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PubMed1996

[Studies on synthetic peptide. XX: the antigenicity and linear epitope map of synthetic peptide hepatitis C virus].

Hepatitis C virus (HCV), the major causative agent of post transfusion non-A, non-B hepatitis (NANB), had been cloned and expressed. According to the protein sequence of HCV-BK and its epitope profiles which combined the hydrophilicity, accessibility, flexibility, antigenicity, charge distribution and HPLC reserve coefficient of protein using the "Goldkey" computer program, we designed and synthesized the following peptides: P1(475-495), P3(449-468), P4(658-663), P5(645-663), P6(484-489), P7(475-489), P15(655-662), P16(230-237), P17(225-237), P18(1220-1240), P19(1694-1735), P24(1230-1240), P25(1482-1493), P26(384-389), P27(2355-2389). The results of ELISA showed that P6(60% positive results) and P19(63% positive results) testing with PT-HC of Gu An, Hebei province were the major antigens in NS1 and in NS4 region, respectively.

Antigens, Viral↗
PubMed1991

Epitope mapping of allergens and antigens of timothy pollen extract.

For the identification of allergens in crude timothy pollen extract, 2D immunoblotting was performed with subsequent use of patients' IgE. Only about 15 of more than 70 detectable components with molecular weights of 55, 38, 35, and 32 kD showed IgE-reactivity. The 55 and 35 kD allergens were identified as glycoproteins by a glycan detection kit. To investigate the protein structure of the allergens, proteins of the pollen extract were cleaved by CNBr and endoproteinases. The cleavage products were identified by patients' IgE and monoclonal antibodies (moab) raised against timothy pollen proteins. While the use of endoproteinases resulted in a complete loss of some allergens, CNBr produced smaller IgE-reactive proteins, e.g. the 38 kD component was split into a 32 kD component and the original 32 kD protein was partially cleaved into IgE-reactive fragments of 30, 21, and 15 kD. In order to investigate the IgE-binding epitopes we intend to determine the amino acid sequence of the cleavage products.

Allergens↗
PubMed1986

Anti-interleukin 2 receptor monoclonal antibodies. Respective role of epitope mapping and monoclonal antibody-receptor interactions in their antagonist effects on interleukin 2-dependent T cell growth.

Functional studies, using mainly interleukin 2 (IL2)-dependent growth of human T cell lines or clones but also mixed lymphocyte cultures and mitogen T cell activation, allowed a collection of locally produced anti-IL2 receptor monoclonal antibodies (mAb) to be classified. They fell into two groups: one with strong to moderate inhibition of IL2, the other without any detectable functional activity in in vitro assays. Direct and sequential immunoprecipitation as well as peptide mapping confirm that all the mAb recognize the same surface molecule. The parameters responsible for such functional dichotomy were characterized: the main parameter was found to be linked to the epitopic cluster recognized on the molecule by the mAb. All functional mAb pertained to a given epitopic cluster and all the nonfunctional ones to an alternative cluster. Studies on mAb receptor and IL2 receptor interactions confirmed these findings and strongly suggest that functional mAb interact with a region on the IL2 receptor identical or very close to the site of ligand-receptor interaction. These data could facilitate the choice of mAb to be used in therapeutical approaches in vivo when ethical objections could be overcome by appropriate committees.

Antibodies, Monoclonal↗
PubMed1992

Epitopic mapping of linear and conformation-dependent antigenic determinants on GP5 of five U.S. bluetongue viruses.

Two distinct antigenic determinants of the major outer capsid protein, GP5, of five U.S. bluetongue viruses have been identified and mapped using monoclonal and oligoclonal antibodies. One antigenic site, identified by oligoclonal antibody AK-15, was found to be common and conserved in all five U.S. BTV serotypes. This linear epitope was located between amino acid residues 175 and 189 (ALQREAAERSEDEIK). The second determinant identified by monoclonal antibody 34.7 was present in BTV-2, -10, -11, and -17 but absent in BTV-13. The binding of this monoclonal antibody to GP5 could be blocked specifically by one of three short synthetic peptides located among amino acid residues 33-42 (KAAERFAESE), 159-168 (EKILKEEDSK), and 206-215 (EIERDGMQEE), indicating that this antigenic determinant is conformation-dependent. Oligoclonal antibody (AK-15) reacted with denatured GP5 immobilized on nitrocellulose membrane after Western transfer as well as with native GP5 present on the surface of purified BTV virions. Monoclonal antibody (34.7) reacted only with denatured GP5 but not native GP5 using an ELISA assay. However, these two antigenic epitopes alone did not elicit detectable neutralizing antibodies as determined by plaque reduction assay.

Amino Acid Sequence↗
PubMed1999

Epitope mapping of anti-rhodopsin antibodies from patients with normal pressure glaucoma.

PURPOSE: The presence of anti-rhodopsin antibodies in patients with normal pressure glaucoma (NPG) has been previously demonstrated with western blot analysis and enzyme-linked immunosorbent assay. To learn more about the characteristics, origin, and possible significance of these antibodies, the epitopic specificity of the anti-rhodopsin antibodies was examined in four NPG patients. METHODS: Antibodies in patient sera were assayed by western blot analysis against purified bovine rhodopsin. Peptides derived from particular segments of the rhodopsin sequence were tested for activity in competing for rhodopsin-antibody binding. RESULTS: Of a series of nine peptides that constitute most of the hydrophilic regions of rhodopsin, only one, consisting of the C-terminal 25 amino acids, prevented binding of the patient antibodies to rhodopsin. Higher resolution mapping using a set of dodecamers of overlapping sequences from the C-terminal region demonstrated that antibody binding is completely dependent on the last two amino acids. Removing the C-terminal alanine alone, or amidating the C terminus carboxyl group, also eliminated antibody binding. CONCLUSIONS: Because four of four patient antibodies examined exhibited the identical epitopic specificity, it is likely that a common mechanism underlies their generation. This may indicate that molecular mimicry has occurred, because several pathogens contain similar C-terminal sequences. Although they may serve as diagnostic markers, and provide evidence that there is an autoimmune component in some patients with glaucoma, the role, if any, that these antibodies play in the pathogenesis of the disease remains unknown.

Animals↗
PubMed1991

Epitope mapping of the receptor-bound agonistic form of human chorionic gonadotropin (hCG) in comparison to the antagonistic form (deglycosylated hCG).

On the surface of free human chorionic gonadotropin (hCG), we can distinguish with our panel of monoclonal antibodies (MCA) 14 topographically distinct epitopes (designated alpha 1 - alpha 5, beta 1 - beta 5, alpha beta 1 - alpha beta 4, depending on the subunit they are attached to). Only 2, i.e. the adjacent beta 3 and beta 5 epitopes, of these 14 are accessible to 125I-labeled MCA binding, when hCG is first allowed to bind to the rat testis hCG receptor. This result indicates that the agonist hCG assumes a defined orientation in its receptor-bound state and that, except for that small area comprising the beta 3 and beta 5 epitopes, most of its surface is masked by the hCG receptor. We therefore asked whether the competitive antagonist deglycosylated hCG (degly-hCG), which, when free, is antigenically (as to number and topography of epitopes) indistinguishable from native hCG, would interact with the receptor differently, that is, in a way that can be discerned by this epitope accessibility paradigm. Here we describe that on receptor-bound degly-hCG the beta 3 and beta 5 epitopes were concealed as were all other epitopes. This observation, together with finding the receptor affinity of degly-hCG to be 4 times higher than that of native hCG, suggests that degly-hCG assumes a signal transduction-incompetent ligand orientation and at the same time interacts with the receptor more intensively, i.e. establishes additional ("antagonist accessory") protein-protein contacts besides those involved in agonist binding. It thus appears that the carbohydrate moieties function to prevent formation of such accessory contacts.

Animals↗
PubMed1992

Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system.

The donor photobleaching method (T. M. Jovin and D. J. Arndt-Jovin. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:271-308.) has been adapted to an ACAS 570 (laser scanning microscope) system to measure fluorescence resonance energy transfer (FRET) on individual human peripheral blood T cells. Photobleaching was completed in approximately 100 ms in our case and it followed double-exponential kinetics. The energy transfer efficiency (E) was approximately 20% between the CD4 epitopes OKT4-FITC and Leu-3a-PE as well as between OKT4E-FITC and OKT4-PE. E was approximately 8% between OKT4-FITC and Leu-4-PE (alpha CD3) and barely detectable (approximately 4%) from OKT4-FITC to Leu-5b-PE (alpha CD2). The E values obtained by the photobleaching method were highly reproducible both in repeated measurement of identical samples and in experiments with different batches of cells and were in agreement with the flow cytometric donor quenching measurements. As expected, E measured between primary and secondary layers of antibodies increased (from approximately 14% to approximately 28%) when F(ab')2 fragments were substituted for whole antibody molecules as the donor. On a T cell line we mapped the distance between the idiotypic determinant of the T cell receptor (TcR) and the Leu-4 epitope of CD3 as proximal as E = 28%, as compared to E = 4% between a framework TcR epitope and Leu-4. In the latter case, however, approximately 40% less Leu-4 was bound suggesting that the antigen binding site of TcR is in close proximity with one of the two CD3 epsilon chains, which hence are not equivalent.

Antibodies, Monoclonal↗
PubMed1991

The antigenic surface of staphylococcal nuclease. I. Mapping epitopes by site-directed mutagenesis.

The analysis of the antigenic surface of staphylococcal nuclease was begun by generating and characterizing a panel of mAb. Twelve mAb were selected from a large number of anti-nuclease mAb and characterized for affinity and isotype, by their ability to block enzyme activity, and by complementation and competitive inhibition assays for the relative location of epitopes. The mAb were placed in complementation groups based on their distinct binding patterns. These groups define a series of eight overlapping epitopes that are estimated to cover a large portion of the nuclease surface. Four mAb blocked the enzyme activity of nuclease. The epitopes defined by two of these four mAb were localized on the surface of nuclease using single amino acid variant Ag generated by site-directed mutagenesis of the cloned nuclease coding sequence. mAb-25 maps to residue 46 which is located at the edge of the enzyme active site consistent with its ability to inhibit enzyme activity. mAb-19, which also blocks enzyme activity and belongs to the same complementation group as mAb-25, was unaffected by the substitution at position 46. This suggests that mAb-19 and mAb-25, if they do react with the same epitope, have differences in fine specificity. mAb-22 blocks enzyme activity and belongs to an overlapping complementation group. The fourth mAb, mAb-1, which belongs to a distinct, nonoverlapping, complementation group, does not blocks enzyme activity, and is directed to a region of nuclease that includes the amino acid at position 133. This residue is located a short distance from the active site in a region that has been suggested to participate in binding of DNA, a substrate for nuclease. Therefore, the four epitopes defined by these mAb are localized at or near the enzyme active site.

Animals↗
PubMed1992

Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase.

Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit. Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity. This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit. In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase. It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase. In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, M., nd Goldfarb, A. (1990) Gene 89, 7-12). The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting. The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876. This region may be important in enzyme assembly or subunit-subunit interaction. The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093. This region may be involved in the catalytic function of RNA polymerase.

Antibodies, Monoclonal↗
PubMed1993

Epitope mapping using bacteriophage peptide libraries.

Vast libraries of random peptides displayed on the surface of bacteriophage potentially allow identification of specific ligands for any peptide receptor of interest. The sequence specificity of antibody-peptide interactions allows these libraries to be used to define and localize continuous epitopes. Remarkably clear-cut results have been obtained with some antibodies; however, the technique has not proved to be generally applicable.

Amino Acid Sequence↗
PubMed1988

Structure and function of macrophage adhesion molecule examined by epitope mapping.

Macrophage adhesion molecule (MAM), a member of the integrin superfamily of heterodimer membrane molecules with adhesive properties, is the guinea pig counterpart of human Mo1 (CD11b/CD18). Earlier work showed that MAM is synthesized as monomeric precursor glycopeptides that assemble to form the heterodimer. The heterodimer and monomer glycopeptides are characterized through the use of twelve mAb in immunoprecipitation, immunoblotting, binding assays, and a quantitative cell adhesion assay. Seven topographic regions are identified, two of which are shown to be critical for adhesion. One adhesion-related topographic region, the M2/M4 region, is on the alpha-subunit, and the other, the M8/M15 region, is on the beta-subunit. Both adhesion-related epitopic regions are not detectable on monomeric glycopeptides but are generated by conformational change on heterodimer formation. It is hypothesized that these structure-function relationships have general applicability to integrin molecules.

Animals↗
PubMed1985

Precise epitope mapping of the murine transformation-associated protein, p53.

Murine p53 cDNA sequences were cloned into an in vitro expression vector, Protem Hind. Four deletion libraries were generated using Bal31 double-stranded exonuclease; two being made from constructs encoding a fusion protein constructed from SV40 small t sequences and the p53 clone, p27.la; and two from the full length p53 clone, pp53-5. Both 5'- and 3'-terminal deletions of the p53 gene were made. Transcription of these constructs using Escherichia coli RNA polymerase holoenzyme, followed by translation in mRNA-dependent rabbit reticulocyte lysate, gave in vitro, truncated protein products which were immunoprecipitated by a panel of anti-p53 monoclonal antibodies. This approach enabled us to map accurately the binding sites of seven different monoclonal antibodies, demonstrating four distinct antigenic sites on p53. A synthetic peptide was constructed corresponding to the predicted amino acid sequence of one of these epitopes. This peptide competes with the epitope on the full length p53 protein for the relevant monoclonal antibodies and dissociates the corresponding p53/antibody complexes.

Animals↗
PubMed1993

Epitope mapping with peptides of Chi t I component III and immunomodulation of the Chi t immune response.

BACKGROUND: Hemoglobins of the Diptera family are known as aggressive inhalant allergens. METHODS: The whole sequence of the insect allergen Chi t I component III was studied for T-cell epitope activity in cell cultures of 15 sensitized patients with a panel of 17 overlapping synthetic peptides and 12 peptides obtained by tryptic cleavage. RESULTS: All but one of the peptides tested were found to be immunogenic. The nonstimulatory sequence 102-109 and two other tryptic peptides (69-90 and 110-135) were used in inhibition experiments. The Chi t I-induced proliferation was downregulated by all tested peptides when compared with stimulation with Chi t I alone. CONCLUSIONS: Because stimulation with phytohemagglutinin or tetanus toxoid was not influenced by the above-mentioned peptides, this mechanism seems to be specific for the antigen. Specific immunomodulation with nonstimulating peptides may offer new therapy approaches.

Adjuvants, Immunologic↗
PubMed1992

Epitope mapping of monoclonal antibodies to the Escherichia coli F1 ATPase alpha subunit in relation to activity effects and location in the enzyme complex based on cryoelectron microscopy.

The interaction of Escherichia coli F1 ATPase (ECF1) with several different monoclonal antibodies (mAbs) specific for the alpha subunit has been examined. The epitopes for each of the mAbs have been localized by using molecular biological approaches to generate fragments of the alpha subunit. The binding of several of the mAbs has also been examined by cryoelectron microscopy of ECF1 Fab complexes. One of the mAbs, alpha II, bound in the region Asn 109-Val 153 without affecting ATPase activity. Most of the mAbs bound in the C-terminal third of the alpha subunit. MAb alpha 1 bound between residues Gln 443 and Trp 513. This mAb activated ATPase activity and was visualized in cryoelectron microscopy, superimposed on the alpha subunit, indicating that the epitope was on the top or bottom of ECF1 in the hexagonal projection. Other mAbs to the C-terminus, including alpha D which also activated the enzyme, reacted between Gly 371 and Trp 513 but failed to bind to small overlapping fragments within this sequence. The epitopes for these mAbs are probably formed by the folded polypeptide which occurs only in Western analysis when long stretches of the alpha subunit are present, suggesting that the C-terminus of alpha is a self-folding domain. In cryoelectron microscopy, Fab fragments for alpha D were seen extending from the sides of the ECF1 complex in hexagonal projection.

Antibodies, Monoclonal↗
PubMed1988

Malaria vaccines: immunogen selection and epitope mapping.

In recent years major efforts have been made to characterize parasite antigens thought to be suitable candidates for malaria vaccines. Many of the relevant plasmodial antigens have been found to contain extensive areas of short amino acid sequences organized in tandem repeats. These are usually strongly antigenic, forming linear epitopes seen by antibodies of the infected host. Several such epitopes have been identified and subunit vaccines are being designed in which synthetic peptides or gene constructs serve as immunogens. However, as an efficient malaria vaccine should give rise to anamnestic T-dependent antibody responses following reinfection after vaccination as well as to antibody independent cell-mediated immunity, efforts are now also being made to identify T-cell epitopes on the vaccine candidate antigens. In this paper the current Plasmodium falciparum sporozoite vaccines and the merozoite antigen Pf155/RESA, a possible candidate for a P. falciparum blood stage vaccine, serve as examples to illustrate recent advances made in this area as well as some of the problems remaining to be resolved.

Animals↗
PubMed1988

Epitope-mapping on the Epstein-Barr virus major capsid protein using systematic synthesis of overlapping oligopeptides.

Systematic solid-phase synthesis of all possible overlapping nonapeptides of the 1381 amino acid sequence of the Epstein-Barr virus major capsid protein (EBV-MCP) was used to identify the position of linear antigen epitopes on this protein as recognised by human polyclonal antisera. Antisera were selected for reactivity with EBV-MCP on immunoblots. The results show that antibodies from different individual donors may recognise EBV-MCP through binding to a variety of different epitopes. These epitopes are localized at random over the protein backbone though some non-binding areas are also present. In addition, ten 'hot-spots' were identified containing closely-spaced reactive peptides (epitope-clusters) recognised by most (greater than or equal to 70%) individuals. No significant correlation was found between the actual location of these epitope-clusters and computer predictions using either hydrophilicity plots, secondary structure plots or a combination of (additional) parameters. Epitope-clusters generally were located in regions of indifferent or hydrophilic nature and mostly contained predicted beta-turn configurations. Only one epitope-cluster was located within a region of sequence homology with the MCPs of herpes simplex virus type 1 and varicella-zoster virus. The present study demonstrates the potential of using systematic peptide synthesis to define serologically relevant linear epitopes on large and relatively unexplored viral polypeptides.

Amino Acid Sequence↗