Academic literature on the topic 'Proteine antigelo'
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Journal articles on the topic "Proteine antigelo"
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Fernández-Quintero, Monica L., Johannes R. Loeffler, Franz Waibl, Anna S. Kamenik, Florian Hofer, and Klaus R. Liedl. "Conformational selection of allergen-antibody complexes—surface plasticity of paratopes and epitopes." Protein Engineering, Design and Selection 32, no. 11 (2019): 513–23. http://dx.doi.org/10.1093/protein/gzaa014.
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Abstract Antibodies have the ability to bind various types of antigens and to recognize different antibody-binding sites (epitopes) of the same antigen with different binding affinities. Due to the conserved structural framework of antibodies, their specificity to antigens is mainly determined by their antigen-binding site (paratope). Therefore, characterization of epitopes in combination with describing the involved conformational changes of the paratope upon binding is crucial in understanding and predicting antibody-antigen binding. Using molecular dynamics simulations complemented with strong experimental structural information, we investigated the underlying binding mechanism and the resulting local and global surface plasticity in the binding interfaces of distinct antibody-antigen complexes. In all studied allergen-antibody complexes, we clearly observe that experimentally suggested epitopes reveal less plasticity, while non-epitope regions show high surface plasticity. Surprisingly, the paratope shows higher conformational diversity reflected in substantially higher surface plasticity, compared to the epitope. This work allows a visualization and characterization of antibody-antigen interfaces and might have strong implications for antibody-antigen docking and in the area of epitope prediction.
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Mavenyengwa, Rooyen T., Johan A. Maeland, and Sylvester R. Moyo. "Putative Novel Surface-Exposed Streptococcus agalactiae Protein Frequently Expressed by the Group B Streptococcus from Zimbabwe." Clinical and Vaccine Immunology 16, no. 9 (2009): 1302–8. http://dx.doi.org/10.1128/cvi.00133-09.
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ABSTRACTGroup B streptococci (GBS) express a variety of surface-exposed and strain-variable proteins which function as phenotypic markers and as antigens which are able to induce protective immunity in experimental settings. Among these proteins, the chimeric and immunologically cross-reacting alpha-like proteins are particularly important. Another protein, R3, which has been less well studied, occurred at a frequency of 21.5% in GBS from Zimbabwe and, notably, occurred in serotype V strains at a frequency of 75.9%. Working with rabbit antiserum raised against the R3 reference strain ATCC 49447 (strain 10/84; serotype V/R3) to detect the expression of the R3 protein, we recorded findings which suggested that strain 10/84 expressed a strain-variable protein antigen, in addition to R3. The antigen was detected by various enzyme-linked immunosorbent assay-based tests by using acid extract antigens or GBS whole-cell coats and by whole-cell-based Western blotting. We named the putative novel antigen the Z antigen. The Z antigen was a high-molecular-mass antigen that was susceptible to degradation by pepsin and trypsin but that was resistant tom-periodate oxidation and failed to show immunological cross-reactivity with any of a variety of other GBS protein antigens. The Z antigen was expressed by 33/121 (27.2%) of strains of a Zimbabwean GBS strain collection and by 64.2% and 72.4% of the type Ib and type V strains, respectively, and was occasionally expressed by GBS of other capsular serotypes. Thus, the putative novel GBS protein named Z showed distinct capsular antigen associations and presented as an important phenotypic marker in GBS from Zimbabwe. It may be an important antigen in GBS from larger areas of southern Africa. Its prevalence in GBS from Western countries is not known.
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Elkon, K. B., and P. W. Jankowski. "Fine specificities of autoantibodies directed against the Ro, La, Sm, RNP, and Jo-1 proteins defined by two-dimensional gel electrophoresis and immunoblotting." Journal of Immunology 134, no. 6 (1985): 3819–24. http://dx.doi.org/10.4049/jimmunol.134.6.3819.
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Abstract Although useful for specific purposes, immunofluorescence, precipitation in agarose gels, and the m.w. estimation of RNA or proteins immunoprecipitated from transformed cells often provide partial or ambiguous definition of autoantibody specificity. We have analyzed organ and cell extracts by one-and two-dimensional electrophoresis together with Western blotting to define the fine specificities of antibodies to the ribonucleoprotein (RNP) antigens Ro, La, Sm, RNP and Jo-1. One-dimensional analysis identified the Ro protein as a 57 kilodalton (kd) protein, although many anti-Ro sera also react with a 50 kd protein. La antisera react with 50 and 43 kd proteins. The 50 kd La protein readily breaks down into 43, 25, and smaller immunoreactive cleavage products. Partial proteolysis of Ro and La proteins in human spleen extracts produces similar immunoreactive products, providing evidence for a common structure. The major immunoreactive Sm antigens defined by human polyclonal antisera and a mouse monoclonal antiserum were doublets of 25/26 and 16/18 kd, whereas anti-RNP sera reacted with a protein of 68 kd. Most Sm-RNP antisera contained antibodies reactive with additional proteins, especially when whole cell extracts were used as a source of antigens. Two-dimensional analysis provided characteristic maps of the antigens. Ro and La were acidic, and La showed a unique set of acidic charge isomers at 50 and 43 kd. Anti-Sm antibodies reacted with discrete dots corresponding to both the acidic and basic regions of the first-dimension (charge) gels, whereas the RNP antigen showed a series of basic charge isomers of 68 kd. Many anti-Sm-RNP sera reacted with other closely spaced proteins of a similar charge and size to the Sm and RNP antigens, suggesting antibody cross-reactivity or reactivity with closely related functional proteins. Although Jo-1 had the same m.w. as the undegraded La antigen, the fingerprints were quite distinctive on two-dimensional electrophoresis. The results of this study indicate how the source and preparation of antigen extracts, as well as protein degradation, influence the m.w. determinations of soluble protein antigens. With these factors taken into account, two-dimensional fractionation with immunoblotting provides a highly discriminating, sensitive, and reproducible method of analysis of autoantibody specificity. This technique can be used to standardize reference antisera and to study protein antigens in normal and abnormal cell and tissue extracts, and could lead to new or more precise correlations with clinical disease.
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Delaney, Kristen N., and Steven B. Mizel. "A vaccine containing recombinant poxvirus proteins and flagellin promotes protective immunity against vaccinia virus (132.17)." Journal of Immunology 182, no. 1_Supplement (2009): 132.17. http://dx.doi.org/10.4049/jimmunol.182.supp.132.17.
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Abstract Bacterial flagellin is a potent adjuvant that enhances adaptive immune responses to a variety of antigens. The vaccinia virus antigens L1R and B5R are highly immunogenic in the context of the parent virus, but recombinant forms of the proteins are only weakly immunogenic. Therefore, we evaluated the response to these antigens when flagellin was used as an adjuvant. We found that flagellin promoted a robust antigen-specific humoral response to poxvirus antigens delivered intranasally and intramuscularly, but intramuscular immunization was more efficient. Flagellin/poxvirus antigen fusion proteins promoted humoral responses that were greater in magnitude than antigen and flagellin as separate proteins. However, there was competition when more than one fusion protein was administered. At least three immunizations with flagellin/poxvirus fusion proteins were required to confer protection in mice against challenge with vaccinia virus. Although mice were protected, they still exhibited significant, but reversible weight loss. This work was supported by a grant from the National Institutes of Health, P01 AI 60642 (to S.B.M.)
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Lichtenwalner, Anne B., Dorothy L. Patton, Wesley C. Van Voorhis, Yvonne T. Cosgrove Sweeney, and Cho-Chou Kuo. "Heat Shock Protein 60 Is the Major Antigen Which Stimulates Delayed-Type Hypersensitivity Reaction in the Macaque Model of Chlamydia trachomatis Salpingitis." Infection and Immunity 72, no. 2 (2004): 1159–61. http://dx.doi.org/10.1128/iai.72.2.1159-1161.2004.
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ABSTRACT Chlamydial delayed-type hypersensitivity antigens were analyzed by using the subcutaneous salpingeal autotransplant model of Macaca nemestrina infected with Chlamydia trachomatis serovar E. Heat shock protein 60 was the only antigen shown to induce delayed-type hypersensitivity among other antigens tested, including UV-inactivated organisms, recombinant major outer membrane protein, purified outer membrane proteins, and heat shock protein 10.
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Yu, Hong, Karuna P. Karunakaran, Xiaozhou Jiang, Caixia Shen, Peter Andersen, and Robert C. Brunham. "Chlamydia muridarum T Cell Antigens and Adjuvants That Induce Protective Immunity in Mice." Infection and Immunity 80, no. 4 (2012): 1510–18. http://dx.doi.org/10.1128/iai.06338-11.
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ABSTRACTMajor impediments to aChlamydiavaccine lie in discovering T cell antigens and polarizing adjuvants that stimulate protective immunity. We previously reported the discovery of three T cell antigens (PmpG, PmpF, and RplF) via immunoproteomics that elicited protective immunity in the murine genital tract infection model againstChlamydiainfection after adoptive transfer of antigen-pulsed dendritic cells. To expand the T cell antigen repertoire necessary for aChlamydiavaccine, we evaluated 10 newChlamydiaT cell antigens discovered via immunoproteomics in addition to the 3 antigens reported earlier as a molecular subunit vaccine. We first tested five adjuvants, including three cationic liposome formulations (dimethyldioctadecylammonium bromide-monophosphoryl lipid A [DDA-MPL], DDA-trehalose 6,6′-dibehenate [DDA-TDB {CAF01}], and DDA-monomycolyl glycerol [DDA-MMG {CAF04}]), Montanide ISA720–CpG-ODN1826, and alum using the PmpG protein as a model T cell antigen in the mouse genital tract infection model. The results showed that the cationic liposomal adjuvants DDA-MPL and DDA-TDB elicited the best protective immune responses, characterized by multifunctional CD4+T cells coexpressing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), and reduced infection by more than 3 logs. Using DDA-MPL as an adjuvant, we found that 7 of 13ChlamydiaT cell antigens (PmpG, PmpE, PmpF, Aasf, RplF, TC0420, and TC0825) conferred protection better than or equal to that of the reference vaccine antigen, major outer membrane protein (MOMP). Pools of membrane/secreted proteins, cytoplasmic proteins, and hypothetical proteins were tested individually or in combination. Immunization with combinations protected as well as the best individual protein in that combination. The T cell antigens and adjuvants discovered in this study are of further interest in the development of a molecularly definedChlamydiavaccine.
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Rennert, Paul, Lan Wu, Lihe Su, Roy Lobb, and Christine Ambrose. "160 Evaluation and development of dual and triple antigen targeting CAR-T Engager proteins for Her2-positive CNS metastases and solid tumors." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (2021): A170. http://dx.doi.org/10.1136/jitc-2021-sitc2021.160.
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BackgroundSolid tumors display pronounced antigen heterogeneity and clinical studies have shown that antigen escape from therapy occurs rapidly, limiting the persistence and efficacy of CAR T cells. Here we present dual and triple-antigen binding proteins that bridge CAR T cells to multiple antigens, allowing a simultaneous attack on tumor antigens by a single CAR T antibody domain. We call these CAR T Engager proteins. CAR T Engager proteins can be encoded into lentiviral vectors for secretion from CAR T cells, can be encoded into oncolytic viral vectors for secretion from transduced tumor cells, or can be engineered as biologics for injection.MethodsCAR T Engagers contain a protein target for a CAR T cell, eg. an anti-CD19 CAR T cell. We have previously presented a Her2-binding CAR T Engager protein with potent in vivo activity against solid tumors. We used this CAR T Engager as the basis for building dual and triple antigen binding proteins. Specifically, we mapped antigen expression for Her2-positive solid tumors, Her2-positive metastases, and primary CNS tumors. Our analysis identified expression patterns of two and three antigens that would essentially saturate the cellular composition of specific solid tumors, greatly reducing the chance of antigen escape from therapy. We created the corresponding CAR T Engagers and have developed single, dual and triple antigen expression cells lines to model the activity and potency of these novel proteins, administered alongside CAR T cells.ResultsCAR T cells plus dual antigen CAR T Engagers that recognize and target Her2 and B7H3 demonstrate potent cytotoxicity against either antigen alone, and synergistic potency (2 pM) if both antigens are expressed. Similarly, triple antigen CART Engagers show single antigen binding and potent cytotoxicity which is enhanced when multiple antigens are expressed on a target cell. All of the cytotoxicity is mediated through one CAR domain expressed on the primary T cells. T cells can be pre-loaded with multi-antigen CAR T Engagers and retain cytotoxic activity. Because the underlying CAR is an anti-CD19 CAR, cell persistence and fitness is further enhanced in the presence of normal B cells.ConclusionsThe CAR T Engager platform is a robust and modular solution for the multi-antigen targeting of solid tumors. Diverse antigens can be readily targeted for diverse indications. Examples of other functional modalities that can be added will be presented.AcknowledgementsWe thank Cancer Research UK for their ongoing support.
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Kim, Ae, Isamu Hartman, and Scheherazade Sadegh-Nasseri. "A cell free antigen processing system identifies immunodominant epitopes (78.19)." Journal of Immunology 182, no. 1_Supplement (2009): 78.19. http://dx.doi.org/10.4049/jimmunol.182.supp.78.19.
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Abstract T cell-mediated responses to protein antigen involve recognition of selected antigenic peptides bound to Major Histocompatibility Complex (MHC) molecules. Proteolytic digestion of protein antigens during antigen processing generates many peptide fragments that can potentially bind to MHC molecules. However, only few selected antigenic epitopes induce T cell responses, defined as "immunodominant". Identifying immunodominant epitopes from a given antigen has wide applications in development of peptide vaccines and therapies against infectious diseases, cancer, autoimmune diseases, and allergy. Currently available methods are not efficient in elucidating CD4+ T cell epitopes. Here, we provide a novel approach for identifying the immunodominant epitopes from protein antigens using a cell-free MHC class II antigen-processing assay. Two different novel antigens, Hemagglutinin (HA1) of the A/Vietnam/1203/2004 (H5N1) virus, and a recombinant form of Plasmodium falciparum liver-stage antigen 1 (LSA-NRC) were used to evaluate our assay. HLA-DR1 restricted epitopes from both proteins were identified by mass spectrometry and were tested for their ability to activate T cells in HLA-DR1 tg mice. We observed that the identified epitopes were indeed immunodominant as CD4+ T cells from mice immunized with the proteins proliferated and produced cytokines upon challenge with the identified epitopes at levels comparable to the whole protein. This cell-free assay provides a useful tool for identification of CD4+ T cell epitopes for specific MHC class II alleles.
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Zou, Jin-Tao, Hai-Ming Jing, Yue Yuan, et al. "Pore-forming alpha-hemolysin efficiently improves the immunogenicity and protective efficacy of protein antigens." PLOS Pathogens 17, no. 7 (2021): e1009752. http://dx.doi.org/10.1371/journal.ppat.1009752.
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Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus to form a HlaH35A-PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that HlaH35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby HlaH35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by HlaH35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.
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Arribillaga, Laura, Maika Durantez, Teresa Lozano, et al. "A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell ResponsesIn Vivo." BioMed Research International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/864720.
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The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with aKd~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβby TLR4-expressing cells, as well as the production of TNF-αby the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.
Dissertations / Theses on the topic "Proteine antigelo"
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MANGIAGALLI, MARCO. "Structural and functional analyses of an ice-binding protein from an Antarctic bacterium." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241269.
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Una proteina in grado di legare i cristalli di ghiaccio è definita proteina legante il ghiaccio o IBP acronimo dall’inglese ice-binding protein. Le IBP grazie alla loro capacità di abbassare il punto di congelamento dell’acqua, aumentando il gap di isteresi termica (TH). Questo intervallo è definito come la differenza tra il punto di fusione e di congelamento dell’acqua. La seconda attività delle IBP è l’inibizione della ricristallizzazione del ghiaccio (ice recrystallization inhibition, IRI). Infatti, queste proteine stabilizzano i piccoli cristalli di ghiaccio impedendo la formazione di cristalli di ghiaccio di grosse dimensioni che sono dannosi per le cellule. Le IBP sono state identificate in numerosi organismi tra cui pesci, insetti, batteri, alghe e lieviti. Queste proteine rappresentano un esempio di evoluzione convergente, infatti tutte le IBP condividono lo stesso meccanismo di legame con il ghiaccio nonostante una sorprendente diversità strutturale e funzionale.
Questo lavoro di tesi è focalizzato sulla caratterizzazione funzionale e strutturale di EfcIBP, una IBP batterica identificata da analisi di metagenomica effettuate sul ciliato Antartico Euplotes focardii e sul consorzio batterico ad esso associato. La struttura 3D di EfcIBP è stata risolta mediante cristallografia ai raggi X e consiste in un β-solenoide con un α-elica parallela all’asse principale della proteina. L’analisi strutturale ha permesso di identificare tre diverse facce del solenoide denominate A, B e C. Simulazioni di docking suggeriscono che EfcIBP è in grado di legare i cristalli di ghiaccio tramite le facce B e C del solenoide. Questa ipotesi è stata verificata attraverso la progettazione razionale di 6 varianti che sono state prodotte e saggiate per la loro attività. In generale, questi risultati indicano che EfcIBP è in grado di legare i cristalli di ghiaccio attraverso le facce B e C del solenoide. Questa peculiarità strutturale si riflette in un’insolita combinazione di attività di IRI e TH. Infatti, EfcIBP presenta una notevole attività di IRI in un intervallo di concentrazione nanomolare e una attività di isteresi termica di 0.53°C alla concentrazione di 50 μM che la rende una IBP moderata. All’interno del gap di TH, i cristalli di ghiaccio presentano una forma esagonale, mentre a temperature al di sotto della temperatura di congelamento presentano una forma a “Saturno". La proteina chimerica formata dalla “green fluorescent protein” e da EfcIBP è stata utilizzata per determinare a quali piani del cristallo di ghiaccio la proteina è in grado di legarsi e con quale cinetica. I dati sperimentali suggeriscono che le peculiarità funzionali di EfcIBP sono dovute alla sua capacità di legare velocemente i piani basali e piramidali del cristallo di ghiaccio. Questi dati, insieme alla presenza di una sequenza segnale per la secrezione, suggeriscono che EfcIBP è secreta e svolge la funzione di mantenere liquido l’ambiente circostante aumentando lo spazio vitale. In conclusione, EfcIBP è un nuovo tipo di IBP con proprietà insolite di legame al ghiaccio e di attività di IRI.
Questo studio ha contribuito ad identificare una nuova classe di IBP moderate che potrebbero essere sfruttate come crioprotettori in diversi settori come la criobiologia e quello alimentare.<br>Ice-binding proteins (IBPs) are characterized by the ability to control the growth of ice crystals. IBPs are active in increasing thermal hysteresis (TH) gap as they decrease the freezing point of water. On the other hand, IBPs can inhibit ice recrystallization (IRI) and stabilize small ice crystals at the expense of the harmful, large ones. IBPs have been identified in several organisms including higher Eukaryotes and microorganisms such as bacteria, yeasts and algae. Although IBPs share the ability to bind ice crystals, proteins from different sources present different 3D structures, from α-helix to β-solenoid proteins.
This thesis is focused on the structural and functional characterization of EfcIBP, a bacterial IBP identified by metagenomic analysis of the Antarctic ciliate Euplotes focardii and the associated consortium of non-cultivable bacteria. The 3D structure of EfcIBP, solved by X-ray crystallography, consists in a β-solenoid with an α-helix aligned along the axis of the β-helix. It is possible to distinguish three different faces: A, B and C. Docking simulations suggest that B and C faces are involved in ice binding. This hypothesis was tested by the rational design of six variants that were produced and assayed for their activity. Overall, these experiments indicate that both solenoid faces contribute to the activity of EfcIBP.
EfcIBP displays remarkable IRI activity at nanomolar concentration and a TH activity of 0.53°C at the concentration of 50 μM. The atypical combination between these two activities could stem from the ability of this protein to bind ice crystals through two faces of the solenoid. In the presence of EfcIBP, ice crystals show a hexagonal trapezohedron shape within the TH gap, and a unique “Saturn-shape” below the freezing point. A chimeric protein consisting of the fusion between EfcIBP and the green fluorescent protein was used to deeper investigate on this aspects by analyses of fluorescence ice plane affinity and binding kinetics. Overall, experimental data suggest that the EfcIBP unique pattern of ice growth and burst are due to its high rate of binding at the basal and the pyramidal near-basal planes of ice crystals. These data, together with the signal sequence for the secretion, suggest that EfcIBP is secreted in local environment where it becomes active in increasing the habitable space.
In conclusion, EfcIBP is a new type of IBP with unusual properties of ice shaping and IRI activity. This study opens new scenarios in the field of IBPs by contributing to identify a new class of moderate IBPs potentially exploitable as cryoprotectants in several fields, such as cryobiology and food science.
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Varelias, Antiopi. "Studies of CD44 variant isoform expression and function on activated human peripheral blood mononuclear cells and in renal transplantation." Title page, summary and contents only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phv293.pdf.
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Winchester, Christopher Charles. "The roles of Hsp70 proteins in antigen processing and presentation." Thesis, University of Oxford, 1997. http://ora.ox.ac.uk/objects/uuid:567dff45-08ce-43b4-b011-d08afea42f76.
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The ability of members of the hsp70 family to bind to peptides in vivo and in vitro suggests that they may be involved in the processing of antigens for binding to Major Histocompatibility (MHC) class I and/or class n molecules. The aims of this thesis have been to provide evidence for the involvement of hsp70s in antigen processing and to characterise the binding of peptides by hspTOs by structural and functional studies. Firstly, the peptide-binding domains of two hsp70s, hsp70hom and PBP74, were expressed in isolation from the rest of the molecule for structure determination. Both of these hsp70s were implicated in antigen processing: hsp70hom in the class I pathway, due to its cytoplasmic localisation and constitutive expression, and the presence of its gene in the MHC; and PBP74 in the class n pathway because published work indicated that it was localised to endosomes and that antibodies against it inhibited antigen processing. The expression and purification of both peptide-binding domains was very successful, and one dimensional NMR experiments indicated that they were folded. However, it was not possible to determine their structures by NMR spectroscopy or X-ray crystallography because they aggregated in solution at high concentrations. Instead, the structure of the C-terminal region of hsp70hom, which includes its peptidebinding domain, was modelled based on the known structure of the equivalent portion of dnaK, the hsp70 of E.coli. The structure of hsp70hom is predicted to be very similar to that of dnaK, and modelling studies suggest that it is likely to bind peptides in a closely related fashion. The modelling of complexes between hsp70hom and two peptides suggest that the peptide-binding groove is very versatile, accounting for the broad peptide-binding specificity of hsp70s. The interactions of hsp70hom and PB74 with peptides were investigated using plate binding assays and isothermattitration calorimetry. A biotinylated peptide bound to the peptide-binding domain of hsp70hom, immobilised in plastic wells, with a Kd of <25 μM, which is within the range of Kds reported for other hsp70-peptide complexes (0.1-100 μM). In solution, isothermal titration calorimetry showed that the binding of peptides to the peptide-binding domains of hsp70hom and PBP74 was likely to be entropically rather than enthalpically driven, and, therefore, the interactions involved are likely to be predominantly hydrophobic. Secondly, PBP74, an hsp70 thought to be involved in the class II antigen processing pathway in endosomes, was localised by immunofluorescence microscopy. It was shown to be a mitochondrial protein, and is, therefore, unlikely to be involved in antigen processing. The presence of other members of the hsp70 family in lysosomes purified from a B cell line by Percoll density gradient centrifugation was investigated using antibodies that reacted with many Afferent members of the hsp70 family. No hsp70s were detected in these late endocytic compartments, even after heat shock or serum starvation. However, the presence of an hsp70 in endosomes, or of a member of this family not detected by the antibodies used, in lysosomes, cannot be ruled out. A third approach investigated the induction of the three hsp70 genes found in the MHC by four cytokines. The hsp70-l and hsp70-2 genes are induced at the mRNA level by IFN-γ and IL- 1, while TNF induces hsp70-2 alone. This data supports a role for the heat-inducible hsp70 in MHC class I antigen processing, as it appears to be coregulated with known members of this antigen processing pathway. The expression of hsp70hom was unaffected by any of the four cytokines examined. In addition, the mitochondrial hsp70 (which is not encoded in the MHC) appears to be induced by IFN-γ at the protein level. The research presented in this thesis provides a greater understanding of the peptide-binding properties of two hsp70s. Further work is necessary to show conclusively whether any of the hsp70s is involved in antigen processing.
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Malhotra, Shikha. "B-cell-antigen receptor endocytosis uses a distinct signaling pathway, involving LAB, Vav, dynamin and Grb2." Oklahoma City : [s.n.], 2009.
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MENTO, ALFREDO. "Unconventional purification and labelling strategies of bioreagents for immunodiagnostic assays." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/309986.
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Gli antigeni e gli anticorpi sono reagenti chiave per lo sviluppo di test immunodiagnostici accurati, riproducibili e sensibili, ampiamente utilizzati per la rilevazione di malattie infettive (HIV, HBV, HCV, ecc.) e per la determinazione di marcatori biologici (vitamine, ormoni, ecc.). Questi bioreagenti devono essere prodotti con un alto grado di purezza, in formulazioni stabili e con sufficiente riproducibilità nel tempo (consistenza da lotto a lotto). Un aspetto spesso trascurato nella produzione di questi reagenti è il loro costo, che deve essere sufficientemente basso da non avere un impatto significativo sul prezzo finale dei saggi immunochimici. Le fasi di purificazione e di marcatura di questi bioreagenti incidono principalmente sul loro costo complessivo poiché vengono utilizzati reagenti e strumentazioni costosi e protocolli complessi che richiedono tempo. Per tutti questi motivi è importante ricercare nuove strategie di purificazione che permettano lo sviluppo di processi più semplici, con minor quantità di reagenti, in tempi più brevi, in modo da ridurre i costi. Pertanto, è necessario sviluppare purificazioni innovative e protocolli di marcatura sito-specifici. Nella prima parte di questo progetto abbiamo sfruttato il sistema ELP-intein. Questo metodo si basa sulla combinazione di due tools tecnologici, Elastin-like-polypeptides (ELP) (tool fisico-chimico) e l'attività dell’inteina MxeGyrA (tool biochimico), appartenente alla famiglia delle cis inteine. Ci siamo concentrati sulla purificazione e la marcatura dell'antigene C33 appartenente al virus dell'epatite C (HCV). L'antigene C33, attualmente utilizzato nel test Diasorin LIAISON® XL Murex HCV per la rilevazione di anticorpi umani contro il virus dell'epatite C, è stato purificato utilizzando un metodo non convenzionale senza passaggi cromatografici. Inoltre, abbiamo realizzato una biotinilazione sito-specifica dell'antigene C33 al suo C-terminale durante la purificazione, sfruttando l'attività biologica inteina di MxeGyrA. Sono stati sviluppati due diversi protocolli; entrambi hanno portato all'ottenimento di un antigene C33 biotinilato con un’elevata purezza e immunoreattività paragonabile a quella dell’antigene attualmente utilizzato nel saggio Diasorin HCV. Alla luce di questi buoni risultati, nella seconda parte del progetto, abbiamo studiato la possibilità di applicare la tecnologia del Protein Trans Splicing (PTS) per eseguire la marcatura sito-specifica di bioreagenti. La tecnologia PTS sfrutta l'attività delle split inteine. In particolare, nei nostri esperimenti abbiamo utilizzato la Cfa split-intein che deriva da un processo di mutagenesi della split-intein naturale Npu che ne ha notevolmente migliorato la cinetica di PTS, stabilità termica e tolleranza agli agenti caotropici. Questa nuova tecnica ci ha permesso di impostare un protocollo di marcatura sito-specifica per la produzione di bioreagenti biotinilati. Sono state utilizzate due proteine modello: lo stesso antigene C33 e una IgG umana ricombinante. Anche l'uso della tecnica PTS ha permesso di ottenere per entrambe le due proteine un'elevata purezza e prestazioni comparabili nei relativi saggi immunodiagnostici. In sintesi, il sistema ELP-inteina ha permesso di purificare l'antigene C33 senza passaggi cromatografici e di marcare in modo sito-specifico la stessa proteina al C-terminale. Inoltre, attraverso l'utilizzo del sistema Cfa split-intein abbiamo ottenuto la biotinilazione sito-specifica dell'antigene C33 e dell'IgG ricombinante. Un aspetto molto rilevante è che tutte queste proteine sono funzionali nella piattaforma LIAISON. In futuro, questi protocolli potrebbero essere utilizzati per la purificazione e / o la marcatura sito-specifica di nuovi bioreagenti utili per lo sviluppo di saggi immunodiagnostici.<br>Antigens and antibodies are key reagents for the development of accurate, reproducible and sensible immunodiagnostic assays, which are widely used for the detection of infectious diseases (HIV,HBV, HCV, etc.) and the determination of biological markers (vitamins, hormones, etc.). These bioreagents need to be produced at a high purity degree, in stable formulations and with sufficient reproducibility over time (lot to lot consistency). An aspect often overlooked in the production of these reagents is their cost, which must be low enough to not have a significant impact on the final price of the immunochemical assays. The purification and labeling steps of these bioreagents mainly affect the overall cost of them since costly reagents and instrumentations and complex and time-consuming protocols are used.For all these reasons it is important to seek new purification strategies that allow the development of simpler processes, with less use of reagents and shorter protocol times, and at the end minor costs. Therefore, it is necessary to develop innovative purifications and site-specific labelling protocols. In the first part of this project we exploited the ELP-intein system. This method is based on the combination of two technological tools, the Elastin-like-polypeptides (ELPs) (a physico-chemical tool) and the MxeGyrA intein activity (a biochemical tool), belonging to the cis intein family. We focused on the purification and labelling of the C33 antigen from Hepatitis C Virus (HCV). The C33 antigen, which is currently used in the Diasorin LIAISON® XL Murex HCV assay for the detection of human antibodies against the Hepatitis C Virus, was purified using a not conventional purification method without chromatographic steps. Moreover, we realized a site-specific biotinylation of C33 antigen at its C-terminus during the purification exploiting the MxeGyrA intein biological activity. Two different protocols were developed; both of them brought to the obtainment of a biotinylated C33 antigen with high purity and a comparable immunoreactivity with the one currently used in the Diasorin LIAISON® XL Murex HCV assay. In light of these good results, in the second part of the project, we investigated the possibility to apply the Protein Trans Splicing (PTS) technology to perform site-specific labelling of bioreagents. PTS technology exploits the split intein activity. In particular, in our experiments we used the Cfa split-intein which derives from a mutagenesis process of the natural Npu split-intein that significantly improved its kinetic of PTS, thermal stability and tolerance at the chaotropic agents. This new technique allowed us to set up a site-specific labelling protocol for the production of biotinylated bioreagents. Two model protein were used: the same C33 antigen and a recombinant human IgG. Also the use of PTS technique permitted to obtain for both of two proteins a high purity and a comparable performance in the immunoassays. In summary, the ELP-intein system allowed to purify the C33 antigen without chromatographic steps and then to site-specific label the same protein at the C-terminus. Moreover, through the use of the Cfa split-intein system we obtained the site-specific biotinylation of the C33 antigen and the recombinant IgG. A very relevant aspects is that all these proteins are functional in the LIAISON platform. In the future, these protocols could be used for the purification and-or the site-specific labelling of new bioreagents useful for the development of immunodiagnostic assays.
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Lot, Perrine. "Les protéines antigel." Paris 5, 1988. http://www.theses.fr/1988PA05P219.
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Schumacher, Dominik. "Site-specific functionalization of antigen binding proteins for cellular delivery, imaging and target modulation." Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18547.
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Antikörper und Antigen-bindende Proteine, die an Fluorophore, Tracer und Wirkstoffe konjugiert sind, sind einzigartige Moleküle, welche die Entwicklung wertvoller diagnostischer und therapeutischer Werkzeuge ermöglichen. Allerdings ist der Konjugationsschritt sehr anspruchsvoll und trotz intensiver Forschung noch immer ein bedeutender Engpass. Zusätzlich sind Antigen-bindende Proteine oftmals nicht dazu in der Lage, die Zellmembran zu durchdringen und im Zellinneren nicht funktionsfähig. Daher ist ihre Verwendung auf extrazelluläre Targets beschränkt, was eine bedeutende Anzahl wichtiger Antigene vernachlässigt. Beide Limitierungen bilden Kernaspekte dieser Arbeit. Mit Tub-tag labeling wurde ein neuartiges und vielseitiges Verfahren für die ortsspezifische Funktionalisierung von Biomolekülen und Antigen-bindenden Proteinen entwickelt, und so die Palette der Proteinfunktionalisierungen bedeutend erweitert. Tub-tag wurde erfolgreich für die ortsspezifische Funktionalisierung verschiedener Proteine und Antigen-bindender Nanobodies angewendet, die für konfokale Mikroskopie, Proteinanreicherung und hochauflösende Mikroskopie eingesetzt wurden. In einem weiteren Projekt wurden zellpermeable Antigen-bindende Nanobodies hergestellt und somit das schon lange Zeit bestehende Ziel, intrazelluläre Targets durch in vitro funktionalisierte Antigen-bindende Proteine zu visualisieren und manipulieren, erreicht. Hierzu wurden zwei verschiedene Nanobodies an ihrem C-Terminus cyclischen zellpenetrierenden Peptiden unter Verwendung von Expressed Protein Ligation funktionalisiert. Diese Peptide ermöglichten die Endozytose-unabhängige Aufnahme der Nanobodies mit sofortiger Bioverfügbarkeit. Mit Tub-tag labeling und der Synthese von zellpermeablen Nanobodies konnten wichtige Bottlenecks im Bereich der Proteinfunktionalisierung und Antikörperforschung adressiert werden und neue Tools für die biochemische und zellbiologische Forschung entwickelt werden.<br>Antibodies and antigen binding proteins conjugated to fluorophores, tracers and drugs are powerful molecules that enabled the development of valuable diagnostic and therapeutic tools. However, the conjugation itself is highly challenging and despite intense research efforts remains a severe bottleneck. In addition to that, antibodies and antigen binding proteins are often not functional within cellular environments and unable to penetrate the cellular membrane. Therefore, their use is limited to extracellular targets leaving out a vast number of important antigens. Both limitations are core aspects of the presented thesis. With Tub-tag labeling, a novel and versatile method for the site-specific functionalization of biomolecules and antigen binding proteins was developed expanding the toolbox of protein functionalization. The method is based on the microtubule enzyme tubulin tyrosine ligase. Tub-tag labeling was successfully applied for the site-specific functionalization of different proteins including antigen binding nanobodies which enabled confocal microscopy, protein enrichment and super-resolution microscopy. In addition to that, cell permeable antigen binding nanobodies have been generated constituting a long thought goal of tracking and manipulating intracellular targets by in vitro functionalized antigen binding proteins. To achieve this goal, two different nanobodies were functionalized at their C-terminus with linear and cyclic cell-penetrating peptides using expressed protein ligation. These peptides triggered the endocytosis independent uptake of the nanobodies with immediate bioavailability. Taken together, Tub-tag labeling and the generation of cell-permeable antigen binding nanobodies strongly add to the functionalization of antibodies and their use in biochemistry, cell biology and beyond.
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Heinrich, Garrett. "A role for CEACAM proteins in energy balance and peripheral insulin action." Toledo, Ohio : University of Toledo, 2010. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1272976279.
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Dissertation (Ph.D.)--University of Toledo, 2010.<br>"Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences." Title from title page of PDF document. "A Dissertation entitled"--at head of file. Bibliography: p. 37-41, 77-82, 102-107, 124-125, 153-160, 195-199, 221-254.
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Eynon, Elizabeth E. "Small B Cells as Antigen Presenting Cells in the Induction of Tolerance to Soluble Protein Antigens: A Dissertation." eScholarship@UMMS, 1991. https://escholarship.umassmed.edu/gsbs_diss/185.
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This thesis proposes a mechanism for the induction of peripheral tolerance to protein antigens. I have investigated the mechanism of tolerance induction to soluble protein antigens by targeting an antigen to small, resting B cells. For this purpose I have used a rabbit antibody directed at the IgD molecule found on the surface of most small, resting B cells but missing or lowered on activated B cells. Intravenous injection of normal mice with 100 μg of an ultracentrifuged Fab fragment of rabbit anti-mouse IgD (Fab anti-δ) makes these mice profoundly tolerant to challenge with nonimmune rabbit Fab (Fab NRG) fragments. This tolerance is antigen specific since treated mice make normal responses to an irrelevant antigen, chicken immunoglobulin (Ig). Fab fragments of rabbit Ig (rabbit Fab) not targeted to B cells do not induce tolerance as well as Fab anti-δ. Evidence suggests that the B cells must remain in a resting state for tolerance to be induced, since injection of F(ab)'2 anti-δ does not induce tolerance. Investigation of the mechanisms of the tolerance, by adoptive transfer, have shown that rabbit Fab specific B cell function has been impaired. The major effect however is in helper T cell function, as shown by adoptive transfer and lack of help for a hapten response. In vitro proliferation experiments show that the T cell response has not been shifted toward activation of different T cell subsets which do not help Ig production, nor is there any change in the Ig isotypes produced. Suppression does not appear to be the major cause of the helper T cell defect as shown by cell mixing experiments. This work shows that an antigen targeted to small B cells can induce tolerance to a soluble protein antigen, and suggests a role for small B cells in tolerance to self-proteins not presented in the thymus.
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Scott, Carol Elizabeth DeWeese. "Molecular modeling and experimental characterization of HLA-DQ proteins and protein/peptide complexes : correlation with insulin-dependent diabetes mellitus (IDDM) /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/8089.
Full textBooks on the topic "Proteine antigelo"
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1929-, Laver William Graeme, Air Gillian, and Cold Spring Harbor Laboratory, eds. Immune recognition of protein antigens. Cold Spring Harbor Laboratory, 1985.
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Z, Atassi M., and Abbott Laboratories, eds. Immunobiology of proteins and peptides IV: T-cell recognition and antigen presentation. Plenum Press, 1987.
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International Symposium on the Immunobiology of Proteins and Peptides (3rd 1984 Tahoe City, Calif.). Immunobiology of proteins and peptides III: Viral and bacterial antigens. Plenum Press, 1985.
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Nitsche, Fiona. Studies on the Epstein-Barr virus antigen leader protein. University of Birmingham, 1998.
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Shand, Geoffrey Harold. Antibiotic resistance and outer membrane protein antigens of Pseudomonas aeruginasa. University of Aston. Department of Pharmaceutical Sciences, 1985.
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1932-, Haber Edgar, ed. Antigen binding molecules: Antibodies and T-cell receptors. Academic Press, 1996.
Find full textBook chapters on the topic "Proteine antigelo"
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Palacio-Castañeda, Valentina, Roland Brock, and Wouter P. R. Verdurmen. "Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen." In Methods in Molecular Biology. Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_8.
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AbstractPhosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1–254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.
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Paxton, Raymond J., and John E. Shively. "Structural Analysis of Carcinoembryonic Antigen (CEA) and a Related Tumor-Associated Antigen (TEX)." In Proteins. Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-1787-6_71.
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Reiss, Errol, and Sandra L. Bragg. "Immunochemical Analysis of Histoplasmin Proteins and Polysaccharide." In Fungal Antigens. Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-0773-0_65.
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Bertina, R. M. "Protein S antigen." In ECAT Assay Procedures A Manual of Laboratory Techniques. Springer Netherlands, 1992. http://dx.doi.org/10.1007/978-94-011-2992-3_12.
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Bertina, R. M. "Protein S antigen." In Laboratory Techniques in Thrombosis - a Manual. Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4722-4_15.
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Kuroda, Daisuke, and Kouhei Tsumoto. "Structural Classification of CDR-H3 in Single-Domain VHH Antibodies." In Computer-Aided Antibody Design. Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2609-2_2.
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AbstractThe immune systems protect vertebrates from foreign molecules or antigens, and antibodies are important mediators of this system. The sequences and structural features of antibodies vary depending on species. Many of antibodies from vertebrates, including camelids, have both heavy and light chain variable domains, but camelids also have antibodies that lack the light chains. In antibodies that lack light chains, the C-terminal variable region is called the VHH domain. Antibodies recognize antigens through six complementarity-determining regions (CDRs). The third CDR of the heavy chain (CDR-H3) is at the center of the antigen-binding site and is diverse in terms of sequence and structure. Due to the importance of antibodies in basic science as well as in medical applications, there have been many studies of CDR-H3s of antibodies that possess both light and heavy chains. However, nature of CDR-H3s of single-domain VHH antibodies is less well studied. In this chapter, we describe current knowledge of sequence–structure–function correlations of single-domain VHH antibodies with emphasis on CDR-H3. Based on the 370 crystal structures in the Protein Data Bank, we also attempt structural classification of CDR-H3 in single-domain VHH antibodies and discuss lessons learned from the ever-increasing number of the structures.
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Ward, Tony Milford. "Carcinoembryonic Antigen." In Proteins and Tumour Markers May 1995. Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-011-0681-8_22.
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Vigneron, Nathalie, Wenbin Ma, Alexandre Michaux, and Benoît J. Van den Eynde. "Identifying Source Proteins for MHC Class I-Presented Peptides." In Antigen Processing. Humana Press, 2012. http://dx.doi.org/10.1007/978-1-62703-218-6_16.
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Bricker, Betsy J., Robert R. Wagner, and Jay W. Fox. "Immunoprotection — A Novel Approach for Mapping Epitopes on an Antigen." In Proteins. Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-1787-6_48.
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Walseng, Even, and Paul A. Roche. "Monitoring Protein Endocytosis and Recycling Using FACS-Based Assays." In Antigen Processing. Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9450-2_20.
Full textConference papers on the topic "Proteine antigelo"
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Beardsley, D. S. "IMMUNE THROMBOCYTOPENIA (ITP) : PLATELET TARGET ANTIGENS OF THE ANTIBODIES IN DIFFERENT CLINICAL SETTINGS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644757.
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Autoantibodies against platelet antigens are important in the pathogenesis of ITP. We have studied the antigenic targets of these autoantibodies by immunoblotting using electrophoretically separated proteins from normal, Glanzmann's Thrombasthenic, and Bernard-Soulier Syndrome platelets immobilized on nitrocellulose paper. Incubation of these proteins with ITP patient serum, immunoglobulin, or F(ab*>2, followed by labeled antiglobulin, allowed identification of the antigenic target glycoproteins (GPfs). Patients with ITP of several categories were studied: A) Chronic ITP (> 1 yr duration) including a subset of patients who hadmild thrombocytopenia but significant hemorrhagic manifestations. B) Acute ITP (<6 mo) following varicella infectionAcute onset ITP unresponsive to any of the usual therapeutic modalities. A majority of patients in group A (32/48) had antibodies directed against a protein with a MW of lOOkD. In some cases, the target antigen was localized to GPIIIa specifically by absence of reactivity with Type I Glanzmann's thrombasthenic platelets known to be totally deficient in GPIIb and Ilia. Two individuals in the subset with unexpectedly severe hemorrhagic symptoms also had anti-GPIIIa antibodies, but the antigenic target was different since only these two antibodies could be shown to interfere with binding of 125j_fibrinogen to normal plateletsIn contrast to the individuals with anti-GPIIIa antibodies, all group B patients (7/7) with acute post-varicella ITP had antibodies directed against an 85kD thrombin sensitive protein, and two of the three group C individuals studied showed antibodies directed against GPIb. These studies indicate that a number of different platelet antigens can be the targets of platelet autoantibodies in ITP. Results suggest that antibodies against a particular antigen may correlate with a similar clinical setting in different patients. Further work will determine whether antigen identification at the time of diagnosis can be used to predict the clinical course for an individual patient
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Church, W., T. Messier, P. Howard, J. Amiral, D. Meyer, and K. Mam. "A SHARED EPITOPE ON HUMAN PROTEIN C, FACTOR X, FACTOR VII, AND PROTTOBIN DEFINED BY A MONOCLONAL ANTIBODY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643937.
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A monoclonal antibody prepared against hunan protein C (HPC) was found to react with several other vitamin K-dependent blood proteins. Using a competitive inhibition solid-phase radioinminoassay with HPC, binding of 125I-HPC to the antibody was inhibited by purified prothrombin, Factor X, and Factor VII in addition to protein C. Other vitamin K-dependent proteins including Factor IX, protein S, and bone-GLA protein did not compete for binding of 125I-HPC to the antibody. The effect of calciun ion on the binding of antibody to 125I-HPC was examined in a solid-phase imnunoassay system with the antibody bound to rabbit anti-mouse inminoglobulin adsorbed to microtiter plates. In the presence of 5 mM calciun ion, radiolabeled protein C did not bind to the antibody; radiolabeled protein C did bind, however, in the presence of 5 nM EDTA suggesting that the epitope is expressed only after removal of calciun ion. The antibody bound to prothrombin and to decarboxylated prothrombin after adsorption of the antigens onto nitrocellulose indicating that the presence of GLA was not required for antibody binding. Iimunoblotting of proteins which were reduced, the peptides separated by SDS-PAGE, and transferred to nitrocellulose showed that the antibody reacts with a determinant found on the light chains of protein C and Factor X and with prothrombin Fragment 1. Comparison of the protein sequences of protein C light chain, Factor X light chain, Factor VII, and prothrombin Fragment 1 identified a segment of amino acid sequence that is highly conserved in all four proteins and might contain the antigenic site. The monoclonal antibody thus defines an antigenic determinant which is masked by calcium ion and is found on the surface of several related, yet different coagulation proteins. This antibody should prove useful in understanding the evolutionary relationships amongst the vitamin K-dependent proteins and also in understanding the effect of calcium ion on the structure of protein C, Factor X, prothrombin, Factor VII and possibly other related proteins. (Supported by NIH grant MHLBI HL35058)
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Hopmeier, P., M. Halbmayer, H. P. Schwarz, F. Heuss, and M. Fischer. "PROTEIN C AND PROTEIN S IN MILD AND MODERATE PREECLAMPSIA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644285.
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In normal pregnancy, total protein S antigen and activity have been reported to be markedly reduced, whereas protein C level was found unaltered. In contrast, in severe preeclampsia protein C antigen was found to be considerably reduced. The presentstudy was done to clarify whether similar changes in protein Cwould alsobe observed for the mildand moderatepreeclamptic state andwhether there would be any effects on the level ofprotein S, since nodata on this cofactor in preeclampsia have been reported to date. 4-0 women in the 3rd trimester of pregnancy - 20 with uncomplicated pregnancies and 20 who had developed a mild (n = 14-) or moderate (n = 6) preeclamptic condition - were included in the study. All groups were well matched in age and gestational age. In addition, 20 healthynon-pregnant women served as controls. All probands had normal liver (SGOT, SGPT) and kidney (BUN, creatinine) values and no other medication than oral vitamins was used. Classification of preeclampsia was done according to a modification of the gestosis index of Goecke using an 11 gradeindex system (0 - 11). ProteinC antigen was measured by an enzyme-linkedimmunosorbent assay and protein S by the Laurell rocket technique.For statistics, the Wilcoxon rank sum test was appliedWe conclude that in comparison tonormal pregnancies, protein S is found elevated at least in the moderate, and protein C in the moderateas well as in the mild preeclamptic state
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Vigano D'Angelo, S., F. Gilardoni, M. P. Seveso, A. Marassi, G. Mari, and A. D'Angelo. "REDUCTION OF THE ANTICOAGULANT ACTIVITY OF PROTEIN C AND PROTEIN S DURING THE POSTOPERATIVE PERIOD." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644287.
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Protein S circulates in plasma as free protein S and in complex with C4b-binding protein, an inhibitor of complement activation. Only free protein S functions as the cofactor for the anticoagulant and profibrinolytic effects of activated protein C. Since isolated reductions of protein C and protein S result in increased thrombotic risk, only measurement of both proteins permits comprehensive evaluation of the antithrombotic potential of the protein C system. No information is available on protein C and protein S functional levels during the postoperative period, an established prothrombotic condition. The plasma changes of protein C, protein S and C4b-binding protein were followed in 40 patients with no malignancy undergoing abdominal surgery. No significant change of protein C and protein S activi ty was observed following minor operations. After major surgery, protein C anticoagulant activity dropped to 80% of preoperative levels during the first postoperative week (p<0.00l). Significant increase of both total protein S antigen (110%, p< 0.01) and C4b-binding protein (130%, p<0.001) were observed after major surgery resulting in reduction of free protein S antigen to 86% of pre operative values (p<0.001). Protein S anticoagulant activity matched the changes of free protein S antigen.Albeit transient and moderate, the observed reductions of both protein C and protein S may act synergistically to cause significant impairment of the antithrombotic potential during the postoperative period. The effect of heparin prophylaxis on protein C and protein S postoperative levels is currently under investigation.
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D'Angelo, A., F. Gilardoni, M. P. Seveso, P. Poli, R. Quintavalle, and C. Manotti. "ANTICOAGULANT AND ANTIGENIC LEVELS OF PROTEIN C AND PROTEIN S IN PATIENTS ON STABILIZED ORAL ANTICOAGULANT TREATMENT." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644286.
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Isolated deficiencies of protein C and protein S, two vitamin K-dependent plasma proteins, constitute about 70% of the congenital abnormalities of blood coagulation observed in patients with recurrent venous thrombosis beLow the age of 40. The laboratory diagnosis of congenital deficiency of these proteins represents a major problem since a large proportion of patients are on oral anticoagulation (OA) at the time the deficiencies are suspected.Under these circumstances the availability of a reference interval obtained in patients on stabilized OA has proven useful.Functional (C) and antigenic levels (Ag) of protein C, protein S, factor IX and II were estimated in 136 patients on stabilized OA, subdivided according to the degree of anticoagulation (Internatio nal Normalized Ratio, INR).The results indicate that with increasing anticoagulation the activity levels of all the vitamin K-dependent factors decrease to a greater extent than the corresponding antigenic levels. At variance with the other factors, total protein S antigen levels are only moderately reduced by OA with protein S anticoagulant activi ty comparing well to factor IX clotting activity. These data suggest the possibility of identifying both quantitative and qualita tive deficiencies of protein C and protein S in patients on oral anticoagulant treatment.
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Comp, P. C., and C. T. Esmon. "Defects in the protein C pathway." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643715.
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Activated protein C functions as an anticoagulant by enzymatically degrading factors Va and Villa in the clotting cascade. Protein C may be converted to its enzymatically active form bythrombin. The rate at which thrombin cleavage of the zymogen occurs is greatly enhanced when thrombin is bound to an endothelial cell receptor protein, thrombomodulin. Activated proteinC has a relatively long half-life in vivo and the formation of activated protein C in response to low level thrombin infusion suggests that the protein C system may provide a feedback mechanism to limit blood clotting. Clinical support for such a physiologic role for activated protein C includes an increased incidence of thrombophlebitis and pulmonary emboli in heterozygous deficient individuals, and severe, often fatal, cutaneous thrombosis in homozygous deficient newborns. A third thrombotic condition associated with protein C deficiency is coumarin induced skin (tissue) necrosis. This localized skin necrosis occurs shortly after the initiation of coumarin therapy and is hypothesized to bedue to the rapid disappearance of protein C activity in the plasma beforean adequate intensity of anticoagulation is achieved. Recent estimates of heterozygous protein C deficiency range as high as 1 in 300 individuals in the general population. Since coumarin compounds are in routine clinical use throughout the world and skin necrosis remains a relatively rare clinical finding, this suggests that factors other than protein C deficiency alone may be involved in the pathogenesis of the skin necrosis.The anticoagulant properties of activated protein C are greatly enhanced by another vitamin K-dependent plasma protein, protein S. Protein S functions by increasing the affinity of activated protein C for cell surfaces.Protein S is found in two forms in plasma: free and in complex with C4b-binding protein, "an inhibitor of the complement system. Free protein S is functionally active and the complexed protein S is not active. Individuals congenitally deficient in protein S ae subject to recurrent thromboembolicevents. At least two classes of protin S deficiency occur.Some patienshavedecreased levels of protein S antigen and reduced protein S functional activity. A second group of deficient individuals have normal levels of protein S antigen but most or all their protein S is complexed to C4b-binding protein and they have little or no functional protein S activity. Such a protein S distribution could result from abnormal forms of protein S or C4b-binding protein or some other abnormal plasma or cellular component. Patients with functionally inactive forms of protein S have yet to be identified. Identification of protein S deficient individuals is complicated by thepossible effect of sex hormones on plasma protein S levels. Total protein S antigen is reduced during pregnancyand during oral contraceptive administration. This finding is of practicalclinical importance since the decrease in protein S which occurs during pregnancy may be an added risk factor for congenitally protein S deficient women and may explain why some proteinS deficient women experience their first episode of thrombosis during pregnancy.In addition to having anticoagulant properties, activated protein C enhances fibrinolysis, at least in part,by inhibiting the inhibitor of tissueplasminogen activator. This profibrinolytic effect is enhanced by protein S and cell surfaces. This protection of plasminogen activator activity suggests that the combination of tissue plasminogen activator and activated protein C may be useful in the treatment of coronary artery thrombi. Tissueplasminogen activator would promote clot lysis while activated protein C protected the plasminogen activatorfrom inhibition and also prevented further clot deposition. There is no evidence at present that fibrinolytic activity is reduced in protein C deficient individuals. The possible clinical relevance of this aspect of protein Cfunction in the predisposition of protein C deficient individuals to thrombosis remains to be defined.
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Klein, Kevin M., Gregory T. Ostrowicki, Andrew Gerwitz, and Suresh K. Sitaraman. "Micro and Nano Thin Film Devices as Bio-Assays for Cancer Diagnosis." In ASME 2006 International Mechanical Engineering Congress and Exposition. ASMEDC, 2006. http://dx.doi.org/10.1115/imece2006-15581.
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Micro and nano Au/Cr and Al thin film devices have been fabricated using DC sputtering and e-beam evaporation in combination with e-beam and photo lithography. These devices can be coated with specific reagents to detect and measure the presence of particular antigens and/or complementary DNA sequences with a smaller sample size and at much earlier stages of disease progression compared to current medical diagnostic technologies. Using the device material stack (Au/Cr/Si), we have assessed the binding affinity of Au, Cr, and Si with Protein G, and antibodies for Prostate Specific Antigen (PSA) and Cancer Antigen 125 (CA125), an ovarian cancer-associated antigen. Based on our experiments, we see that the thin gold layer of the Au/Cr/Si samples, provides increased bio-material binding affinity, and the chromium layer has a similar, if not less, binding affinity compared to the silicon chip alone.
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Novikova, L. I., S. S. Bochkareva, A. V. Aleshkin, et al. "DYNAMICS OF ANTIBODIES TO VARIOUS ANTIGENS OF THE SARS-COV-2 CORONAVIRUS IN PATIENTS WITH CONFIRMED COVID-19 INFECTION." In Molecular Diagnostics and Biosafety. Federal Budget Institute of Science 'Central Research Institute for Epidemiology', 2020. http://dx.doi.org/10.36233/978-5-9900432-9-9-159.
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Presence of IgG and IgM antibodies in venous blood of 76 patients with confirmed presence of SARS-CoV-2 was determined. The study was carried out by ELISA using Russian test systems. Revealed different levels of IgM antibodies to N-protein and RBD (receptor binding domain of the Spike protein). The level of IgM to RBD did not reach high values, while the level of IgM to N-protein sharply increased in a short period of time to high values by the 3rd week of the disease and decreased only by the 8th week. The dynamics of IgG antibodies to the whole virion antigen and the recombinant spikes was similar, reaching high values at 4-5 weeks of the disease, however, the dynamics of IgG to the N-protein differed, showing a slight increase by the 1st week of the disease and a low level throughout the entire period of observation. Different dynamics of production of IgG and IgM antibodies to N-protein and RBD were noted. The amount of IgM to the N-protein increased sharply and reached a high level, while the amount of IgG increased smoothly and did not show a high level. The opposite picture was observed for antibodies to RBD. The characteristic dynamics of IgG, measured using test systems withsorbed whole virion or recombinant spike proteins, suggests duration of the disease
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Sugo, T., S. Tanabe, K. Shinoda, and M. Matsuda. "MONOCLONAL ANTIBODIES THAT RECOGNIZE Ca2+-INDUCED CONFORMER OF PROTEIN C, INDEPENDENT OF GLA RESIDUES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643644.
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Monoclonal antibodies (MCA’s) were prepared against human protein C (PC) according to Köhler & Milstein, and those that recognize the Ca2+-dependent PC conformers were screened by direct ELISA in the presence of 2 mM either CaCl2 or EDTA. Out of nine MCAߣs thus screened, five MCA's designated as HPC-1˜5, respectively, were found to react with PC in the presence of Ca2+ but not EDTA. By SDS-PAGE coupled with Western Blotting performed in the presence of 2 mM CaCl2, we found that two MCA’s HPC-1 and 2, recognized the light chain, and two others, HPC-3 and 4, recognized the heavy chain of PC. But another MCA, HPC-5 was found to react with only non-reduced antigens. Further study showed that HPC-1 and 5 failed to react with the Gla-domainless PC, i.e. PC from which the N-terminal Gla-domain of the light chain had been cleaved off by α-chymotrypsin. However, all the other three MCA's retained the reactivity with the antigen in the presence of Ca2+ even after the Gla-domain had been removed. The binding of these MCA’s to PC in the presence of Ca2+ was found to be saturable with respect to the Ca2+ concentration and the half maximal binding for each MCA was calculated to be about 0.5+mM. Moreover, many other divalent cations such as Mg2+, Mn2+ , Ba2+, Zn2+, Co2+, Sr2+, were found to substitute for Ca2+ in inducing the metal ion-dependent but Gla-domain-independent conformer of PC.Cross-reactivity to other vitamin K-aependent plasma proteins was examined by direct ELISA; HPC-2 and 3 reacted solely to PC, but HPC-1 and 4 also reacted with prothrombin and HPC-5 with both prothrombin and factor X.These findings indicated that there are two or more metal binding sites besides the Gla-domain, possibly one in the light chain and the other(s) in the heavy chain. The presence of these metal binding sites may contribute to the unique conformer of vitamin K-dependent plasma proteins including protein C.
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Mallakin, Ali, Kazushi Inoue, and Martin Guthold. "In-Situ Quantitative Analysis of Tumor Suppressor Protein (hDMP1) Using a Nanomechanical Cantilever Beam." In ASME 2005 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference. ASMEDC, 2005. http://dx.doi.org/10.1115/detc2005-84503.
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This study is focused on testing “immuno-sensors” of micro-cantilever beams for the purpose of future design of high-throughout bioassays. We currently study the aberrant expression, deletion and mutation of hDMP1 (Human DMP1) in human lung cancer. Lung cancer is the leading cause of cancer deaths among men and women in North America. There are four major histological subtypes of human lung cancer: small-cell carcinoma (SCC), adenocarcinoma (AC), squamous cell carcinoma (SCC), and large-cell carcinoma (LCC). The hDMP1 locus is localized on chromosome 7q21, a region frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of acute myeloid leukemia and myelodysplastic syndrome. Recent data demonstrate the critical role of Dmp1 in Ras-Raf-Arf signaling and cellular senescence. In order to study the interactions of hDMP1 gene product and selected tumor markers with BioMEMS devices, protein coating (Antibody) conduct on cantilevers with silicon nitride (SiNx) surfaces. Silicon nitride surface has the potential to provide a good interface for BioMEMS devices, due to enhanced adherence of substances on this surface. The cantilever biosensors coated with hDMP1 antibody were used for the detection of hDMP1 antigen, which is known to be a tumor suppressor protein. Results revealed that the changes in nano-mechanical forces provided sufficient differential torque to bend the cantilever beam upon injection of the antigen. Theoretical models have been developed for the prediction of the vibrational responses of atomic force microscope (AFM) cantilevers before and after antigen/antibody interaction. Exposure of the proteins to micro-cantilever (MC) resulted in un-reversible large stress. Static deflection of micro-cantilever appeared as a result of the surface stresses that are induced by changes upon antibody-antigen interaction. This indicated that the micro-cantilever approach is useful for detecting proteins and tumor markers, and this system is applicable as a model to design miniaturized biosensor systems. We also applied gene micro-array to identify unknown targets for hDMP1 and extend our observation of the complicated process of carcinogenesis.
Reports on the topic "Proteine antigelo"
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Bercovier, Herve, Raul Barletta, and Shlomo Sela. Characterization and Immunogenicity of Mycobacterium paratuberculosis Secreted and Cellular Proteins. United States Department of Agriculture, 1996. http://dx.doi.org/10.32747/1996.7573078.bard.
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Our long-term goal is to develop an efficient acellular vaccine against paratuberculosis based on protein antigen(s). A prerequisite to achieve this goal is to analyze and characterize Mycobacterium paratuberculosis (Mpt) secreted and cellular proteins eliciting a protective immune response. In the context of this general objective, we proposed to identify, clone, produce, and characterize: the Mpt 85B antigen and other Mpt immunoreactive secreted proteins, the Mpt L7/L12 ribosomal protein and other immunoreactive cellular proteins, Mpt protein determinants involved in invasion of epithelial cells, and Mpt protein antigens specifically expressed in macrophages. Paratuberculosis is still a very serious problem in Israel and in the USA. In the USA, a recent survey evaluated that 21.6% of the dairy herd were infected with Mpt resulting in 200-250 million dollars in annual losses. Very little is known on the virulence factors and on protective antigens of Mpt. At present, the only means of controlling this disease are culling or vaccination. The current vaccines do not allow a clear differentiation between infected and vaccinated animals. Our long-term goal is to develop an efficient acellular paratuberculosis vaccine based on Mpt protein antigen(s) compatible with diagnostic tests. To achieve this goal it is necessary to analyze and characterize secreted and cellular proteins candidate for such a vaccine. Representative Mpt libraries (shuttle plasmid and phage) were constructed and used to study Mpt genes and gene products described below and will be made available to other research groups. In addition, two approaches were performed which did not yield the expected results. Mav or Mpt DNA genes that confer upon Msg or E. coli the ability to invade and/or survive within HEp-2 cells were not identified. Likewise, we were unable to characterize the 34-39 kDa induced secreted proteins induced by stress factors due to technical difficulties inherent to the complexity of the media needed to support substantial M. pt growth. We identified, isolated, sequenced five Mpt proteins and expressed four of them as recombinant proteins that allowed the study of their immunological properties in sensitized mice. The AphC protein, found to be up regulated by low iron environment, and the SOD protein are both involved in protecting mycobacteria against damage and killing by reactive oxygen (Sod) and nitrogen (AhpC) intermediates, the main bactericidal mechanisms of phagocytic cells. SOD and L7/L12 ribosomal proteins are structural proteins constitutively expressed. 85B and CFP20 are both secreted proteins. SOD, L7/L12, 85B and CFP20 were shown to induce a Th1 response in immunized mice whereas AphC was shown by others to have a similar activity. These proteins did not interfere with the DTH reaction of naturally infected cows. Cellular immunity provides protection in mycobacterial infections, therefore molecules inducing cellular immunity and preferentially a Th1 pathway will be the best candidate for the development of an acellular vaccine. The proteins characterized in this grant that induce a cell-mediated immunity and seem compatible with diagnostic tests, are good candidates for the construction of a future acellular vaccine.
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Becker, Yechiel, Richard Witter, and Mertyn Malkinson. Studies on Marek's Disease Virus Antigen B Proteins and Gene. United States Department of Agriculture, 1992. http://dx.doi.org/10.32747/1992.7599674.bard.
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Vakharia, Vikram, Shoshana Arad, Yonathan Zohar, Yacob Weinstein, Shamila Yusuff, and Arun Ammayappan. Development of Fish Edible Vaccines on the Yeast and Redmicroalgae Platforms. United States Department of Agriculture, 2013. http://dx.doi.org/10.32747/2013.7699839.bard.
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Betanodaviruses are causative agents of viral nervous necrosis (VNN), a devastating disease of cultured marine fish worldwide. Betanodavirus (BTN) genome is composed of two single-stranded, positive-sense RNA molecules. The larger genomic segment, RNA1 (3.1 kb), encodes the RNA-dependent RNA polymerase, while the smaller genomic segment, RNA 2 (1.4kb), encodes the coat protein. This structural protein is the host-protective antigen of VNN which assembles to form virus-like particles (VLPs). BTNs are classified into four genotypes, designated red-spotted grouper nervous necrosis virus (RGNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), and striped jack nervous necrosis virus (SJNNV), based on phylogenetic analysis of the coat protein sequences. RGNNV type is quite important as it has a broad host-range, infecting warm-water fish species. At present, there is no commercial vaccine available to prevent VNN in fish. The general goal of this research was to develop oral fish vaccines in yeast and red microalgae (Porphyridium sp.) against the RGNNV genotype. To achieve this, we planned to clone and sequence the coat protein gene of RGNNV, express the coat protein gene of RGNNV in yeast and red microalgae and evaluate the immune response in fish fed with recombinantVLPs antigens produced in yeast and algae. The collaboration between the Israeli group and the US group, having wide experience in red microalgae biochemistry, molecular genetics and large-scale cultivation, and the development of viral vaccines and eukaryotic protein expression systems, respectively, was synergistic to produce a vaccine for fish that would be cost-effective and efficacious against the betanodavirus infection.
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Oldstone, Michael B. Proteins of Human Immunodeficiency Virus that Cross-React with Human 'Self' Antigens. Defense Technical Information Center, 1991. http://dx.doi.org/10.21236/ada246936.
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McElwain, Terry F., Eugene Pipano, Guy H. Palmer, Varda Shkap, Stephn A. Hines, and Wendy C. Brown. Protection of Cattle against Babesiosis: Immunization against Babesia bovis with an Optimized RAP-1/Apical Complex Construct. United States Department of Agriculture, 1999. http://dx.doi.org/10.32747/1999.7573063.bard.
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Previous research and current efforts at control of babesiosis fall short of meeting the needs of countries where the disease is endemic, such as Israel, as well as the needs of exporting countries and countries bordering on endemic areas, such as the U.S. Our long-term goal is to develop improved methods of immunization against bovine babesiosis based on an understanding of the molecular mechanisms of immune protection and parasite targets of a protective immune response. In our previous BARD project, we established the basis for focusing on rhoptry antigens as components of a subunit vaccine against bovine babesiosis, and for additional research to better characterize rhoptry associated protein-1 (RAP-1) as a target of protective immunity. In this continuation BARD project, our objectives were to [1] optimize the immune response against RAP-1, and [2] identify additional rhoptry candidate vaccine antigens. The entire locus encoding B. bovis RAP-1 was sequenced, and the rap-1 open reading frame compared among several strains. Unlike B. bigemina, in which multiple gene copies with variant domains encode RAP-1, the B. bovis RAP-1 locus contains only two identical genes which are conserved among strains. Through testing of multiple truncated constructs of rRAP-1, one or more immunodominant T cell epitopes were mapped to the amino terminal half of RAP-1. At least one linear and one conformational B cell epitope have been demonstrated in the same amino terminal construct, which in B. bigemina RAP-1 also contains an epitope recognized by neutralizing antibody. The amine terminal half of the molecule represents the most highly conserved part of the gene family and contains motifs conserved broadly among the apicomplexa. In contrast, the carboxy terminal half of B. bovis RAP-1 is less well conserved and contains multiple repeats encoding a linear B cell epitope potentially capable of inducing an ineffective, T cell independent, type 2 immune response. Therefore, we are testing an amino terminal fragment of RAP-1 (RAP-1N) in an immunization trial in cattle. Cattle have beer immunized with RAP-1N or control antigen, and IL-12 with Ribi adjuvant. Evaluation of the immune response is ongoing, and challenge with virulent B. bovis will occur in the near future. While no new rhoptry antigens were identified, our studies did identify and characterize a new spherical body antigen (SBP3), and several heat shock proteins (HSP's). The SBP3 and HSP21 antigens stimulate T cells from immune cattle and are considered new vaccine candidates worthy of further testing. Overall, we conclude that a single RAP-1 vaccine construct representing the conserved amino terminal region of the molecule should be sufficient for immunization against all strains of B. bovis. While results of the ongoing immunization trial will direct our next research steps, results at this time are consistent with our long term goal of designing a subunit vaccine which contains only the epitopes relevant to induction of protective immunity. Parallel studies are defining the mechanisms of protective immunity. Apicomplexan protozoa, including babesiosis and malaria, cause persistent diseases for which control is inadequate. The apical organelles are defining features of these complex protozoa, and have been conserved through the evolutionary process, Past and current BARD projects on babesiosis have established the validity and potential of exploiting these conserved organelles in developing improved control methods applicable to all apicomplexan diseases.
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Lillehoj, Hyun, Dan Heller, and Mark Jenkins. Cellular and molecular identification of Eimeria Acervulina Merozoite Antigens eliciting protective immunity. United States Department of Agriculture, 1992. http://dx.doi.org/10.32747/1992.7561056.bard.
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Coccidiosis, ubiquitous diseases of poultry, seriously impair the growth and feed utilization of livestock and poultry. Coccidiosis causes over $600 million annual losses world-wide and no vaccine is currently available. The goal of this study was to investigate the cellular and molecular mechanisms controlling protective immune responses to coccidia parasites in order to develop immunological control strategy against coccidiosis. The major findings of this study were: 1) cell-mediated immunity plays a major role in protection against coccidiosis, 2) when different genetic lines showing different levels of disease susceptibility were compared, higher T-cell response was seen in the strains of chickens showing higher disease resistance, 3) early interferon secretion was observed in more coccidia-resistant chicken strains, 4) both sporozoite and merozoite antigens were able to induce interferon production, and 5) chicken monoclonal antibodies which detect immunogenic coccidia proteins have been developed. This study provided a good background work for future studies toward the development of recombinant coccidial vaccine. Availability of chicken monoclonal antibodies which detect immunogenic coccidia proteins will enhance our ability to identify potential coccidial vaccine antigens.
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McClure, Michael A., Yitzhak Spiegel, David M. Bird, R. Salomon, and R. H. C. Curtis. Functional Analysis of Root-Knot Nematode Surface Coat Proteins to Develop Rational Targets for Plantibodies. United States Department of Agriculture, 2001. http://dx.doi.org/10.32747/2001.7575284.bard.
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The goal of this research was to provide a better understanding of the interface between root-knot nematodes, Meloidogyne spp., and their host in order to develop rational targets for plantibodies and other novel methods of nematode control directed against the nematode surface coat (SC). Specific objectives were: 1. To produce additional monoclonal SC antibodies for use in Objectives 2, 3, and 4 and as candidates for development of plantibodies. 2. To determine the production and distribution of SC proteins during the infection process. 3. To use biochemical and immunological methods to perturbate the root-knot nematode SC in order to identify SC components that will serve as targets for rationally designed plantibodies. 4. To develop SC-mutant nematodes as additional tools for defining the role of the SC during infection. The external cuticular layer of nematodes is the epicuticle. In many nematodes, it is covered by a fuzzy material termed "surface coat" (SC). Since the SC is the outermost layer, it may playa role in the interaction between the nematode and its surroundings during all life stages in soil and during pathogenesis. The SC is composed mainly of proteins, carbohydrates (which can be part of glycoproteins), and lipids. SC proteins and glycoproteins have been labeled and extracted from preparasitic second-stage juveniles and adult females of Meloidogyne and specific antibodies have been raised against surface antigens. Antibodies can be used to gain more information about surface function and to isolate genes encoding for surface antigens. Characterization of surface antigens and their roles in different life-stages may be an important step towards the development of alternative control. Nevertheless, the role of the plant- parasitic nematode's surface in plant-nematode interaction is still not understood. Carbohydrates or carbohydrate-recognition domains (CROs) on the nematode surface may interact with CROs or carbohydrate molecules, on root surfaces or exudates, or be active after the nematode has penetrated into the root. Surface antigens undoubtedly play an important role in interactions with microorganisms that adhere to the nematodes. Polyclonal (PC) and monoclonal (MC) antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes, were used to characterize the surface coat and secreted-excreted products of M. javanica and M. incognita. Some of the MC and PC antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Further characterization, in planta, of the epitopes recognized by the antibodies, showed that they were present in the parasitic juvenile stages and that the surface coat is shed during root penetration by the nematode and its migration between root cells. At the molecular level, we have followed two lines of experimentation. The first has been to identify genes encoding surface coat (SC) molecules, and we have isolated and characterized a small family of mucin genes from M. incognita. Our second approach has been to study host genes that respond to the nematode, and in particular, to the SC. Our previous work has identified a large suite of genes expressed in Lycopersicon esculentum giant cells, including the partial cDNA clone DB#131, which encodes a serine/threonine protein kinase. Isolation and predicted translation of the mature cDNA revealed a frame shift mutation in the translated region of nematode sensitive plants. By using primers homologous to conserved region of DB#131 we have identified the orthologues from three (nematode-resistant) Lycopersicon peruvianum strains and found that these plants lacked the mutation.
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Pleva, Christina M., Tracey A. Hamilton, John P. Petrali, and Robert K. Kan. Determining Optimal Microwave Antigen Retrieval Conditions for Microtubule-Associated Protein 2 Immunohistochemistry in the Guinea Pig Brain. Defense Technical Information Center, 2002. http://dx.doi.org/10.21236/ada417833.
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Gershoni, Jonathan M., David E. Swayne, Tal Pupko, et al. Discovery and reconstitution of cross-reactive vaccine targets for H5 and H9 avian influenza. United States Department of Agriculture, 2015. http://dx.doi.org/10.32747/2015.7699854.bard.
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Research objectives: Identification of highly conserved B-cell epitopes common to either H5 or H9 subtypes of AI Reconstruction of conserved epitopes from (1) as recombinantimmunogens, and testing their suitability to be used as universal vaccine components by measuring their binding to Influenza vaccinated sera of birds Vaccination of chickens with reconstituted epitopes and evaluation of successful vaccination, clinical protection and viral replication Development of a platform to investigate the dynamics of immune response towards infection or an epitope based vaccine Estimate our ability to focus the immune response towards an epitope-based vaccine using the tool we have developed in (D) Summary: This study is a multi-disciplinary study of four-way collaboration; The SERPL, USDA, Kimron-Israel, and two groups at TAU with the purpose of evaluating the production and implementation of epitope based vaccines against avian influenza (AI). Systematic analysis of the influenza viral spike led to the production of a highly conserved epitope situated at the hinge of the HA antigen designated “cluster 300” (c300). This epitope consists of a total of 31 residues and was initially expressed as a fusion protein of the Protein 8 major protein of the bacteriophagefd. Two versions of the c300 were produced to correspond to the H5 and H9 antigens respectively as well as scrambled versions that were identical with regard to amino acid composition yet with varied linear sequence (these served as negative controls). The recombinantimmunogens were produced first as phage fusions and then subsequently as fusions with maltose binding protein (MBP) or glutathioneS-transferase (GST). The latter were used to immunize and boost chickens at SERPL and Kimron. Furthermore, vaccinated and control chickens were challenged with concordant influenza strains at Kimron and SEPRL. Polyclonal sera were obtained for further analyses at TAU and computational bioinformatics analyses in collaboration with Prof. Pupko. Moreover, the degree of protection afforded by the vaccination was determined. Unfortunately, no protection could be demonstrated. In parallel to the main theme of the study, the TAU team (Gershoni and Pupko) designed and developed a novel methodology for the systematic analysis of the antibody composition of polyclonal sera (Deep Panning) which is essential for the analyses of the humoral response towards vaccination and challenge. Deep Panning is currently being used to monitor the polyclonal sera derived from the vaccination studies conducted at the SEPRL and Kimron.
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Brayton, Kelly A., Varda Shkap, Guy H. Palmer, Wendy C. Brown, and Thea Molad. Control of Bovine Anaplasmosis: Protective Capacity of the MSP2 Allelic Repertoire. United States Department of Agriculture, 2014. http://dx.doi.org/10.32747/2014.7699838.bard.
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Anaplasmosis is an arthropod-borne disease of cattle caused by the rickettsia Anaplasmamarginale and is an impediment to efficient production of healthy livestock in both Israel and the United States. Currently, the only effective vaccines are derived from the blood of infected cattle. The risk of widespread transmission of both known and newly emergent pathogens has prevented licensure of live blood-based vaccines in the U.S. and is a major concern for their continued use in Israel. Consequently, development of a safe, effective vaccine is a high priority. Despite its drawbacks as a live, blood-based vaccine, the Israel vaccine strain protects against disease upon challenge with wild-type A. marginale in extensive experimental trials and during 50 years of deployment in Israel. Field studies in Australia and Argentina indicate that this protection is broadly effective. Thus, to identify antigens for development of a safe and effective recombinant vaccine, we have used a comparative genomics approach by sequencing the Israel vaccine strain and searching for shared surface antigens with sequenced wild-type U.S. strains. We have focused on Msp2, the immune-dominant but antigenically variable surface protein, based on shared structure among strains and demonstration that antibody from cattle immunized with the Israel vaccine strain binds Msp2 from the genetically and geographically distinct U.S. St. Maries strain, consistent with the ability to protect against St. Maries challenge. Importantly, we have defined the full repertoire of Msp2 simple variants encoded by the vaccine strain and hypothesize that a recombinant vaccine encoding this full repertoire will induce protection equivalent to that induced by the live vaccine strain. Any escape from immunity by generation of complex Msp2 variants is predicted to carry a severe fitness cost that prevents high-level bacteremia and disease— consistent with the type of protection induced by the live vaccine strain. We tested the hypothesis that the Msp2 simple variant repertoires in wild-type A. marginale strains are recognized by antibody from cattle immunized with the Israel vaccine strain and that immunization with the vaccine strain Msp2 repertoire can recapitulate the protection provided by the vaccine strain upon challenge with Israel and U.S. strains of A. marginale. Our findings demonstrate that a set of conserved outer membrane proteins are recognized by immune serum from A. centrale vaccinated animals but that this set of proteins does not include Msp2. These findings suggest that “subdominant” immunogens are required for vaccine induced protection.