Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Epitope Mapping”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,261 records · Page 70Linked to original sources
PubMed1995

Autoreactive epitopes within the human alpha-enolase and their recognition by sera from patients with endometriosis.

Patients with endometriosis significantly develop autoantibodies directed against endometrial proteins, which may be involved in the aetiology of this gynaecological disease. Based on standard Western blot analysis, a 48 kDa protein was localized in the soluble protein extract of endometrial adenocarcinoma cells using sera from patients with clinically staged endometriosis and identified as the glycolytic enzyme alpha-enolase. The corresponding cDNA coding for the human alpha-enolase was isolated from a human endometrial cDNA library and cloned into the vector pH6EX3, allowing the efficient expression of recombinant human alpha-enolase with an N-terminal histidine-hexapeptide as affinity ligand in Escherichia coli. The purified recombinant human alpha-enolase was evaluated as a specific antigenic tool for the diagnostic measurement of antiendometrial antibodies in sera from patients with endometriosis. With selected endometriosis sera, two linear autoreactive epitopes were localized within the recombinant human alpha-enolase using epitope mapping techniques, and they were characterized.

Adenocarcinoma↗
PubMed1993

Approaches for studying the pathogenic T cells in autoimmune patients.

Our provisional conclusions from this work are as follows. (1) For screening responses of established lines, native human AChR is not prohibitively scarce, especially if it is concentrated onto beads, and class II-transfected TE671 cells may be useful too; both may give vital evidence of AChR-specificity, but it is still crucial to confirm that with synthetic peptides. (2) For mapping epitopes, panels of full-length and shorter recombinant human polypeptides, and of synthetic peptides, are invaluable complementary material: longer peptides tend to stimulate particularly strongly. (3) Initial selection with pooled synthetic peptides can easily generate interesting lines from both patients and controls, but they may depend on the artificial processing sites that are an inevitable consequence of arbitrarily chosen start and stop points. Of course, these might conceivably be employed in unusual antigen-presenting cells (such as thymic myoid cells), so we cannot totally dismiss such "cryptic" epitopes. This system can sometimes select T cells responding to "natural" epitopes too, as now reported for tetanus toxin. Nevertheless, for these and other reasons, at present, we strongly favor using the longest human recombinant material possible, because it is apparently processed more naturally. This must be combined with rigorous screening for reactivity to E. coli-derived contaminants plus concomitant mapping of epitopes as above. Use of intact AChR for initiating lines may yet become feasible. (4) The T cells thus isolated and characterized so far are proving to be heterogeneous in the epitopes and presenting class II molecules they recognize, and in their T-cell receptor gene usage. It is premature to claim key myasthenogenic epitopes or clonotypes, but HLA-DR3 and the linked -DQw2 do not appear to monopolize presentation. (5) Assessing the disease-relevance of these T cells is a separate problem, highlighted by their apparent similarity in healthy controls. In the meantime, to test their potential pathogenicity, we are assaying their cytokine profiles and ability to help specific antibody production in vitro. In the hope that they do prove to be relevant, we are also using some of them to test possible therapeutic strategies that might prove applicable in the patients.

Amino Acid Sequence↗
PubMed1999

Analysis of monoclonal antibodies to prostate-specific antigen: reactivity with native and recombinant prostate-specific antigen.

Epitope mapping analysis was performed on 53 antibodies submitted to the ISOBM TD-3 PSA Workshop. Western blotting and N-terminal amino acid sequencing, using both native and recombinant human prostate-specific antigen (rPSA), identified four different epitope groups for native PSA. Under reducing conditions native PSA was not recognized by 18/53 antibodies suggesting they reacted with conformation-dependent epitopes. Nine other antibodies reacted with a rPSA polypeptide doublet of 34-35 kD corresponding to different rPSA glycoforms. From sequence mapping studies 22/53 antibodies bound epitopes within amino acid residues 25-85, 3/53 antibodies bound to epitopes within residues 86-220, while 10/53 antibodies bound epitopes within residues 221-261. These results indicate that there are multiple immunogenic epitopes localized on the PSA molecule.

Antibodies, Monoclonal↗
PubMed1998

Cellular and molecular characterization of a major soybean allergen.

Soybean proteins share a large number of cross-reacting allergens with other members of the legume family; however, soy-allergic patients rarely react clinically to other members of the legume family. Gly m Bd 30K, an IgE-binding protein with a molecular weight of 30 kD, was identified in soybean extracts by Western IgE-immunoblot analysis. This monomeric allergen was shown to have an N-terminal amino acid sequence and amino acid composition identical to that of the seed 34-kD protein, P34, a thiol protease of the papain family. Electron-microscopic immunolocalization of P34 monoclonal antibodies and IgE binding to sections of soybean seeds showed dense staining throughout the vacuolar bodies, localizing the allergens in protein storage vacuoles of seed cotyledons. We used pooled serum from soybean-sensitive patients to determine the linear IgE-specific epitopes in the 34-kD allergen amino acid sequence. B-cell epitope mapping revealed 10 regions of IgE-binding activity using an overlapping peptide strategy of 15-mers offset by 8 amino acids throughout the P34 sequence. Smaller overlapping peptides, 10-mers offset by 2 amino acids, revealed 16 distinct linear epitopes, 9 of which were mapped to the mature protein. No obvious amino acid sequence motifs could be identified by the smaller IgE-binding epitopes. Using individual patient serum, 5 immunodominant epitopes were identified in this allergen.

Allergens↗
PubMed2003

Human T-cells recognise N-terminally Fmoc-modified peptide.

We aimed to generate T-cell clones specific for human pre-proinsulin. An HLA DQ8, CD4+ T-cell clone that recognised a 10mer (C65-A9) peptide from pre-proinsulin was isolated. Further analysis revealed that the clone responded neither to recombinant proinsulin nor to re-synthesised C65-A9 peptide. Analysis of the original peptide revealed minor contamination (<0.5%) with an N-terminal Fmoc adduct. This peptide was synthesised and shown to stimulate the clone. Thus, Fmoc-modified peptides, which are common contaminants in synthetic peptides, can stimulate human CD4+ T-cells. This finding has important implications for the use of synthetic peptides in screening and epitope mapping studies and their use as vaccines in humans.

Autoantibodies↗
PubMed1998

Analysis of the processing of Plasmodium falciparum rhoptry-associated protein 1 and localization of Pr86 to schizont rhoptries and p67 to free merozoites.

The processing and localization of Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) products were examined using polyclonal and monoclonal antibodies raised to a recombinant protein containing residues 1-294 of RAP-1. Immunoblot and epitope mapping results with antibodies that selectively bound epitopes in the RAP-1 products Pr86, p82, and p67 showed that p82 and p67 are formed from Pr86 by progressive removal of epitopes from the amino-terminus of the RAP-1 coding sequence. The capacity of Pr86 to form complexes was revealed after size fractionation of parasite proteins radiolabeled in the presence of brefeldin A to prevent processing of Pr86. Fractions containing complexed Pr86 also contained the RAP-2 product p39 and the RAP-3 product p37, suggesting that Pr86, p39 and p37 may form complexes similar to complexes previously reported for p82 and p67 with p39 or p37. Immunofluorescence localization and immunoblot studies revealed that Pr86 is present in the rhoptries, but only transiently, and that it is not detected in segmenting schizonts or extracellular merozoites. p67 and p82, on the other hand, were shown to be major RAP-1 components in purified merozoites. Neither p67 nor p82 were relocalized from the intracellular rhoptries to the merozoite surface under conditions that promoted relocalization of the rhoptry protein PF83/apical membrane antigen 1. These results suggest that processing of Pr86 begins after Pr86 complexes are transported to the forming rhoptries and that two site-selective processing reactions occur in the rhoptries, a rapid cleavage of Pr86 to p82 and a delayed cleavage of p82 to p67. Since p67 is missing from ring-stage parasites (Howard et al., Am J Trop Med Hyg, 1984;33:1055 59), the present results indicate there is a narrow time during which p67 may play a role in merozoite invasion of erythrocytes.

Animals↗
PubMed1996

Studies on the epitope of pig zona pellucida recognized by a fertilization-blocking monoclonal antibody.

Heat solubilized pig zona pellucida (pZP) was separated into four major glycoprotein families (pZP1, pZP2, pZP3, pZP4). Cloning of a full-length cDNA coding for pZP1 revealed that the coding sequence for pZP4 was immediately followed by the sequence for the amino-terminal residue of pZP2 and that they shared a significant similarity with mouse and human ZP2, which are considered to serve as a secondary sperm receptor in sperm-zona interactions. One of the monoclonal antibodies, mAb-5H4, produced against pZP4 inhibited the binding of boar and human spermatozoa to each of the homologous oocytes. The epitope recognized by mAb-5H4 was determined to be present on a 10 amino acid sequence of pZP1(50-59) by using epitope mapping and analysis of chain flexibility of pZP4. A synthetic 18mer peptide corresponding to pZP1(50-67) reacted with mAb-5H4, and mouse antisera raised to the synthetic 18mer peptide recognized intact pig zona pellucida and strongly inhibited the fertilization of pig oocytes with boar spermatozoa in vitro. These results suggest that the pZP4 peptide (50-59) identified as an epitope for mAb-5H4 could be a promising candidate in the development of a contraceptive vaccine.

Animals↗
PubMed2006

Characterization of monoclonal antibodies directed against the rat neurotensin receptor NTS1.

G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a variety of ligands and represent major drug targets. Despite their medical importance, detailed structural information is limited because only one GPCR has been crystallized and its structure determined. To develop tools to aid in the formation of well-ordered crystals, we generated monoclonal antibodies with high affinity to the rat neurotensin receptor. All antibodies bound to the C-terminus of the receptor, which may reflect the selection strategy used to identify high-affinity binders. Further characterization revealed that some antibodies bound to the receptor in a sodium chloride sensitive manner, but others did not. Epitope mapping revealed distinct antigenic regions within the receptor C-terminus. Tight binding of Fab fragments to the receptor was verified by size exclusion chromatography.

Animals↗
PubMed2001

Analysis of prostate specific antigen and alpha1-antichymotrypsin interaction using antipeptide monoclonal antibodies.

PURPOSE: The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs). MATERIALS AND METHODS: Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane. RESULTS: Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA. CONCLUSIONS: The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.

Animals↗
PubMed2004

Basic immunological aspects of botulinum toxin therapy.

Previous studies in this laboratory had mapped the immune recognition profile of the regions recognized antibodies (Abs) and by T cells on the protective H(C) domain (C-terminal fragment corresponding to residues 855-1296 of the heavy chain) of botulinum neurotoxin serotype A (BoNT/A). The localization of these regions has several potential applications and has provided a basis for the understanding of immunoresistance to treatment. We briefly outline these localized regions and discuss the impact of these findings on the immunotherapeutic applications of BoNT/A. Immunoresistance to toxin therapy can appear in some patients after a few injections with the toxin. Our epitope mapping studies have shown that several factors can influence the immune response to the toxin. These factors include dose, duration of treatment, frequency of immunization, and quality of the toxin. The immune response to the whole toxin is under genetic control, and the response to each epitope is under separate genetic control. Therefore, the appearance of blocking Abs (i.e., immunoresistance) in patients might be controlled by the major histocompatability of the host. Once a patient becomes immunoresistant to one toxin then switching to another toxin will most often be of limited and short-lived benefit, because the patient becomes rapidly immunoresistant to the second toxin. Finally, because of the considerable structural homology between tetanus neurotoxin (TeNT) and BoNTs, it is possible, although not certain, that a prior active immune response to TeNT might play some role in the early appearance on anti-BoNT Abs in some patients.

Amino Acid Sequence↗
PubMed1991

Monoclonal antibodies define linear and conformational epitopes of HIV-1 pol gene products.

Purified recombinant reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to raise 21 monoclonal antibodies with anti-RT specificities. The antibodies were characterized using Western blotting against native virus and recognized either the p66 or p66, p51 components of RT. Further immunoblotting using either cyanogen bromide fragmented RT or truncated mutants of RT along with cross-competition studies enabled the location of various immunogenic regions of RT to be identified. Three antibodies recognized a linear epitope in the N-terminal region (amino acids 128-176). Also, a neutralizing RT antibody recognized a conformational epitope in this region. Three monoclonals had epitopes mapped to linear sequences in the RNase H region at the C-terminus of the RT. Another neutralizing antibody, also requiring folding of the RT protein had its epitope more centrally located (231-353). Of the remaining 13 monoclonals, 7 were roughly located in the C-terminal region and required folding of the protein for epitope recognition and only three of the remaining six could be mapped to conformational epitopes in N-terminal and central regions of the RT. None of the antibodies tested recognized HIV-2 RT products p68 and p55 in Western blot.

Animals↗
PubMed2004

Atomic mapping of the interactions between the antiviral agent cyanovirin-N and oligomannosides by saturation-transfer difference NMR.

The minimum oligosaccharide structure required for binding to the potent HIV-inactivating protein cyanovirin-N (CV-N) was determined by saturation-transfer difference (STD) NMR spectroscopy. Despite the low molecular mass of the protein (11 kDa), STD-NMR spectroscopy allowed the precise atomic mapping of the interactions between CV-N and various di- and trimannosides, substructures of Man-9, the predominant oligosaccharide on the HIV viral surface glycoprotein gp120. Contacts with mannosides containing the terminal Manalpha(1-->2)Manalpha unit of Man-9 were observed, while (1-->3)- and (1-6)-linked di- and trimannosides showed no interactions, demonstrating that the terminal Manalpha(1-->2)Manalpha structure plays a key role in the interaction. Precise epitope mapping revealed that, for Manalpha(1-->2)ManalphaOMe, Manalpha(1-->2)Manalpha(1-->3)ManalphaOMe, and Manalpha(1-->2)Manalpha(1-->6)ManalphaOMe, the protein is in close contact with H2, H3, and H4 of the nonreducing terminal mannose unit. In contrast, the STD-NMR spectrum of the CV-N/trisaccharide Manalpha(1-->2)Manalpha(1-->2)ManalphaOMe complex was markedly different, with resonances on all sugar units displaying equal enhancements, suggesting that CV-N is able to discriminate between the three structurally related trisaccharides.

Anti-HIV Agents↗
PubMed1996

Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

The structure of the small hepatitis B virus surface antigen (HBsAg) was investigated by epitope mapping of four anti-HBsAg monoclonal antibodies (mAbs). Amino acid sequences of epitopes were derived from affinity-enrichment experiments (biopanning) using a filamentous phage peptide library. The library consists of 10(9) different clones bearing a 30-residue peptide fused to gene III. Sequence homologies between peptides obtained from panning the library against the antibodies and the native HBsAg sequence allowed for precise description of the binding regions. Three of four mAbs were found to bind to distinct discontinuous epitopes between amino acid residues 101 and 207 of HBsAg. The fourth mAb was demonstrated to bind to residues 121-124. The sequence data are supported by ELISA assays demonstrating the binding of the HBsAg-specific peptides on filamentous phage to mAbs. The sequence data were used to map the surface of HBsAg and to derive a topological model for the alpha-carbon trace of the 101-207 region of HBsAg. The approach should be useful for other proteins for which the crystal structure is not available but a representative set of mAbs can be obtained.

Amino Acid Sequence↗
PubMed2000

The 300-kDa intermediate filament-associated protein (IFAP300) is a hamster plectin ortholog.

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin.

Amino Acid Sequence↗
PubMed1994

The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function.

Synergistic sites in the central cell-adhesive domain of fibronectin (FN) substantially enhance cell adhesion mediated by the alpha 5 beta 1 integrin receptor for fibronectin. We characterized a critical minimal sequence needed for synergistic activity using site-directed mutagenesis and homology scanning using intramolecular chimeras. The minimal cell-binding domain of FN consisting of the 9th and 10th type III FN repeat was expressed in an Escherichia coli expression system. This protein retained high biological activity when assayed using a competitive inhibition assay for FN-mediated adhesion of baby hamster kidney or HT-1080 cells. In contrast, a construct consisting of the 8th and 10th repeat displayed very low biological activity. By replacing various portions of the 8th repeat with homologous 9th repeat segments, we mapped the synergistic region to the center of the 9th repeat. When a very short peptide sequence, Pro-His-Ser-Arg-Asn (PHSRN), from the 9th repeat was substituted for the homologous pentapeptide site in the 8th repeat sequence, the recombinant protein showed markedly enhanced activity. Further mutagenesis analysis suggested that the arginine residue of this pentapeptide sequence is important for function. We also identified a weaker adjacent synergy region other than the PHSRN region. Epitope mapping of an anti-FN monoclonal antibody that inhibits FN-mediated adhesion identified the same critical regions. A synthetic peptide containing the PHSRN sequence showed neither competitive inhibitory activity in solution nor synergy with a soluble RGD-containing peptide. However, when the same synthetic peptide was positioned via a covalent bond at the corresponding site of the normally inactive 8th repeat, it mediated an enhancement of adhesive activity. These results identify a pentapeptide site that synergistically enhances the cell-adhesive activity of the FN RGD sequence.

Amino Acid Sequence↗
PubMed2007

Fine specificity mapping of autoantigens targeted by anti-centromere autoantibodies.

Autoantibodies to centromeric proteins are commonly found in sera of limited scleroderma and other rheumatic disease patients. To better understand the inciting events and possible pathogenic mechanisms of these autoimmune responses, this study identified the common antigenic targets of CENP-A in scleroderma patient sera. Utilizing samples from 263 anti-centromere immunofluorescence positive patients, 93.5% were found to have anti-CENP-A reactivity and 95.4% had anti-CENP-B reactivity by ELISA. Very few patient samples exclusively targeted CENP-A (2.7%) or CENP-B (4.2%). Select patient sera were tested for reactivity with solid phase overlapping decapeptides of CENP-A. Four distinct epitopes of CENP-A were identified. Epitopes 2 and 3 were confirmed by additional testing of 263 patient sera by ELISA for reactivity with these sequences constructed as multiple antigenic peptides. Inhibition CENP-A Western blots also confirmed the specificity of these humoral peptide immune responses in a subset of patient sera. The first three arginine residues (aa 4-6) of CENP-A appear essential for antibody recognition, as replacing these arginines with glycine residues reduced antibody binding to the expressed CENP-A protein by an average of 93.2% (range 80-100%). In selected patients with serial samples spanning nearly a decade, humoral epitope binding patterns were quite stable and showed no epitope spreading over time. This epitope mapping study identifies key antigenic targets of the anti-centromere response and establishes that the majority of the responses depend on key amino-terminal residues.

Adult↗
PubMed2004

Unexpected diversity in the fine specificity of monoclonal antibodies that use the same V region gene to glucuronoxylomannan of Cryptococcus neoformans.

Most mAbs to the capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans are generated from the same VH and VL gene families. Prior Ab studies have assessed protective efficacy, Id structure and binding to capsular polysaccharides, and peptide mimetics. These data have been interpreted as indicating that most mAbs to GXM have the same specificity. A new approach to Ab specificity analysis was investigated that uses genetic manipulation to generate C. neoformans variants with structurally different capsules. C. neoformans mutants expressing GXM with defective O-acetylation were isolated and complemented by the C. neoformans gene CAS1, which is necessary for the O-acetylation of GXM. The mAbs exhibited differences in their binding to the GXM from these mutant strains, indicating previously unsuspected differences in specificity. Analysis of three closely related IgMs revealed that one (mAb 12A1) bound to an epitope that did not require O-acetylation, another (mAb 21D2) was inhibited by O-acetylation, and the third (mAb 13F1) recognized an O-acetylation-dependent conformational epitope. Furthermore, an IgG Ab (mAb 18B7) in clinical development retained binding to de-O-acetylated polysaccharide; however, greater binding was observed to O-acetylated GXM. Our findings suggest that microbial genetic techniques can provide a new approach for epitope mapping of polysaccharide-binding Abs and suggest that this method may applicable for studying the antigenic complexity of polysaccharide Ags in other capsulated microorganisms.

Antibodies, Fungal↗
PubMed1996

Immunochemical study of human immunoglobulin G Fc region.

Fifteen murine monoclonal antibodies (mAb) were produced that reacted with conformational epitopes present on the Fc portion of all human IgG subclasses (PAN-Fc). Inhibition and sandwich ELISA were used to elucidate overlapping and distinct epitopes. The epitopes were aligned to form a continuous "chain" of overlapping determinants from the CH2-CH3 interface to the direction of hinge. The five more hinge-proximal epitopes demonstrated high lability both in competitive and sandwich assays, being completely or partially destroyed when any of these 15 other mAb bound the IgG first. Heat-inactivation of IgG caused full disruption of these epitopes. In contrast, epitopes situated at the opposite distal of the "chain" were more stable and mAb binding could only be affected by occupying an overlapping epitope. Under heat-inactivation these epitopes were affected, but not completely destroyed. Human IgG class anti-DNA autoantibodies were bound to insolubilized dsDNA and their reaction with PAN-Fc mAb was studied. mAb titration plots on IgG and dsDNA-IgG were compared. Five epitopes proved to be altered by antigen (dsDNA) binding. Two of these were the labile hinge-proximal epitopes and the other three were situated near the CH2-CH3 interface. Cross-reactivity of mAb with xenogeneic IgG was also studied. An "epitope map" of the crystallographic model of human IgG Fc portion drawn was based on these experimental data and printed matter, concerning the location of subclass-specific amino acids and homology regions of human and animal IgG.

Animals↗