ranged from 3 to 20 kGy. After treatment with pepsin and trypsin, the binding ability of PSA in extracts of the irradiated sausages was decreased more relative to that of intact sausages and showed no significant differences when the period of trypsin treatment is increased or when the dose of irradiation is increased. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicated that there was no visible change in the intensity of the PSA band in extracts of the irradiated sausages. After pepsin and trypsin treatment, the intensity of PSA band faded with increasing doses of irradiation. In conclusion, antigenicity of PSA in pork sausages could be reduced by gamma irradiation. - Highlights: → Change in antigenicity of PSA in irradiated sausage extract (ISE) was examined. → Binding ability of PSA in ISE was decreased compared to intact extract. → Binding ability of PSA in ISE after enzyme treatments was also further decreased. → Intensity of PSA band in ISE after enzyme treatments became weak.

  17. Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

    Directory of Open Access Journals (Sweden)

    Edith S Málaga-Machaca

    2017-11-01

    Full Text Available Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease.Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA.Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

  18. Ultraviolet light-induced suppression of antigen presentation

    International Nuclear Information System (INIS)

    Spellman, C.W.; Tomasi, T.B.

    1983-01-01

    Ultraviolet (UV) light irradiation of animals results in the development of specific T suppressor cells that inhibit antitumor immune responses. It is thought that suppression may arise as a consequence of altered antigen presentation by UV-irradiated epidermal cells. This hypothesis is based on evidence demonstrating that specific lymphoid tissues from UV-irradiated hosts exhibit impaired antigen-presenting function and that animals cannot be contact sensitized when antigens are applied to a UV-irradiated skin site. Langerhans cells of the skin are likely candidates as targets of UV-induced defects in antigen presentation as they bear Fc and C3b receptors, express Ia antigens, are of bone marrow origin, and are capable of presenting antigen in vitro. We speculate on the possible clinical usefulness of UV-induced tolerance to specific antigens such as those encountered in monoclonal antibody therapy and tissue transplantation

  19. Review of Mycobacteriumavium subsp. paratuberculosis antigen candidates with diagnostic potential

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose

    2011-01-01

    antigens, heat shock antigens and hypothetical antigens. Strategies for evaluation of novel antigen candidates are discussed critically. Relatively few of the described antigens were evaluated for their use in CMI based diagnostic assays and so far, no obvious candidate has been identified...... to development of antibodies and shedding of detectable amounts of MAP. At present, available diagnostic assays are limited by the lack of MAP specific antigens included in these assays resulting in poor specificity. The objective of this review is to provide a systematic overview of diagnostic MAP antigen...... faeces; however, these diagnostic tools are often not applicable until years after infection. Detection of MAP specific cell-mediated immune (CMI) responses can serve as an alternative and be implemented in a diagnostic tool. CMI responses can be measured at an early stage of infection, prior...

  20. Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1.

    Science.gov (United States)

    Knight, J M; Zingales, B; Bottazzi, M E; Hotez, P; Zhan, B

    2014-12-01

    Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N-terminal 65 kDa portion of Trypomastigote surface antigen-1 (TSA-1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA-1 has not previously been addressed. To assess the variation in TSA-1, we cloned and sequenced TSA-1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA-1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA-A 02 allele suggests that interlineage variation in TSA-1 would not impair the range and efficacy of a vaccine containing TSA-1. © 2014 John Wiley & Sons Ltd.

  1. Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

    Science.gov (United States)

    Han, Zhe-Yi; Wu, Kai-Chun; He, Feng-Tian; Han, Quan-Li; Nie, Yong-Zhan; Han, Ying; Liu, Xiao-Nan; Zheng, Jian-Yong; Xu, Mei-Hong; Lin, Tao; Fan, Dai-Ming

    2003-01-01

    AIM: Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pIII in order to get some information on mimotopes. METHODS: Through affinity enrichment and ELISA screening, positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced) By blocking test, specificity of the mimic phage epitopes was identified. RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced. According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-III. The percentage of blocking was from (21.0 ± 1.6)% to (39.0 ± 2.7)%. CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines. PMID:12970876

  2. Neuronal surface antigen antibodies in limbic encephalitis

    Science.gov (United States)

    Graus, F; Saiz, A; Lai, M; Bruna, J; López, F; Sabater, L; Blanco, Y; Rey, M J.; Ribalta, T; Dalmau, J

    2008-01-01

    Objective: To report the frequency and type of antibodies against neuronal surface antigens (NSA-ab) in limbic encephalitis (LE). Methods: Analysis of clinical features, neuropathologic findings, and detection of NSA-ab using immunochemistry on rat tissue and neuronal cultures in a series of 45 patients with paraneoplastic (23) or idiopathic (22) LE. Results: NSA-ab were identified in 29 patients (64%; 12 paraneoplastic, 17 idiopathic). Thirteen patients had voltage-gated potassium channels (VGKC)-ab, 11 novel NSA (nNSA)-ab, and 5 NMDA receptor (NMDAR)-ab. nNSA-ab did not identify a common antigen and were more frequent in paraneoplastic than idiopathic LE (39% vs 9%; p = 0.03). When compared with VGKC-ab or NMDAR-ab, the nNSA associated more frequently with intraneuronal antibodies (11% vs 73%; p = 0.001). Of 12 patients (9 nNSA-ab, 2 VGKC-ab, 1 NMDAR-ab) with paraneoplastic LE and NSA-ab, concomitant intraneuronal antibodies occurred in 9 (75%). None of these 12 patients improved with immunotherapy. The autopsy of three of them showed neuronal loss, microgliosis, and cytotoxic T cell infiltrates in the hippocampus and amygdala. These findings were compatible with a T-cell mediated neuronal damage. In contrast, 13 of 17 (76%) patients with idiopathic LE and NSA-ab (8 VGKC-ab, 4 NMDAR-ab, 1 nNSA-ab) and 1 of 5 (20%) without antibodies had clinical improvement (p = 0.04). Conclusions: In paraneoplastic limbic encephalitis (LE), novel antibodies against neuronal surface antigens (nNSA-ab) occur frequently, coexist with antibodies against intracellular antigens, and these cases are refractory to immunotherapy. In idiopathic LE, the likelihood of improvement is significantly higher in patients with NSA-ab than in those without antibodies. GLOSSARY GAD = glutamic acid decarboxylase; LE = limbic encephalitis; NMDAR = N-methyl-D-aspartate receptor; NSA = neuronal surface antigens; nNSA = novel NSA; SCLC = small-cell lung cancer; VGKC = voltage-gated potassium channels

  3. Improved pan-specific MHC class I peptide-binding predictions using a novel representation of the MHC-binding cleft environment

    DEFF Research Database (Denmark)

    Carrasco Pro, S.; Zimic, M.; Nielsen, Morten

    2014-01-01

    of the current state-of-the-art methods for MHC class I is NetMHCpan, which has a core ingredient for the representation of the MHC class I molecule using a pseudo-sequence representation of the binding cleft amino acid environment. New and large MHC-peptide-binding data sets are constantly being made available...... of different MHC data sets including human leukocyte antigen (HLA), non-human primates (chimpanzee, macaque and gorilla) and other animal alleles (cattle, mouse and swine). From these constructs, we showed that by focusing on MHC sequence positions found to be polymorphic across the MHC molecules used to train...

  4. In silico and cell-based analyses reveal strong divergence between prediction and observation of T-cell-recognized tumor antigen T-cell epitopes.

    Science.gov (United States)

    Schmidt, Julien; Guillaume, Philippe; Dojcinovic, Danijel; Karbach, Julia; Coukos, George; Luescher, Immanuel

    2017-07-14

    Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

    Science.gov (United States)

    Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

    2014-02-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

  6. Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens.

    Science.gov (United States)

    Restrepo, B I; Obregón-Henao, A; Mesa, M; Gil, D L; Ortiz, B L; Mejía, J S; Villota, G E; Sanzón, F; Teale, J M

    2000-05-01

    Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

  7. Limitations of the radioimmunoprecipitation polyethylene glycol assay (RIPEGA) for detection of filarial antigens in serum

    International Nuclear Information System (INIS)

    Hamilton, R.G.; Alexander, E.; Adkinson, N.F.

    1984-01-01

    The performance of the radioimmunoprecipitation polyethylene glycol assay (RIPEGA) was examined for quantitation of filarial antigens (Brugia malayi and Dirofilaria immitis) in serum from infected human and animal hosts and non-infected controls. Multiple PEG concentrations were employed to determine the level of non-specific binding (NSB) in non-exposed human sera (NEHS) containing no filarial antigen. The NSB observed when 3 different 125 I-labelled IgG antibodies were added to 26 NEHS varied 3-fold and was correlated significantly with total serum IgM (r = 0.80, P 2 fragment of the 125 I-labelled antibody was used, but the correlation of NSB with total serum IgM remained significant (r = 0.57, P < 0.01). The presence of rheumatoid factor in NEHS sera also significantly increased NSB by an average of 3-fold. These effects eliminated the assay's ability to detect in sera from infected hosts filarial antigen the presence of which could be readily demonstrated by an immunoradiometric assay. The RIPEGA's precision (intra-assay coefficient of variation (CV) = 21% at 35% Bsub(max)) and reproducibility (inter-assay CV = 29% at 35% Bsub(max)) are less satisfactory than many alternative immunoassays. (Auth.)

  8. High-throughput identification of potential minor histocompatibility antigens by MHC tetramer-based screening

    DEFF Research Database (Denmark)

    Hombrink, Pleun; Hadrup, Sine R; Bakker, Arne

    2011-01-01

    the technical feasibility of high-throughput analysis of antigen-specific T-cell responses in small patient samples. However, the high-sensitivity of this approach requires the use of potential epitope sets that are not solely based on MHC binding, to prevent the frequent detection of T-cell responses that lack......T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T......MHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1(IMA) antigen demonstrates that identification of MiHA through this approach is in principle...

  9. TIA-1 RRM23 binding and recognition of target oligonucleotides.

    Science.gov (United States)

    Waris, Saboora; García-Mauriño, Sofía M; Sivakumaran, Andrew; Beckham, Simone A; Loughlin, Fionna E; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C J; Wilce, Jacqueline A

    2017-05-05

    TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Immunochemical similarity of GTP-binding proteins from different systems

    International Nuclear Information System (INIS)

    Kalinina, S.N.

    1986-01-01

    It was found that antibodies against the GTP-binding proteins of bovine retinal photoreceptor membranes blocked the inhibitory effect of estradiol on phosphodiesterase from rat and human uterine cytosol and prevented the cumulative effect of catecholamines and guanylyl-5'-imidodiphosphate on rat skeletal muscle adenylate cyclase. It was established by means of double radial immunodiffusion that these antibodies form a precipitating complex with purified bovine brain tubulin as well as with retinal preparations obtained from eyes of the bull, pig, rat, frog, some species of fish, and one reptile species. Bands of precipitation were not observed with these antibodies when retinal preparations from invertebrates (squid and octopus) were used as the antigens. The antibodies obtained interacted with the α- and β-subunits of GTP-binding proteins from bovine retinal photoreceptor membranes

  11. Molecular Characteristics of Carcinoembryonic Antigen and Nonspecific Cross-reacting Antigen(Clinical Application of Tumor Antigen)

    OpenAIRE

    内山, 一晃; Uchiyama, Kazuaki

    1990-01-01

    Carcinoembryonic antigen (CEA) is one of the most famous laboratory tests of tumor markers. CEA was first reported in 1965, but molecular structure of CEA was not clear untill recent years. Amino acid sequence of CEA was reported in 1987, by the success of cDNA clonig of CEA. The CEA molecule is composed of five major domains, called domain N, I, II, III, C from the -NH_2 terminal. But sugar chains of CEA are complicated and have much variety, so there are few informations about them. If CEA ...

  12. Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

    Science.gov (United States)

    Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

    2016-07-01

    Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Mohammed, M. E. A.

    2010-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Mohammed, M E. A. [University of Khartoum, Khartoum (Sudan)

    2010-02-15

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

  15. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

    Directory of Open Access Journals (Sweden)

    Lorenzo Asti

    2016-04-01

    Full Text Available The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6, outperforming other sequence- and structure-based models.

  16. Relationship between laminin binding capacity and laminin expression on tumor cells sensitive or resistant to natural cell-mediated cytotoxicity

    International Nuclear Information System (INIS)

    Laybourn, K.A.; Varani, J.; Fligiel, S.E.G.; Hiserodt, J.C.

    1986-01-01

    Previous studies have identified the presence of laminin binding sites on murine NK and NC sensitive tumor cells by 125 I-laminin binding and laminin induced cell-cell aggregation. The finding that the addition of exogenous laminin inhibits NK/NC binding to sensitive tumor cells suggests laminin binding sites may serve as target antigens for NK cells. The present study extends earlier reports by analyzing a large panel of tumor cells for laminin binding capacity, laminin expression and sensitivity to NK/NC killing. The data indicate that all tumor cells which bind to NK/NC cells (8 lines tested) express laminin binding sites. All of these tumor cells were capable of competing for NK lysis of YAC-1 cells in cold target competition assays, and all bound enriched NK cells in direct single cell binding assays. In contrast, tumor cells expressing high levels of surface laminin (B16 melanomas, C57B1/6 fibrosarcomas, and RAS transfected 3T3 fibroblasts) but low levels of laminin binding capacity did not bind NK/NC cells and were resistant to lysis. These data support the hypothesis that expression of laminin/laminin binding sites may contribute to tumor cell sensitivity to NK/NC binding and/or killing

  17. Protamine-based nanoparticles as new antigen delivery systems.

    Science.gov (United States)

    González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

    2015-11-01

    The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  19. Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

    Science.gov (United States)

    Ong, E K; Knox, R B; Singh, M B

    1995-03-01

    The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

  20. Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus.

    Science.gov (United States)

    Gao, Xiang; Esseili, Malak A; Lu, Zhongyan; Saif, Linda J; Wang, Qiuhong

    2016-05-15

    Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed

  1. Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

    NARCIS (Netherlands)

    Burgess, Janette K.; Lopez, Jose A.; Gaudry, Leonie E.; Chong, Beng H.

    2000-01-01

    The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because

  2. Shared peptide binding of HLA Class I and II alleles associate with cutaneous nevirapine hypersensitivity and identify novel risk alleles

    DEFF Research Database (Denmark)

    Pavlos, Rebecca; McKinnon, Elizabeth J.; Ostrov, David A.

    2017-01-01

    Genes of the human leukocyte antigen (HLA) system encode cell-surface proteins involved in regulation of immune responses, and the way drugs interact with the HLA peptide binding groove is important in the immunopathogenesis of T-cell mediated drug hypersensitivity syndromes. Nevirapine (NVP), is...

  3. The original Pathologische Anatomie Leiden-Endothelium monoclonal antibody recognizes a vascular endothelial growth factor binding site within neuropilin-1

    NARCIS (Netherlands)

    Jaalouk, Diana E.; Ozawa, Nfichael G.; Sun, Jessica; Lahdenranta, Johanna; Schlingemann, Reinier O.; Pasqualini, Renata; Arap, Wadih

    2007-01-01

    For two decades, the antigen recognized by the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody, a standard vascular endothelial cell marker, has remained elusive. Here, we used a combinatorial phage display-based approach ("epitope mapping") to select peptides binding to the

  4. CD4+ T-cell epitope prediction using antigen processing constraints.

    Science.gov (United States)

    Mettu, Ramgopal R; Charles, Tysheena; Landry, Samuel J

    2016-05-01

    T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. [Limbic encephalitis with antibodies against intracellular antigens].

    Science.gov (United States)

    Morita, Akihiko; Kamei, Satoshi

    2010-04-01

    Limbic encephalitis is a paraneoplastic syndrome that is often associated with small cell lung cancer (SCLC), breast cancer, testicular tumors, teratoma, Hodgkin's lymphoma and thymoma. The common clinical manifestations of limbic encephalitis are subacute onset, cognitive dysfunction, seizures and psychiatric symptoms. Paraneoplastic neurological disorders are considered to occur because of cytotoxic T cell responses and antibodies against target neuronal proteins that are usually expressed by an underlying tumor. The main intracellular antigens related to limbic encephalitis are Hu, Ma2, and less frequently CV2/CRMP5 and amphiphysin. The anti-Hu antibody, which is involved in cerebellar degeneration and extensive or multifocal encephalomyelitis such as limbic encephalitis is closely associated with a history of smoking and SCLC. The anti-Ma2 antibody is associated with encephalitis of the limbic system, hypothalamus and brain-stem. For this reason, some patients with limbic encephalitis have sleep disorders (including REM sleep abnormalities), severe hypokinesis and gaze palsy in addition to limbic dysfunction. In men aged less than 50 years, anti-Ma2 antibody encephalitis is almost always associated with testicular germ-cell tumors that are occasionally difficult to detect. In older men and women, the most common tumors are non-SCLC and breast cancer. Limbic encephalitis associated with cell-surface antigens (e.g., voltage-gated potassium channels, NMDA receptors) is mediated by antibodies and often improves after a reduction in the antibody titer and after tumor resection. Patients with antibodies against intracellular antigens, except for those with anti-Ma2 antibodies and testicular tumors, are less responsive. Early diagnosis and treatment with immunotherapy, tumor resection or both are important for improving or stabilizing the condition of limbic encephalitis.

  6. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Carboplatin binding to histidine

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M. [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom); Diederichs, Kay [University of Konstanz, D-78457 Konstanz (Germany); Kroon-Batenburg, Loes M. J. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Levy, Colin [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Schreurs, Antoine M. M. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  8. Prostate specific antigen and its clinical application

    International Nuclear Information System (INIS)

    Xu Yang

    2000-01-01

    Prostate-Specific Antigen (PSA), a serine proteases, is a glycoprotein consisting of a single polypeptide chain. Secreted exclusively by epithelial cells of the prostate gland, PSA is found largely in seminal plasma. Only a small amount of PSA can be found in normal serum. Serum PSA levels are found to be, considerably increased in prostate cancer patients. A number of studies on PSA have made great achievement on its biochemistry, analytical method and clinical application. PSA as one of the most important tumor marker, is used to help diagnosis and monitor the therapeutic efficacy of prostate cancer

  9. Interference of heparin in carcinoembryonic antigen radioimmunoassays

    International Nuclear Information System (INIS)

    Wu, J.T.

    1983-01-01

    A false Roche carcinoembryonic antigen (CEA) activity could be detected in all commercial and noncommercial heparin preparations examined. The possibility of 'due to contamination' has been ruled out. Using the Roche procedure, heparin solutions, in the absence of CEA, gave positive CEA activity; on the other hand, no CEA activity was detected in solutions containing only heparin when the Abbott Kit was used. When heparin was present in specimens containing CEA, the Abbott Kit underestimated the CEA activity, whereas the Roche Kit gave false elevated values. However, the negative effect of heparin could be reduced by heat treatment in the presence of plasma proteins. (Auth.)

  10. Antigen Presentation Keeps Trending in Immunotherapy Resistance.

    Science.gov (United States)

    Kalbasi, Anusha; Ribas, Antoni

    2018-04-19

    Through a gain-of-function kinome screen, MEX3B was identified as a mediator of resistance to T-cell immunotherapy not previously identified using CRISPR-based screens. MEX3B is a posttranscriptional regulator of HLA-A, validating the critical role of tumor-intrinsic antigen presentation in T-cell immunotherapy and indicating a new putative molecular target. Clin Cancer Res; 24(14); 1-3. ©2018 AACR. See related article by Huang et al., p. xxxx . ©2018 American Association for Cancer Research.

  11. The glycosphingolipid P₁ is an ovarian cancer-associated carbohydrate antigen involved in migration.

    Science.gov (United States)

    Jacob, F; Anugraham, M; Pochechueva, T; Tse, B W C; Alam, S; Guertler, R; Bovin, N V; Fedier, A; Hacker, N F; Huflejt, M E; Packer, N; Heinzelmann-Schwarz, V A

    2014-10-14

    The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data

  12. Protein antigenic structures recognized by T cells: potential applications to vaccine design.

    Science.gov (United States)

    Berzofsky, J A; Cease, K B; Cornette, J L; Spouge, J L; Margalit, H; Berkower, I J; Good, M F; Miller, L H; DeLisi, C

    1987-08-01

    In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic

  13. Topographic study of the ADP/ATP transport protein. Localization of ADP and atractyloside fixation sites. Identification of the antigenic domains

    International Nuclear Information System (INIS)

    Boulay, Francois

    1983-01-01

    The objectives of this research thesis were: to determine the intramolecular localisation of binding sites of atractyloside and adenine-nucleotides; to determine whether antibodies obtained against the ADP/ATP carrier protein and isolated from beef heart mitochondria possess a reactivity specific to the organ or the species, where antigenic determinants are localized and whether there is conservation of the antigenic structure from one species to the other; to study how to follow and interpret conformational changes of the protein under the effect of ADP and inhibitors (carboxy-atractyloside or bongkrekic acid), and where the SH group unmasked by ADP and bongkrekic acid is localized [fr

  14. Optical Binding of Nanowires

    Czech Academy of Sciences Publication Activity Database

    Simpson, Stephen Hugh; Zemánek, Pavel; Marago, O.M.; Jones, P.H.; Hanna, S.

    2017-01-01

    Roč. 17, č. 6 (2017), s. 3485-3492 ISSN 1530-6984 R&D Projects: GA ČR GB14-36681G Grant - others:AV ČR(CZ) CNR-16-12 Program:Bilaterální spolupráce Institutional support: RVO:68081731 Keywords : optical binding nanowires * Brownian motion * self-organization * non-equilibrium thermodynamics * non-equilibrium steady state * spin-orbit coupling * emergent phenomena Subject RIV: BH - Optics, Masers, Lasers OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 12.712, year: 2016

  15. Preclinical assessment of viral vectored and protein vaccines targeting the Duffy-binding protein region II of Plasmodium vivax

    Directory of Open Access Journals (Sweden)

    Simone C de Cassan

    2015-07-01

    Full Text Available Malaria vaccine development has largely focused on Plasmodium falciparum; however a reawakening to the importance of P. vivax has spurred efforts to develop vaccines against this difficult to treat and at times severe form of relapsing malaria, which constitutes a significant proportion of human malaria cases worldwide. The almost complete dependence of P. vivax red blood cell invasion on the interaction of the P. vivax Duffy-binding protein region II (PvDBP_RII with the human Duffy antigen receptor for chemokines (DARC, makes this antigen an attractive vaccine candidate against blood-stage P. vivax. Here, we generated both preclinical and clinically-compatible adenoviral and poxviral vectored vaccine candidates expressing the Salvador I allele of PvDBP_RII – including human adenovirus serotype 5 (HAdV5, chimpanzee adenovirus serotype 63 (ChAd63 and modified vaccinia virus Ankara (MVA vectors. We report on the antibody and T cell immunogenicity of these vaccines in mice or rabbits, either used alone in a viral vectored prime-boost regime, or in ‘mixed-modality’ adenovirus prime – protein-in-adjuvant boost regimes (using a recombinant protein PvDBP_RII protein antigen formulated in Montanide®ISA720 or Abisco®100 adjuvants. Antibodies induced by these regimes were found to bind to native parasite antigen from P. vivax infected Thai patients and were capable of inhibiting the binding of PvDBP_RII to its receptor DARC using an in vitro binding inhibition assay. In recent years, recombinant ChAd63 and MVA vectors have been quickly translated into human clinical trials for numerous antigens from P. falciparum as well as a growing number of other pathogens. The vectors reported here are immunogenic in small animals, elicit antibodies against PvDBP_RII and have recently entered clinical trials which will provide the first assessment of the safety and immunogenicity of the PvDBP_RII antigen in humans.

  16. Identification of immunogenic Salmonella enterica serotype Typhi antigens expressed in chronic biliary carriers of S. Typhi in Kathmandu, Nepal.

    Directory of Open Access Journals (Sweden)

    Richelle C Charles

    Full Text Available Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage.To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT, to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479, an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70% chronic carriers, 0 of 8 bile culture-negative controls (0%, 0 of 8 healthy Bangladeshis (0%, and 1 of 8 (12.5% Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present.Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic

  17. Overview of Plant-Made Vaccine Antigens against Malaria

    Directory of Open Access Journals (Sweden)

    Marina Clemente

    2012-01-01

    Full Text Available This paper is an overview of vaccine antigens against malaria produced in plants. Plant-based expression systems represent an interesting production platform due to their reduced manufacturing costs and high scalability. At present, different Plasmodium antigens and expression strategies have been optimized in plants. Furthermore, malaria antigens are one of the few examples of eukaryotic proteins with vaccine value expressed in plants, making plant-derived malaria antigens an interesting model to analyze. Up to now, malaria antigen expression in plants has allowed the complete synthesis of these vaccine antigens, which have been able to induce an active immune response in mice. Therefore, plant production platforms offer wonderful prospects for improving the access to malaria vaccines.

  18. Tissue distribution of histo-blood group antigens

    DEFF Research Database (Denmark)

    Ravn, V; Dabelsteen, Erik

    2000-01-01

    carrier carbohydrate chains. Histo-blood group antigens are found in most epithelial tissues. Meanwhile, several factors influence the type, the amount, and the histological distribution of histoblood group antigens, i.e. the ABO, Lewis, and saliva-secretor type of the individual, and the cell- and tissue......The introduction of immunohistochemical techniques and monoclonal antibodies to specific carbohydrate epitopes has made it possible to study in detail the tissue distribution of histo-blood group antigens and related carbohydrate structures. The present paper summarizes the available data...... concerning the histological distribution of histo-blood group antigens and their precursor structures in normal human tissues. Studies performed have concentrated on carbohydrate antigens related to the ABO, Lewis, and TTn blood group systems, i.e. histo-blood group antigens carried by type 1, 2, and 3 chain...

  19. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA

  20. Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

    Science.gov (United States)

    Poirier, Danielle; Renaud, Frédéric; Dewar, Vincent; Strodiot, Laurent; Wauters, Florence; Janimak, Jim; Shimada, Toshio; Nomura, Tatsuya; Kabata, Koki; Kuruma, Koji; Kusano, Takayuki; Sakai, Masaki; Nagasaki, Hideo; Oyamada, Takayoshi

    2017-11-01

    Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50 °C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45 °C and only a 10% loss was observed after 6 months at 50 °C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Expression of peanut agglutinin-binding mucin-type glycoprotein in human esophageal squamous cell carcinoma as a marker

    Directory of Open Access Journals (Sweden)

    Balakrishnan Ramathilakam

    2003-11-01

    Full Text Available Abstract Background The TF (Thomson – Friedenreich blood group antigen behaves as an onco-foetal carcinoma-associated antigen, showing increased expression in malignancies and its detection and quantification can be used in serologic diagnosis mainly in adenocarcinomas. This study was undertaken to analyze the sera and tissue level detectable mucin-type glycoprotein (TF-antigen by Peanut agglutinin (PNA and its diagnostic index in serum as well tissues of human esophageal squamous cell carcinoma as marker. Results We examined 100 patients for serological analysis by Enzyme Linked Lectin Assay (ELISA and demonstrated a sensitivity of 87.5%, specificity of 90% and a positive predictive value of 95%. The immuno-histochemical localization of TF antigen by Fluorescence Antigen Technique (FAT in 25 specimens of normal esophageal squamous epithelium specimens and 92 specimens with different grades of, allowed a quicker and more precise identification of its increased expression and this did not correlate with gender and tumor size. There was a positive correlation between membrane bound TF antigen expression with different histological progression, from well differentiated to poorly differentiated, determined by PNA binding. Specimens showed morphological changes and a pronounced increase in PNA binding in Golgi apparatus, secretory granules of the cytosol of well differentiated and an increased cell membrane labeling in moderately and poorly differentiated, when compared with ESCC and normal tissues. Conclusion The authors propose that the expression of TF-antigen in human may play an important role during tumorigenesis establishing it as a chemically well-defined carcinoma-associated antigen. Identification of the circulating TF-antigen as a reactive form and as a cryptic form in the healthy individuals, using PNA-ELLA and Immunohistochemical analysis of TF antigen by FAT is positively correlated with the different histological grades as a simple

  2. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

    Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

  3. Re-purification of labelled ferritin antigen with HPLC

    International Nuclear Information System (INIS)

    Zhang Haoyi; Jin Lichun

    2002-01-01

    Objective: To improve the quality of long-term stored labelled ferritin antigen with HPLC. Methods: The antigen was analyzed and purified with HPLC and again analyzed with RIA afterwards. Results: Ferritin antigen underwent significant polymerization after long-term (aggregation) storage. After re-purification with HPLC, its immuno-activity and labelled specific radioactivity were both significantly improved. Conclusion: Quality of stored ferritin RIA kit could be greatly improved after re-purification with HPLC

  4. Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

    OpenAIRE

    Jiménez, T; Díaz, A M; Zlotnik, H

    1990-01-01

    Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Myco...

  5. Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro.

    Directory of Open Access Journals (Sweden)

    Priya Saikumar Lakshmi

    Full Text Available Tuberculosis (TB caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6 kDa early secretory antigenic target and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39 fused with cholera toxin B-subunit (CTB and LipY (a cell wall protein were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential

  6. Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bio-encapsulation, stability and functional evaluation in vitro.

    Science.gov (United States)

    Lakshmi, Priya Saikumar; Verma, Dheeraj; Yang, Xiangdong; Lloyd, Bethany; Daniell, Henry

    2013-01-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6 kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long

  7. Monoclonal antibodies to carcino-embryonic antigen

    International Nuclear Information System (INIS)

    Teh, Jinghee; McKenzie, I.F.C.

    1990-01-01

    With the aim of producing new MoAb to colorectal carcinoma, immunization with cell suspensions of a fresh colonic tumour was performed and MoAb 17C4 was obtained. To produce other MoAb to colon cancer, an immunization protocol using fresh tumour, colonic cell lines and sera from patients with colonic tumours was employed and resulted in MoAb JGT-13, LK-4 and XPX-13. MoAb I-1 and O-1 were raised against sera from patients with colon cancer to produce MoAb directed against circulating tumour associated antigens. The six antibodies gave a range of reactions with normal and malignant tissues, indicating that they most likely reacted with different epitopes. Thus, apart from the reactions of 17C4, LK-4 and XPX-13 with fresh and formalin-fixed granulocytes, none of the antibodies reacted with formalin-fixed normal tissues. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to carcino-embryonic antigen polyclonal antibodies, that is the MoAb gave no obvious advantage. 9 refs., 1 tab., 3 figs

  8. Immunoregulation by Taenia crassiceps and Its Antigens

    Directory of Open Access Journals (Sweden)

    Alberto N. Peón

    2013-01-01

    Full Text Available Taenia crassiceps is a cestode parasite of rodents (in its larval stage and canids (in its adult stage that can also parasitize immunocompromised humans. We have studied the immune response elicited by this helminth and its antigens in mice and human cells, and have discovered that they have a strong capacity to induce chronic Th2-type responses that are primarily characterized by high levels of Th2 cytokines, low proliferative responses in lymphocytes, an immature and LPS-tolerogenic profile in dendritic cells, the recruitment of myeloid-derived suppressor cells and, specially, alternatively activated macrophages. We also have utilized the immunoregulatory capabilities of this helminth to successfully modulate autoimmune responses and the outcome of other infectious diseases. In the present paper, we review the work of others and ourselves with regard to the immune response induced by T. crassiceps and its antigens, and we compare the advances in our understanding of this parasitic infection model with the knowledge that has been obtained from other selected models.

  9. Autoantibodies and their antigens in autoimmune hepatitis.

    Science.gov (United States)

    Bogdanos, Dimitrios P; Mieli-Vergani, Giorgina; Vergani, Diego

    2009-08-01

    Autoantibody detection assists in the diagnosis and allows differentiation of autoimmune hepatitis (AIH) type 1 (AIH-1), characterized by antinuclear antibody (ANA) and/or smooth muscle antibody (SMA), and type 2 (AIH-2), distinguished by the presence of antibodies to liver-kidney microsome type 1 (anti-LKM1) and/or antibodies to liver cytosol type 1 (anti-LC1). Detection of atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA) and anti-soluble liver antigen (SLA) antibodies can act as an additional pointer toward the diagnosis of AIH, particularly in the absence of the conventional autoantibodies. Routine autoantibody testing by indirect immunofluorescence has been recently complemented by molecular assays based on purified or recombinant antigens. Although the AIH-1-specific ANA and SMA targets need better definition, those of anti-LKM1 and anti-LC1 in AIH-2 have been clearly identified; the fine specificity of antibody reactivity and its clinical relevance to disease pathogenesis are the focus of ongoing investigation. This article critically discusses the current knowledge of the diagnostic and clinical significance of AIH-related autoantibody reactivities, focusing on key issues that the physician needs to be aware of to be able to request the appropriate testing and to interpret correctly the laboratory results within the clinical context of the patient. Copyright Thieme Medical Publishers.

  10. Identification of protective antigens for vaccination against systemic salmonellosis

    Directory of Open Access Journals (Sweden)

    Dirk eBumann

    2014-08-01

    Full Text Available There is an urgent medical need for improved vaccines with broad serovar coverage and high efficacy against systemic salmonellosis. Subunit vaccines offer excellent safety profiles but require identification of protective antigens, which remains a challenging task. Here, I review crucial properties of Salmonella antigens that might help to narrow down the number of potential candidates from more than 4000 proteins encoded in Salmonella genomes, to a more manageable number of 50-200 most promising antigens. I also discuss complementary approaches for antigen identification and potential limitations of current pre-clinical vaccine testing.

  11. I-125 input into antibodies molecules specific to australian antigen

    International Nuclear Information System (INIS)

    Abdukayumov, A. M.; Chistyakov, P.G.; Garajshina, G. R.

    1999-01-01

    There are experimental data on I-125 input into antibodies molecules specific to superficial antigen of hepatitis B virus (australian antigen). Three ways of input are submitted: with the help of T chloramine usage, Bolton-Hunter Reagent and with the help of iodogen. There are also comparative characteristics of iodized products obtained: molar radioactivity, radiochemical frequency, immuno - reactivity. The report also discusses advantages and disadvantages of the used methods for inputting I-125 into antibodies to australian antigen in order to study the possibility of creating radio immunological test system for detecting superficial antigen of B hepatitis

  12. ANTIGENICITY OF COW'S MILK PROTEINS IN TWO ANIMAL MODELS

    OpenAIRE

    T.R. Neyestani; M. Djalali M. I'ezeshki

    2000-01-01

    Antigenicity of proteins found in cow's milk is age dependent. This is primarily due to infants possessing a more permeable intestinal wall than that in adults. Thus infants may acquire cow's milk allergy during their first year of life. While milk antigen specific IgE may cause allergy in susceptible subjects, there is some evidence indicating that milk antigen specific IgG may play some role in chronic disease development. The puropose of this study was to determine the antigenicity of cow'...

  13. Radioimmunoassay for the detection of Australia-SH antigen

    Energy Technology Data Exchange (ETDEWEB)

    Gerhardt, H [Giessen Univ. (Germany, F.R.). Zentrum fuer Innere Medizin

    1974-06-01

    Among infectious diseases, hepatitis presents a great problem in all countries with a high medical standard. The number of Australia antigen-positive cases rises from year to year, due to the increase in drug-fixer hepatitis and blood transfusions. Highly sensitive and at the same time practicable methods are therefore required for the identification of Australia antigen carriers and their elimination as blood donors. The most sensitive of all currently used tests for the detection of Australia antigen is the 'solid phase' radioimmunoassay since it permits an objective and quantitative measurement of the antigen.

  14. Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

    Science.gov (United States)

    Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

    2017-11-01

    The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Meningococcal factor H-binding protein vaccines with decreased binding to human complement factor H have enhanced immunogenicity in human factor H transgenic mice.

    Science.gov (United States)

    Rossi, Raffaella; Granoff, Dan M; Beernink, Peter T

    2013-11-04

    Factor H-binding protein (fHbp) is a component of a meningococcal vaccine recently licensed in Europe for prevention of serogroup B disease, and a second vaccine in clinical development. The protein specifically binds human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity. There are conflicting data from studies in human fH transgenic mice on whether binding of human fH to fHbp vaccines decreases immunogenicity, and whether mutant fHbp vaccines with decreased fH binding have enhanced immunogenicity. fHbp can be classified into two sub-families based on sequence divergence and immunologic cross-reactivity. Previous studies of mutant fHbp vaccines with low fH binding were from sub-family B, which account for approximately 60% of serogroup B case isolates. In the present study, we evaluated the immunogenicity of two mutant sub-family A fHbp vaccines containing single substitutions, T221A or D211A, which resulted in 15- or 30-fold lower affinity for human fH, respectively, than the corresponding control wild-type fHbp vaccine. In transgenic mice with high serum concentrations of human fH, both mutant vaccines elicited significantly higher IgG titers and higher serum bactericidal antibody responses than the control fHbp vaccine that bound human fH. Thus, mutations introduced into a sub-family A fHbp antigen to decrease fH binding can increase protective antibody responses in human fH transgenic mice. Collectively the data suggest that mutant fHbp antigens with decreased fH binding will result in superior vaccines in humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response.

    Science.gov (United States)

    Lee, Yuan-Cho; Zhou, Qing; Chen, Junjie; Yuan, Jingsong

    2016-12-19

    ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Carbohydrate epitopes on Haemonchus contortus antigens

    NARCIS (Netherlands)

    Schallig, H. D.; van Leeuwen, M. A.

    1996-01-01

    Extracts of infective larvae and adults of the trichostrongylid Haemonchus contortus were studied for the presence of carbohydrate moieties. Several different lectin-binding sites were demonstrated in both stages using a panel of nine lectins. The carbohydrate specificity of the lectins used

  18. Genetic variants related to disease susceptibility and immunotolerance in the Duffy antigen receptor for chemokines (DARC, Fy) gene in the black lion tamarin (Leontopithecus chrysopygus, primates).

    Science.gov (United States)

    Ansel, Ashley; Lewis, James D; Melnick, Don J; Martins, Cristiana; Valladares-Padua, Claudio; Perez-Sweeney, Beatriz

    2017-10-01

    The DARC (Duffy antigen receptor for chemokines) gene encodes the DARC protein, which serves multiple roles in the immune system, as a binding site for the malarial parasites Plasmodium vivax and Plasmodium knowlesi, a promiscuous chemokine receptor and a blood group antigen. Variation in DARC may play particularly significant roles in innate immunity, immunotolerance and pathogen entry in callitrichines, such as the black lion tamarin (Leontopithecus chrysopygus). We compared amino acid sequences of DARC in the black lion tamarin (BLT) to non-human Haplorhine primates and Homo sapiens. Consistent with prior studies in other Haplorhines, we observed that the chemokine receptor experiences two opposing selection forces: (1) positive selection on the Plasmodium binding site and (2) purifying selection. We observed also that D21N, F22L, and V25L differentiated BLT from humans at a critical site for P. vivax and P. knowlesi binding. One amino acid residue, F22L, was subject to both positive selection and fixation in New World monkeys, suggesting a beneficial role as an adaptive barrier to Plasmodium entry. Unlike in humans, we observed no variation in DARC among BLTs, suggesting that the protein does not play a role in immunotolerance. In addition, lion tamarins differed from humans at the blood compatibility Fy a /Fy b antigen-binding site 44, as well as at the putative destabilizing residues A61, T68, A187, and L215, further supporting a difference in the functional role of DARC in these primates compared with humans. Further research is needed to determine whether changes in the Plasmodium and Fy a /Fy b antigen-binding sites disrupt DARC function in callitrichines. © 2017 Wiley Periodicals, Inc.

  19. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G

    1998-01-01

    GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

  20. Preliminary crystallographic analysis of the RNA-binding domain of HuR and its poly(U)-binding properties

    International Nuclear Information System (INIS)

    Wang, Hong; Li, Heng; Shi, Hui; Liu, Yang; Liu, Huihui; Zhao, Hui; Niu, Liwen; Teng, Maikun; Li, Xu

    2011-01-01

    Here, the recombinant ARE-binding region of HuR (residues 18–186) was crystallized in space group P2 1 2 1 2, with unit-cell parameters a = 41.2, b = 133.1, c = 31.4 Å. Human antigen R (HuR), a ubiquitously expressed member of the Hu protein family, is an important post-transcriptional regulator which has three RNA-recognition motif (RRM) domains. The two tandem N-terminal RRM domains can selectively bind to the AU-rich element (ARE), while the third one interacts with the poly(A) tail and other proteins. Here, the recombinant ARE-binding region of HuR (residues 18–186) was crystallized in space group P2 1 2 1 2, with unit-cell parameters a = 41.2, b = 133.1, c = 31.4 Å. X-ray diffraction data were collected to a resolution of 2.8 Å. Mutagenesis analysis and SPR assays revealed its poly(U)-binding properties

  1. Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

    Directory of Open Access Journals (Sweden)

    Yukiko Kiniwa

    Full Text Available Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+ T helper (Th cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1 as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+ T cell-mediated immunotherapy in melanoma.

  2. Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

    Science.gov (United States)

    Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E; Wang, Rong-Fu; Wang, Helen Y

    2015-01-01

    Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+) T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+) T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+) T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+) Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+) T cell-mediated immunotherapy in melanoma.

  3. Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray.

    Directory of Open Access Journals (Sweden)

    Daniel S Chen

    2005-10-01

    Full Text Available In contrast to many animal model studies, immunotherapeutic trials in humans suffering from cancer invariably result in a broad range of outcomes, from long-lasting remissions to no discernable effect.In order to study the T cell responses in patients undergoing a melanoma-associated peptide vaccine trial, we have developed a high-throughput method using arrays of peptide-major histocompatibility complexes (pMHC together with antibodies against secreted factors. T cells were specifically immobilized and activated by binding to particular pMHCs. The antibodies, spotted together with the pMHC, specifically capture cytokines secreted by the T cells. This technique allows rapid, simultaneous isolation and multiparametric functional characterization of antigen-specific T cells present in clinical samples. Analysis of CD8+ lymphocytes from ten melanoma patients after peptide vaccination revealed a diverse set of patient- and antigen-specific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-gamma (IFNgamma and tumor necrosis factor-alpha (TNFalpha in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNgamma and TNFalpha did so.Such multiparametric analysis of T cell antigen specificity and function provides a valuable tool with which to dissect the molecular underpinnings of immune responsiveness and how this information correlates with clinical outcome.

  4. Comparison of allele frequencies of Plasmodium falciparum merozoite antigens in malaria infections sampled in different years in a Kenyan population.

    Science.gov (United States)

    Ochola-Oyier, Lynette Isabella; Okombo, John; Wagatua, Njoroge; Ochieng, Jacob; Tetteh, Kevin K; Fegan, Greg; Bejon, Philip; Marsh, Kevin

    2016-05-06

    Plasmodium falciparum merozoite antigens elicit antibody responses in malaria-endemic populations, some of which are clinically protective, which is one of the reasons why merozoite antigens are the focus of malaria vaccine development efforts. Polymorphisms in several merozoite antigen-encoding genes are thought to arise as a result of selection by the human immune system. The allele frequency distribution of 15 merozoite antigens over a two-year period, 2007 and 2008, was examined in parasites obtained from children with uncomplicated malaria. In the same population, allele frequency changes pre- and post-anti-malarial treatment were also examined. Any gene which showed a significant shift in allele frequencies was also assessed longitudinally in asymptomatic and complicated malaria infections. Fluctuating allele frequencies were identified in codons 147 and 148 of reticulocyte-binding homologue (Rh) 5, with a shift from HD to YH haplotypes over the two-year period in uncomplicated malaria infections. However, in both the asymptomatic and complicated malaria infections YH was the dominant and stable haplotype over the two-year and ten-year periods, respectively. A logistic regression analysis of all three malaria infection populations between 2007 and 2009 revealed, that the chance of being infected with the HD haplotype decreased with time from 2007 to 2009 and increased in the uncomplicated and asymptomatic infections. Rh5 codons 147 and 148 showed heterogeneity at both an individual and population level and may be under some degree of immune selection.

  5. Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9 M to 7.0×10(-10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

  6. Antigenic determinants of prostate-specific antigen (PSA) and development of assays specific for different forms of PSA.

    OpenAIRE

    Nilsson, O.; Peter, A.; Andersson, I.; Nilsson, K.; Grundstr?m, B.; Karlsson, B.

    1997-01-01

    Monoclonal antibodies were raised against prostate-specific antigen (PSA) by immunization with purified free PSA, i.e. not in complex with any protease inhibitor (F-PSA) and PSA in complex with alpha1-anti-chymotrypsin (PSA-ACT). Epitope mapping of PSA using the established monoclonal antibody revealed a complex pattern of independent and partly overlapping antigenic domains in the PSA molecule. Four independent antigenic domains and at least three partly overlapping domains were exposed both...

  7. Activation of the ATR kinase by the RPA-binding protein ETAA1

    DEFF Research Database (Denmark)

    Haahr, Peter; Hoffmann, Saskia; Tollenaere, Maxim A X

    2016-01-01

    Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway...... in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically...

  8. Multiplexed evaluation of capture agent binding kinetics using arrays of silicon photonic microring resonators.

    Science.gov (United States)

    Byeon, Ji-Yeon; Bailey, Ryan C

    2011-09-07

    High affinity capture agents recognizing biomolecular targets are essential in the performance of many proteomic detection methods. Herein, we report the application of a label-free silicon photonic biomolecular analysis platform for simultaneously determining kinetic association and dissociation constants for two representative protein capture agents: a thrombin-binding DNA aptamer and an anti-thrombin monoclonal antibody. The scalability and inherent multiplexing capability of the technology make it an attractive platform for simultaneously evaluating the binding characteristics of multiple capture agents recognizing the same target antigen, and thus a tool complementary to emerging high-throughput capture agent generation strategies.

  9. Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus

    Directory of Open Access Journals (Sweden)

    Guus Koch

    2013-04-01

    Full Text Available Avian influenza A virus comprises sixteen hemagglutinin (HA and nine neuraminidase (NA subtypes (N1–N9. To isolate llama single-domain antibody fragments (VHHs against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6 or were enzymatically inactive (rN1, rN5 in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1 describes methods for isolating NA binding VHHs, (2 illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3 describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology.

  10. Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands

    Science.gov (United States)

    2015-01-01

    Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors. PMID:25073654

  11. Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics

    Science.gov (United States)

    Gul, Ahmet; Erman, Burak

    2018-03-01

    Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

  12. Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Vesa Kirjavainen

    Full Text Available Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS O-antigen (O-ag and outer core (OC do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp, an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

  13. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  14. Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

    Directory of Open Access Journals (Sweden)

    Darryl J Hill

    Full Text Available The human pathogen Neisseria meningitides (Nm attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf, a trimeric autotransporter, binds to the activated form of vitronectin (aVn to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

  15. Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

    Science.gov (United States)

    Hill, Darryl J; Griffiths, Natalie J; Borodina, Elena; Andreae, Clio A; Sessions, Richard B; Virji, Mumtaz

    2015-01-01

    The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

  16. Endothelial cells present antigens in vivo

    Directory of Open Access Journals (Sweden)

    Tellides George

    2004-03-01

    Full Text Available Abstract Background Immune recognition of vascular endothelial cells (EC has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli β-galactosidase (β-gal in two lines of transgenic mice that express β-gal exclusively in their EC. TIE2-lacZ mice express β-gal in all EC and VWF-lacZ mice express β-gal in heart and brain microvascular EC. Results Transgenic and congenic wild type FVB mice immunized with β-gal expression vector DNA or β-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express β-gal, and their blood vessels showed no histological abnormalities. In response to β-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-γ. Infection with recombinant vaccinia virus encoding β-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express β-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost β-gal+ EC and the hosts developed β-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained β-gal+ EC and no antisera developed, suggesting a tolerant host immune system. Conclusion Resting, β-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against β-gal expressed by EC within

  17. Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

    Science.gov (United States)

    Cole, David K.; Bulek, Anna M.; Dolton, Garry; Schauenberg, Andrea J.; Szomolay, Barbara; Trimby, Andrew; Jothikumar, Prithiviraj; Fuller, Anna; Skowera, Ania; Rossjohn, Jamie; Zhu, Cheng; Miles, John J.; Wooldridge, Linda; Rizkallah, Pierre J.; Sewell, Andrew K.

    2016-01-01

    The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease. PMID:27183389

  18. Tissue polypeptide antigen activity in cerebrospinal fluid

    DEFF Research Database (Denmark)

    Bach, F; Söletormos, Georg; Dombernowsky, P

    1991-01-01

    Tissue polypeptide antigen (TPpA) in the cerebrospinal fluid (CSF) was measured in 59 consecutive breast cancer patients with suspected central nervous system (CNS) metastases. Subsequently, we determined that 13 patients had parenchymal brain metastases, 10 had leptomeningeal carcinomatosis......, and 36 had no CNS involvement. The concentration of TPpA, which is a nonspecific marker for cell proliferation, was significantly higher in patients with CNS metastases than in those without it (P less than .0001; Mann-Whitney test). A tentative cutoff value for CNS metastases was set at 95 U/L TPp...... metastases, no correlation was found between TPpA activity in corresponding CSF and blood samples (correlation coefficient, Spearman's rho = .4; P greater than .1). In three patients treated for leptomeningeal carcinomatosis, the measurements of CSF TPpA showed correlation between the presence of tumor cells...

  19. Chemiluminescence immunoassay for prostate-specific antigen

    International Nuclear Information System (INIS)

    Zhang Xuefeng; Liu Yibing; Jia Juanjuan; Xu Wenge; Li Ziying; Chen Yongli; Han Shiquan

    2008-01-01

    The chemiluminescence immunoassay (CLIA) for serum total prostate-specific antigen (T-PSA) was developed. The reaction of luminol with hydrogen peroxide was introduced into this chemiluminescence system. The detection limit is established as 0.12 μg/L (n=10, mean of zero standard + 2SD) and the analytical recovery of PSA is 83.8%-118.7%. The intra-assay and inter-assay CVs vary from 4.4%-5.0% and 6.2%-11.7%, respectively. The experimental correlation coefficient of dilution is found to be 0.999. Compared with immunoradiometric assay (IRMA) kits, the correlative equation is y=1.07x+0.68, and correlation coefficient r=0.97. The standard range for the method is 1.5-80 μg/L, and it presents good linearity. (authors)

  20. T cell recognition of breast cancer antigens

    DEFF Research Database (Denmark)

    Petersen, Nadia Viborg; Andersen, Sofie Ramskov; Andersen, Rikke Sick

    Recent studies are encouraging research of breast cancer immunogenicity to evaluate the applicability ofimmunotherapy as a treatment strategy. The epitope landscape in breast cancer is minimally described, thus it is necessary to identify T cell targets to develop immune mediated therapies.......This project investigates four proteins commonly upregulated in breast cancer and thus probable tumor associated antigens (TAAs). Aromatase, prolactin, NEK3, and PIAS3 contribute to increase growth, survival, and motility of malignant cells. Aspiring to uncover novel epitopes for cytotoxic T cells, a reverse...... recognition utilizing DNA barcode labeled MHC multimers to screen peripheral blood lymphocytes from breast cancer patients and healthy donor samples. Signif-icantly more TAA specific T cell responses were detected in breast cancer patients than healthy donors for both HLA-A*0201 (P

  1. Engineering antigen-specific immunological tolerance.

    Energy Technology Data Exchange (ETDEWEB)

    Kontos, Stephan; Grimm, Alizee J.; Hubbell, Jeffrey A.

    2015-05-01

    Unwanted immunity develops in response to many protein drugs, in autoimmunity, in allergy, and in transplantation. Approaches to induce immunological tolerance aim to either prevent these responses or reverse them after they have already taken place. We present here recent developments in approaches, based on engineered peptides, proteins and biomaterials, that harness mechanisms of peripheral tolerance both prophylactically and therapeutically to induce antigenspecific immunological tolerance. These mechanisms are based on responses of B and T lymphocytes to other cells in their immune environment that result in cellular deletion or ignorance to particular antigens, or in development of active immune regulatory responses. Several of these approaches are moving toward clinical development, and some are already in early stages of clinical testing.

  2. Statistical prediction of immunity to placental malaria based on multi-assay antibody data for malarial antigens

    DEFF Research Database (Denmark)

    Siriwardhana, Chathura; Fang, Rui; Salanti, Ali

    2017-01-01

    Background Plasmodium falciparum infections are especially severe in pregnant women because infected erythrocytes (IE) express VAR2CSA, a ligand that binds to placental trophoblasts, causing IE to accumulate in the placenta. Resulting inflammation and pathology increases a woman’s risk of anemia...... to 28 malarial antigens and used the data to develop statistical models for predicting if a woman has sufficient immunity to prevent PM. Methods Archival plasma samples from 1377 women were screened in a bead-based multiplex assay for Ab to 17 VAR2CSA-associated antigens (full length VAR2CSA (FV2), DBL...... in the following seven statistical approaches: logistic regression full model, logistic regression reduced model, recursive partitioning, random forests, linear discriminant analysis, quadratic discriminant analysis, and support vector machine. Results The best and simplest model proved to be the logistic...

  3. Detection and partial characterization of a midlamina lucida-hemidesmosome-associated antigen (19-DEJ-1) present within human skin

    DEFF Research Database (Denmark)

    Fine, J D; Horiguchi, Y; Jester, J

    1989-01-01

    , esophagus, cervix, and cornea, and BMs surrounding smooth muscle in medium-sized vessels, placenta, uterus, and esophagus. When 16 human fetal skins (aged 54-142 gestational days) were examined, the antigen was first detected at 81 days. Using immunoperoxidase and immunogold staining techniques, indirect......A murine anti-human monoclonal antibody (19-DEJ-1) has been produced that binds to basement membranes (BMs) of the dermoepidermal junction and arrector pili muscles but not to either dermal glandular or vascular BMs. 19-DEJ-1 also recognizes BMs underneath epithelia of buccal mucosa, tongue......-specific proteoglycan that is present within BMs along the epithelial-connective tissue interface and around smooth muscle in skin and other selected organs. Its unique ultrastructural localization suggests the possibility that 19-DEJ-1 may recognize an antigenic epitope of either anchoring filaments or alternatively...

  4. Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species.

    Science.gov (United States)

    Schirrmeier, Horst; Strebelow, Günther; Depner, Klaus; Hoffmann, Bernd; Beer, Martin

    2004-12-01

    The genus Pestivirus within the family Flaviviridae currently consists of four different main species: Classical swine fever virus, Bovine viral diarrhea virus types 1 and 2 and Border disease virus. A fifth tentative species is represented by an isolate from a giraffe. In this study, a completely new pestivirus, isolated from a batch of fetal calf serum that was collected in Brazil, is described. It is proposed that the isolate D32/00_'HoBi' may constitute a novel sixth pestivirus species, because it is genetically, as well as antigenically, markedly different from all other pestiviruses. Based on the entire N(pro)- and E2-encoding sequences, identities of pestivirus species were determined. Similarly, cross-neutralization and binding studies using antisera and mAbs revealed marked antigenic differences between D32/00_'HoBi' and all other pestiviruses.

  5. Recruitment of PfSET2 by RNA polymerase II to variant antigen encoding loci contributes to antigenic variation in P. falciparum.

    Directory of Open Access Journals (Sweden)

    Uchechi E Ukaegbu

    2014-01-01

    Full Text Available Histone modifications are important regulators of gene expression in all eukaryotes. In Plasmodium falciparum, these epigenetic marks regulate expression of genes involved in several aspects of host-parasite interactions, including antigenic variation. While the identities and genomic positions of many histone modifications have now been cataloged, how they are targeted to defined genomic regions remains poorly understood. For example, how variant antigen encoding loci (var are targeted for deposition of unique histone marks is a mystery that continues to perplex the field. Here we describe the recruitment of an ortholog of the histone modifier SET2 to var genes through direct interactions with the C-terminal domain (CTD of RNA polymerase II. In higher eukaryotes, SET2 is a histone methyltransferase recruited by RNA pol II during mRNA transcription; however, the ortholog in P. falciparum (PfSET2 has an atypical architecture and its role in regulating transcription is unknown. Here we show that PfSET2 binds to the unphosphorylated form of the CTD, a property inconsistent with its recruitment during mRNA synthesis. Further, we show that H3K36me3, the epigenetic mark deposited by PfSET2, is enriched at both active and silent var gene loci, providing additional evidence that its recruitment is not associated with mRNA production. Over-expression of a dominant negative form of PfSET2 designed to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment. RNA pol II is known to transcribe non-coding RNAs from both active and silent var genes, providing a possible mechanism by which it could recruit PfSET2 to var loci. This work unifies previous reports of histone modifications, the production of ncRNAs, and the promoter activity of var introns into a mechanism that contributes to antigenic variation by malaria parasites.

  6. The 2.5 Å Structure of CD1c in Complex with a Mycobacterial Lipid Reveals an Open Groove Ideally Suited for Diverse Antigen Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Scharf, Louise; Li, Nan-Sheng; Hawk, Andrew J.; Garzón, Diana; Zhang, Tejia; Fox, Lisa M.; Kazen, Allison R.; Shah, Sneha; Haddadian, Esmael J.; Gumperz, Jenny E.; Saghatelian, Alan; Faraldo-Gómez, José D.; Meredith, Stephen C.; Piccirilli, Joseph A.; Adams, Erin J. (Harvard); (UC); (MXPL-G); (UW-MED)

    2011-08-24

    CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 {angstrom} resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-{beta}1-phosphomycoketide (MPM). CD1c accommodated MPM's methylated alkyl chain exclusively in the A pocket, aided by a unique exit portal underneath the {alpha}1 helix. Most striking was an open F pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical major histocompatibility complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.

  7. Dissecting antigen processing and presentation routes in dermal vaccination strategies

    NARCIS (Netherlands)

    Platteel, Anouk C M; Henri, Sandrine; Zaiss, Dietmar M; Sijts, Alice J A M

    2017-01-01

    The skin is an attractive site for vaccination due to its accessibility and presence of immune cells surveilling this barrier. However, knowledge of antigen processing and presentation upon dermal vaccination is sparse. In this study we determined antigen processing routes that lead to CD8(+) T cell

  8. Protein antigen adsorption to the DDA/TDB liposomal adjuvant

    DEFF Research Database (Denmark)

    Hamborg, Mette; Jorgensen, Lene; Bojsen, Anders Riber

    2013-01-01

    Understanding the nature of adjuvant-antigen interactions is important for the future design of efficient and safe subunit vaccines, but remains an analytical challenge. We studied the interactions between three model protein antigens and the clinically tested cationic liposomal adjuvant composed...... of dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB)....

  9. Protein modeling of apical membrane antigen-1(AMA-1) of ...

    African Journals Online (AJOL)

    Apical membrane Antigen-1(AMA-1), an asexual blood stage antigen of Plasmodium cynomolgi, is an important candidate for testing as a component of malarial vaccine. The degree of conservation of. AMA-1 sequences implies a conserved function for this molecule across different species of Plasmodium. Since the AMA-1 ...

  10. Identification of Surface Exposed Elementary Body Antigens of ...

    African Journals Online (AJOL)

    This study sought to identify the surface exposed antigenic components of Cowdria ruminantium elementary body (EB) by biotin labeling, determine effect of reducing and non-reducing conditions and heat on the mobility of these antigens and their reactivity to antibodies from immunized animals by Western blotting.

  11. Antigen Loss Variants: Catching Hold of Escaping Foes.

    Science.gov (United States)

    Vyas, Maulik; Müller, Rolf; Pogge von Strandmann, Elke

    2017-01-01

    Since mid-1990s, the field of cancer immunotherapy has seen steady growth and selected immunotherapies are now a routine and preferred therapeutic option of certain malignancies. Both active and passive cancer immunotherapies exploit the fact that tumor cells express specific antigens on the cell surface, thereby mounting an immune response specifically against malignant cells. It is well established that cancer cells typically lose surface antigens following natural or therapy-induced selective pressure and these antigen-loss variants are often the pop