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Epitope Mapping (epitope + mapping)
Selected AbstractsBet,v,1, the major birch pollen allergen, initiates sensitization to Api,g,1, the major allergen in celery: evidence at the T,cell levelEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2003Barbara Bohle Abstract Due to IgE cross-reactivity, birch pollen-allergic individuals frequently develop type,I hypersensitivity reactions to celery tuber. We evaluated the T,cell response to the major allergen in celeriac, Api,g,1, and the cellular cross-reactivity with its homologous major allergen in birch pollen, Bet,v,1. Api,g,1-specific T,cell lines (TCL) and clones (TCC) were established from peripheralblood mononuclear cells of allergic patients. Epitope mapping of Api,g,1 with overlapping Api,g,1-derived peptides revealed one dominant T,cell-activating region, Api,g,1109,126. TCL and TCC generated with Api,g,1 cross-reacted with the birch pollen allergen and, although initially stimulated with the food allergen, cellular responses to Bet,v,1 were stronger than to Api,g,1. Epitopemapping with Bet,v,1-derived peptides revealed that T,cells specific for several distinct epitopes distributed over the complete Bet,v,1 molecule could be activated by Api,g,1. Bet,v,1109,126 was identified as the most important T,cell epitope for cross-reactivity with Api,g,1. This epitope shares 72% amino acid sequence similarity with the major T,cell-activating region of the food allergen, Api,g,1109,126. Our data provide evidence that humoral as well as cellular reactivity to the major celery allergen is predominantly based on cross-reactivity with the major birch pollen allergen. The activation of Bet,v,1-specific Th2 cells by Api,g,1, in particular outside the pollen season, may have consequences for birch pollen-allergic individuals. [source] A new panel of NS1 antibodies for easy detection and titration of influenza A virus,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2010Zhihao Tan Abstract The non-structural protein NS1 of the influenza A virus is a good target for the development of diagnostic assays. In this study, three NS1 monoclonal antibodies (mAbs) were generated by using recombinant NS1 protein of H5N1 virus and found to bind both the native and denatured forms of NS1. Two of the mAbs, 6A4 and 2H6, bind NS1 of three different strains of influenza A virus, namely H1N1, H3N2, and H5N1. Epitope mapping revealed that residues 42,53 of H5N1 NS1 are essential for the interaction with both mAbs. Between the three strains, there is only one amino acid difference in this domain, which is consistent with the observed cross-reactivities. On the other hand, mAb 1G1 binds to residues 206,215 of H5N1 NS1 and does not bind NS1 of H1N1 or H3N2. Furthermore, all three mAbs detected NS1 proteins expressed in virus infected MDCK cells and indirect immunofluorescence staining with mAbs 6A4 and 2H6 provided an alternative method for viral titer determination. Quantifying the numbers of fluorescent foci units yielded viral titers for three different isolates of H5N1 virus that are highly comparable to that obtained by observing cytopathic effect induced by virus infection. Importantly, this alternative method yields results at 1 day post-infection while the conventional method using cytopathic effect yields results at 3 days post-infection. The results showed that this new panel of NS1 antibodies can detect NS1 protein expressed during viral infection and can be used for fast and easy titration of influenza A virus. J. Med. Virol. 82:467,475, 2010. © 2010 Wiley-Liss, Inc. [source] Epitope mapping of the neuronal growth inhibitor Nogo-A for the Nogo receptor and the cognate monoclonal antibody IN-1 by means of the SPOT techniqueJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2007Hilke Zander Abstract Nogo-A is a potent inhibitor of axonal outgrowth in the central nervous system of adult mammals, where it is expressed as a membrane protein on oligodendrocytes and in myelin. Here we describe an attempt to identify linear peptide epitopes in its sequence that are responsible for the interaction either with the Nogo receptor (NgR) or with the neutralizing monoclonal antibody IN-1. Analysis of an array of immobilized overlapping 15,mer peptides covering the entire amino acid sequence of human Nogo-A (1192 residues) revealed a single epitope with prominent binding activity both towards the recombinant NgR and the IN-1 Fab fragment. Further truncation and substitution analysis yielded the minimal epitope sequence 'IKxLRRL' (x,,,P), which occurs within the so-called Nogo66 region (residues 1054,1120) of Nogo-A. The bacterially produced Nogo66 fragment exhibited binding activity both for the recombinant NgR and for the IN-1 Fab fragment on the Western blot as well as in ELISA. Unexpectedly, the synthetic epitope peptide and the recombinant Nogo66 showed cross-reactivity with the 8-18C5 Fab fragment, which is directed against myelin oligodendrocyte glycoprotein (MOG) as a structurally unrelated target. On the other hand, the recombinant N-terminal domain of Nogo-A (residues 334,966) was shown to specifically interact on the Western blot and in an ELISA with the IN-1 Fab fragment but not with the recombinant NgR, which is in agreement with previous results. Hence, our data suggest that there is a distinct binding site for the Nogo receptor in the Nogo66 region of Nogo-A, whereas its interaction with NgR is less specific than anticipated before. Although there probably exists a non-linear epitope for the neutralizing antibody IN-1 in the N-terminal region of Nogo-A, which is likely to be accessible from outside the cell, a previously postulated second binding site for NgR in this region (called Nogo-A-24) remains elusive. Copyright © 2007 John Wiley & Sons, Ltd. [source] Epitope mapping of canine distemper virus phosphoprotein by monoclonal antibodiesMICROBIOLOGY AND IMMUNOLOGY, Issue 12 2009Akihiro Sugai ABSTRACT The gene for phosphoprotein (P) of CDV encodes three different proteins, P, V, and C. The P protein is involved in viral gene transcription and replication. In the present study, we produced MAbs against a unique domain of the CDV-P protein, from aa 232 to 507, and determined their antigenic sites. By immunizing BALB/c mice with the recombinant P protein-specific fragment, we obtained six MAbs. Competitive binding inhibition assays revealed that they recognized two distinct regions of the P protein. Western blot analysis and immunofluorescence assays using deletion mutants of the unique C-terminus of the CDV-P protein revealed that all MAbs recognized a central short region (aa 233,303) of the CDV-P protein. In addition, linear and conformational epitopes have been determined, and at least four antigenic sites exist in the P protein central region. Furthermore, four of the MAbs were found to react with the P protein of recent Japanese field isolates but not with that of the older CDV strains, including a vaccine strain. Thus, these MAbs could be clinically useful for quick diagnosis during the CDV outbreaks. [source] Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved proteinPROTEIN SCIENCE, Issue 6 2002Abel Baerga-Ortiz Abstract The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen. [source] Antiplectin autoantibodies in subepidermal blistering diseasesBRITISH JOURNAL OF DERMATOLOGY, Issue 4 2009J.J.A. Buijsrogge Summary Background, Hemidesmosomal proteins may become targets of autoimmunity in subepidermal blistering diseases. Well-known recognized autoantigens are the intracellular plaque protein BP230, the transmembrane BP180 and its shed ectodomain LAD-1. Objectives, To establish the prevalence of autoimmunity against plectin, another intracellular plaque protein, and to investigate its antigenic sites. Methods, Two hundred and eighty-two patients with subepidermal blistering diseases, investigated by routine immunoblot analysis for possible antiplectin antibodies, were included in the study. Epitope mapping was performed using recombinantly produced overlapping plectin domains from the actin-binding domain to the rod domain. The COOH-terminal region of plectin was not included in the study. Results, In 11 of 282 (3·9%) patients an immunoblot staining pattern identical to that of antiplectin monoclonal antibody HD121 was found. Affinity-purified antibodies bound back to normal human skin in a pattern typical for plectin, i.e. to the epidermal basement membrane zone as well as to keratinocytes in the epidermis, and to myocytes. No binding was seen to plectin-deficient skin of a patient with epidermolysis bullosa simplex with muscular dystrophy. Epitope mapping of the plectin molecule showed that the central coiled-coil rod domain is an immunodominant hotspot as 92% of the sera with antiplectin antibodies reacted with it. Most patients with antiplectin antibodies also had antibodies to other pemphigoid antigens. Conclusions, Plectin is a minor pemphigoid antigen with an immunodominant epitope located on the central rod domain. [source] Immunological characterization of a Blo t 12 isoallergen: identification of immunoglobulin E epitopesCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2009J. Zakzuk Summary Background Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens. Objective To evaluate the differences in the IgE response against two Blo t 12 isoallergens. Methods The IgE-binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross-inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin-binding domain of the two isoallergens. Results The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE-binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin-binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes. Conclusion In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE-reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy. [source] Random mutagenesis of Legionella pneumophila reveals genes associated with lipopolysaccharide synthesis and recognition by typing monoclonal antibodiesJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2007C. Wagner Abstract Aims:, To use random mutagenesis for the characterization of Legionella pneumophila lipopolysaccharide (LPS) components and serotypes. Methods and Results:, Five strains belonging to different serogroups and/or monoclonal subgroups were mutagenized using a mini-Tn10 transposon. Exactly 11 819 mutants were checked for alterations in LPS using at least 11 monoclonal antibodies (mAbs) that define L. pneumophila serotypes. Among the mutants, five different mini-Tn10 insertions were identified. Four mutants originating from serogroup-1 did not lose their serogroup-specific epitope, but did sustain subtler changes that resulted in switches to different mAb subgroups. In contrast, a mutant from serogroup-6 lost its serogroup-specific epitope, while retaining a serogroup-cross-reacting epitope. Conclusions:, Random mutagenesis is a valuable tool for LPS epitope mapping. While some characteristics of L. pneumophila LPS can be altered, others appear resistant to mutagenesis. This underscores both the flexibility and rigidity of LPS architecture in L. pneumophila. Significance and Impact of the Study:, Losses of L. pneumophila LPS epitopes can result in new serotypes, changes that might escape detection by current DNA-based typing schemes. But, as the frequency of these changes is rare, based upon our observations, serotyping should remain an important tool for identifying L. pneumophila in water systems that are implicated in human infection. [source] Systematic epitope analysis of the p26 EIAV core proteinJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2007Adriana Soutullo Abstract The major core protein of equine infectious anemia virus (EIAV), p26, is one of the primary immunogenic structural proteins during a persistent infection of horses and is highly conserved among antigenically variants of viral isolates. In order to investigate its immune profile in more detail for a better diagnostic, an epitope mapping was carried out by means of two libraries of overlapping peptide fragments prepared by simultaneous and parallel SPPS on derivatized cellulose membranes (SPOT synthesis). Polyclonal equine sera from infected horses were used for the biological assay. Particularly two promising continuous epitopes (NAMRHL and MYACRD) were localized on the C-terminal extreme of p26, region 194,222. A cyclic synthetic fragment of 29 amino acid residues containing the identified epitopes was designed and studied. A significant conformational change towards a helical structure was observed when the peptide was cyclized by a bridge between Cys198 and Cys218. This observation correlated with an improvement of its ability to be recognized by specific antibodies in an EIA (Enzyme-linked Immunosorbent assay). These results suggest that the conformationally restricted synthetic antigen adequately mimics the native structure of this region of p26 core protein. Copyright © 2007 John Wiley & Sons, Ltd. [source] Discovery of novel mechanisms and molecular targets for the inhibition of activated thrombin activatable fibrinolysis inhibitorJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2008K. HILLMAYER Summary.,Background:,Thrombin activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis and an attractive target to develop profibrinolytic drugs. Objective:,To analyze the (inhibitory) properties of five monoclonal antibodies (mAbs) directed towards rat TAFI (i.e. MA-RT13B2, MA-RT30D8, MA-RT36A3F5, MA-RT36B2 and MA-RT82F12). Methods and results:,Direct interference of the mAb with rat activated TAFI (TAFIa) activity was assayed using a chromogenic activity assay. This revealed reductions of 79% ± 1%, 54% ± 4%, and 19% ± 2% in activity in the presence of a 16-fold molar excess of MA-RT13B2, MA-RT36A3F5, and MA-RT82F12, respectively whereas MA-RT30D8 and MA-RT36B2 had no direct inhibitory effect. Additionally, MA-RT13B2 and MA-RT36A3F5 reduced rat TAFIa half-life by 56% ± 2% and 61% ± 3%. Tissue-type plasminogen activator mediated in vitro clot lysis was determined using rat plasma. Compared to potato tuber carboxypeptidase inhibitor, MA-RT13B2, MA-RT30D8, MA-RT36A3F5, and MA-RT82F12 reduced clot lysis times by 86% ± 14%, 100% ± 5%, 100% ± 10%, and 100% ± 11%, respectively. During epitope mapping, Arg227 and Ser251 were identified as major residues interacting with MA-RT13B2. Arg188 and His192 contribute to the interaction with MA-RT36A3F5. Arg227, Ser249, Ser251, and Tyr260 are involved in the binding of MA-RT30D8 and MA-RT82F12 with rat TAFI(a). The following mechanisms of inhibition have been deduced: MA-RT13B2 and MA-RT36A3F5 have a destabilizing effect on rat TAFIa whereas MA-RT30D8 and MA-RT82F12 partially block the access to the active site of TAFIa or interact with the binding of TAFIa to the blood clot. Conclusions:,The described inhibitory mAb towards rat TAFIa will facilitate TAFI research in murine models. Additionally, we reveal novel molecular targets for the direct inhibition of TAFIa through different mechanisms. [source] Classical sheep transmissible spongiform encephalopathies: pathogenesis, pathological phenotypes and clinical diseaseNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 4 2007M. Jeffrey Scrapie is a prion disease or transmissible spongiform encephalopathy (TSE) of sheep, goats and moufflon. As with its human counterparts, pathology consists of vacuolation, gliosis and accumulations of abnormal forms of a host prion protein (PrPd) in the brain of affected individuals. Immunohistochemical methods can be used to identify both the intracellular truncation sites of PrPd in different cell types (PrPd epitope mapping) and the different morphological patterns of accumulation (PrPd profiling). Differences in the inferred truncation sites of PrPd are found for different strains of sheep TSEs and for different infected cell types within individual strains. Immunochemical methods of characterizing strains broadly correspond to PrPd mapping discriminatory results, but distinct PrPd profiles, which provide strain- and source-specific information on both the cell types which sustain infection (cellular tropisms) and the cellular processing of PrPd, have no immunoblotting counterparts. The cause of neurological dysfunction in human is commonly considered to be neuronal loss secondary to a direct or indirect effect of the accumulation of PrPd. However, in sheep scrapie there is no significant neuronal loss, and relationships between different magnitudes, topographical and cytological forms of PrPd accumulation and clinical signs are not evident. PrPd accumulation also occurs in lymphoid tissues, for which there is indirect evidence of a pathological effect, in the peripheral nervous system and in other tissues. It is generally assumed that neuroinvasion results from infection of the enteric nervous system neurones subsequent to amplification of infectivity in lymphoid tissues and later spread via sympathetic and parasympathetic pathways. The evidence for this is, however, circumstantial. Accumulation of PrPd and presence of infectivity in tissues other than the nervous and lymphoreticular systems gives insights on the ways of transmission of infection and on food safety. [source] Human T and B cell epitope mapping of Taenia solium paramyosinPARASITE IMMUNOLOGY, Issue 11 2001José Vázquez-Talavera Taenia solium paramyosin is an immunodominant antigen in human and porcine cysticercosis that has shown promise as a vaccine candidate against schistosomiasis and some filariasis. There are few studies to identify the immunologically relevant regions of paramyosin. In this work, we characterize the humoral and cellular response of neurocysticercotic patients against T. solium paramyosin. Western blots using different recombinant fragments of T. solium paramyosin, showed that the sera from neurocysticercotic patients were strongly reactive against the carboxyl end region, with poor recognition of the central and amino regions. In contrast, the cellular immune response of patients did not show preferential recognition of any region of paramyosin. [source] Early targets of nuclear RNP humoral autoimmunity in human systemic lupus erythematosusARTHRITIS & RHEUMATISM, Issue 3 2009Brian D. Poole Objective The U1 small nuclear RNPs are common targets of autoantibodies in lupus and other autoimmune diseases. However, the etiology and progression of autoimmune responses directed against these antigens are not well understood. The aim of this study was to use a unique collection of serial samples obtained from patients before and after the development of nuclear RNP (nRNP) antibodies to investigate early humoral events in the development of anti-nRNP autoimmunity. Methods Lupus patients with sera available from both before and after the development of nRNP antibody precipitin were identified from the Oklahoma Clinical Immunology Serum Repository. Antibodies in the serial samples were analyzed by enzyme-linked immunosorbent assay, Western blotting, solid-phase epitope mapping, and competition assays. Results The first-detected nRNP antibodies targeted 6 common initial epitopes in nRNP A, 2 in nRNP C, and 9 in nRNP 70K. The initial epitopes of nRNP A and nRNP C were significantly enriched for proline and shared up to 95% sequence homology. The initial nRNP 70K humoral epitopes differed from those of nRNP A and nRNP C. The initial antibodies to nRNP A and nRNP C were cross-reactive with the SmB,-derived peptide PPPGMRPP. Antibody binding against all 3 nRNP subunits diversified significantly over time. Conclusion Autoantibodies to nRNP A and nRNP C initially targeted restricted, proline-rich motifs. Antibody binding subsequently spread to other epitopes. The similarity and cross-reactivity between the initial targets of nRNP and Sm autoantibodies identifies a likely commonality in cause and a focal point for intermolecular epitope spreading. [source] | ||||||||||||||||