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Epitope Analysis (epitope + analysis)
Selected AbstractsAntibody response against NY-ESO-1 in CHP-NY-ESO-1 vaccinated patientsINTERNATIONAL JOURNAL OF CANCER, Issue 10 2007Ryohei Kawabata Abstract NY-ESO-1 specific humoral responses are frequently observed in patients with various types of NY-ESO-1 antigen expressing tumors. In a large proportion of NY-ESO-1 antibody-positive patients of NY-ESO-1-specific CD8 T-cells can also be detected suggesting that monitoring of the NY-ESO-1 specific humoral immune response may be a relevant and more practical surrogate for estimating the overall immune response against NY-ESO-1 in clinical vaccine studies. We have immunized 9 cancer patients with full length NY-ESO-1 protein formulated with cholesterol-bearing hydrophobized pullulan (CHP-NY-ESO-1) and investigated the humoral immune responses against NY-ESO-1. Seven patients were NY-ESO-1 antibody-negative and 2 patients were positive prior to vaccination. Vaccination with CHP-NY-ESO-1 resulted in the induction or increase of NY-ESO-1 antibody responses in all 9 patients immunized. Epitope analysis revealed 5 regions in the NY-ESO-1 protein molecule that were recognized by antibodies induced after vaccination. The 5 regions were also recognized by antibodies present in nonvaccinated, NY-ESO-1 antibody-positive cancer patients. A peptide spanning amino acids 91,108 was recognized in 6 out of 9 vaccinated patients and in 8 out of 9 nonvaccinated, sero-positive patients, being the most dominant antigenic epitope in NY-ESO-1 for antibody recognition in cancer patients. In conclusion, we showed that CHP-NY-ESO-1 protein vaccination had a potent activity for inducing humoral immune responses against NY-ESO-1 antigen in cancer patients. The antigenic epitopes recognized by antibodies in the vaccinated patients were similar to those recognized in cancer patients with spontaneous humoral immunity against NY-ESO-1. © 2007 Wiley-Liss, Inc. [source] Non-enzymatic developmental functions of acetylcholinesterase , the question of redundancyFEBS JOURNAL, Issue 20 2008Glynis Johnson Despite in vitro demonstrations of non-enzymatic morphogenetic functions in acetylcholinesterase (AChE), the AChE knockout phenotype is milder than might be expected, casting doubt upon the relevance of such functions in vivo. Functional redundancy is a possible explanation. Using in vitro findings that AChE is able to bind to laminin-111, together with detailed information about the interaction sites, as well as an epitope analysis of adhesion-inhibiting anti-AChE mAbs, we have used molecular docking and bioinformatics techniques to explore this idea, investigating structurally similar molecules that have a comparable spatiotemporal expression pattern in the embryonic nervous system. On this basis, molecules with which AChE could be redundant are the syndecans, glypicans, perlecan, the receptor tyrosine kinase Mer, and the low-density lipoprotein receptor. It is also highly likely that AChE may be redundant with the homologous neuroligins, although there is no evidence that the latter are expressed before synaptogenesis. AChE was observed to dock with Gas6, the ligand for Mer, as well as with apolipoprotein E3 (but not apolipoprotein E4), both at the same site as the laminin interaction. These findings suggest that AChE may show direct functional redundancy with one or more of these molecules; it is also possible that it may itself have a unique function in the stabilization of the basement membrane. As basement membrane molecules are characterized by multiple molecular interactions, each contributing cumulatively to the construction and stability of the network, this may account for AChE's apparently promiscuous interactions, and also for the survival of the knockout. [source] Investigations into the development of catalytic activity in anti-acetylcholinesterase idiotypic and anti-idiotypic antibodiesJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009Glynis Johnson Abstract We have previously described anti-acetylcholinesterase antibodies that display acetylcholinesterase-like catalytic activity. No evidence of contaminating enzymes was found, and the antibodies are kinetically and apparently structurally distinct from both acetylcholinesterase (AChE) and butyrylcholinesterase. We have also mimicked the antibody catalytic sites in anti-anti-idiotypic (Ab3) antibodies. Independently from us, similar acetylcholinesterase-like antibodies have been raised as anti-idiotypic (Ab2) antibodies against a non-catalytic anti-acetylcholinesterase antibody, AE-2. In this paper, we describe an epitope analysis, using synthetic peptides in ELISA and competition ELISA, and a peptide array, of five catalytic anti-acetylcholinesterase antibodies (Ab1s), three catalytic Ab3s, as well as antibody AE-2 and a non-catalytic Ab2. The catalytic Ab1s and Ab3s recognized three Pro- and Gly-containing sequences (40PPMGPRRFL, 78PGFEGTE, and 258PPGGTGGNDTELVAC) on the AChE surface. As these sequences do not adjoin in the AChE structure, recognition would appear to be due to cross-reaction. This was confirmed by the observation that the sequences superimpose structurally. The non-catalytic antibodies, AE-2 and the Ab2, recognized AChE's peripheral anionic site (PAS), in particular, the sequence 70YQYVD, which contains two of the site's residues. The crystal structure of the AChE tetramer (Bourne et al., 1999) shows direct interaction and high complementarity between the 257CPPGGTGGNDTELVAC sequence and the PAS. Antibodies recognizing the sequence and the PAS may, in turn, be complementary; this may account for the apparent paradox of catalytic development in both Ab1s and Ab2s. The PAS binds, but does not hydrolyze, substrate. The catalytic Ab1s, therefore, recognize a site that may function as a substrate analog, and this, together with the presence of an Arg-Glu salt bridge in the epitope, suggests mechanisms whereby catalytic activity may have developed. In conclusion, the development of AChE-like catalytic activity in anti-AChE Ab1s and Ab2s appears to be the result of a combination of structural complementarity to a substrate-binding site, charge complementarity to a salt bridge, and specific structural peculiarities of the AChE molecule. Copyright © 2008 John Wiley & Sons, Ltd. [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] Highly frequent anti-idiotype antibody in cynomolgus monkeys developed against mouse-derived regions of anti-Fas antibody humanized by complementarity determining region graftingBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2009M Saito-Yabe Background and purpose:, We investigated the immunogenicity of a humanized anti-human Fas monoclonal antibody, R-125224, in cynomolgus monkeys to estimate its efficacy, as well as its toxicity in clinical situations. Experimental approach:, R-125224 was intravenously administered to cynomolgus monkeys at single doses of 0.4, 1.2, 6 and 30 mg·kg,1, and the plasma concentrations of R-125224 and anti-R-125224 antibody (ARA) were measured. We conducted a competitive enzyme-linked immunosorbent assay to determine which part of R-125224 was recognized by ARA. We also examined the retention of radioactivity in mononuclear cells and granulocytes after the injection of [125I]-R-125224 to a collagen-induced arthritis monkey model. Key results:, After i.v. administration of R-125224, the elimination of the plasma R-125224 concentrations was accelerated at around 10 days post-dose, and 10 of 12 monkeys were ARA positive. From an epitope analysis of ARA, the ARA produced in monkeys recognized the mouse-derived regions located in complementarity determining regions, but could not recognize the human IgG. After the injection of [125I]-R-125224 to a collagen-induced arthritis monkey model, a significantly longer retention of the radioactivity in mononuclear cells compared to granulocytes was observed. Conclusions and implications:, In monkeys, the development of antibodies against R-125224 is rapid and highly frequent. Our hypothesis is that this highly frequent development of ARA might be due to the binding of R-125224 to immune cells, and its circulation in monkey blood might contribute to an increase in its chances of being recognized as an immunogen. [source] Identification of immunoglobulin E (IgE)-binding epitopes and recombinant IgE reactivities of a latex cross-reacting Indian jujube Ziz m 1 allergenCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2008M. F. Lee Summary Ziz m 1 is a major Indian jujube (Zizyphus mauritiana) allergen involved in latex-fruit syndrome, and cDNA of the allergen has been cloned, sequenced and expressed in yeast by our laboratory previously. In this study, we performed an immunoglobulin E (IgE)-binding epitope analysis of Ziz m 1 using overlapping recombinant fragments. Eight overlapping recombinant fragments were generated from the recombinant Ziz m 1 allergen. The fragments were expressed in Escherichia coli and IgE-binding activities were evaluated by sera of latex,Indian jujube-allergic subjects and normal subjects using immunoblotting. Human allergic sera are not able to recognize fragments consisting of amino acid sequences 26,71, 119,280 and 119,291. However, residues at positions 26,199, 26,105, 26,86, 119,320 and 238,330 were found relevant in the IgE-binding. Our results indicate that 72NISGHCSDCTFLGEE86 and 292VWNRYYDLKTNYSSSIILEYVNSGTKYLP320 of Ziz m 1 are the sequences required for human IgE binding. Four corresponding peptides, 72NISGHCSDCTE86, 292VWNRYYDLKT301, 300KTNYSSSIILEY311 and 309LEYVNSGTKYLP320, were synthesized, and these peptides reacted with 70%, 100%, 70% and 70% of 10 allergic sera tested, as revealed by enzyme-linked immunosorbent assay. Sensitization to 292VWNRYYDLKT301 correlated significantly with the presence of allergic symptoms (P < 0·001). These findings will be useful in designing diagnostic and therapeutic approaches, thereby contributing to the development of specific immunotherapy for subjects with latex,fruit syndrome. [source] | |||||||||||||