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PubMed1990

Epitope mapping of the PreS1 domain of the hepatitis B virus large surface protein.

The large (L) surface glycoprotein of hepatitis B virus is an important component of the virion envelope derived from translation initiation at the 5' end of the PreS1 domain of the surface antigen open reading frame. Since key roles in virion assembly and infectivity have been postulated for this protein, further understanding of its structure and topology is important. To this end we have mapped the epitopes recognized by a panel of monoclonal antibodies specific for this polypeptide by examining their reactivity with a series of deletion mutants of the PreS1 region expressed in cultured cells. On the basis of this and other techniques, the antibodies fall into two groups mapping to two distinct epitopes spanning residues 27-35 and 72-78, respectively. Immunoprecipitation studies indicate that both regions are exposed on the surface of HBV-encoded particles.

Antibodies, Monoclonal↗
PubMed2003

T cell epitope identification for bovine vaccines: an epitope mapping method for BoLA A-11.

T cell responses play an important role in immunity to parasites and other microbial agents of infectious diseases, therefore a number of T cell-directed vaccines are in development. Computer-driven algorithms that facilitate the discovery of T cell epitopes from protein and genome sequences are now being used to accelerate preclinical studies of human vaccines. Similar tools are not yet available for predicting T cell epitopes for animal vaccines, but there may be sufficient data available to begin the process of compiling the algorithms. We describe the construction of a novel mathematical 'matrix' that describes the properties of bovine major histocompatibility complex (BoLA) system antigen (BoLA) A-11 peptide ligands, developed for use with EpiMatrix, an existing T cell epitope-mapping algorithm. An alternative means of developing BoLA matrices, using the pocket profile method, is also discussed. Matrices such as the one described here may be used to develop T cell epitope-mapping tools for cattle and other ruminants. Epitope-mapping algorithms offer a significant advantage over other methods of epitope selection, such as the screening of synthetic overlapping peptides, because high throughput screening can be performed in silico, followed by ex vivo confirmatory studies. Furthermore, using epitope-mapping algorithms, putative T cell epitopes can be derived directly from genomic sequences, allowing researchers to circumvent labor-intensive cloning steps in the genome-to-vaccine discovery pathway.

Algorithms↗
PubMed1984

A gel electrophoresis method for epitope mapping studies with monoclonal antibodies.

The linear polyacrylamide gradient gel electrophoresis method of Lambin and Fine (1979, Anal. Biochem. 98, 160-168) has been adapted for estimation of the molecular weights of antigen-antibody complexes, thereby providing information useful in epitope mapping studies with monoclonal antibodies. The method has been applied to mapping of the epitopes recognized by four different monoclonal antibodies raised against rat brain hexokinase (1984, Finney et al., J. Biol. Chem., 259, 8232-8237). The results obtained with the gel electrophoresis method were in agreement with epitope mapping studies conducted by the molecular sieve high-performance liquid chromatographic method of Crawford et al., (1982, Proc. Natl. Acad. Sci. USA 79, 7031-7035). Epitope mapping by the gel electrophoretic method offers several advantages in terms of speed, convenience, and economy when compared with alternative procedures that have been described.

Animals↗
PubMed1998

Mapping epitopes for 20 monoclonal antibodies to CR1.

Complement receptor type one (CR1; CD35) binds and processes C3b and C4b opsonized immune complexes and regulates complement activation. We have characterized the epitopes of 13 previously reported and seven new MoAbs to human CR1. The MoAbs formed seven groups based on their reactivity with a panel of deletion forms of CR1. Seventeen of the MoAbs reacted with CR1 at more than one site, a consequence of its repetitive sequence. All five of the MoAbs recognizing epitopes in the nearly identical repeats 3, 10, and 17, as well as one MoAb which reacted with repeats 8 or 1/2 of 9 and 15 or 1/2 of 16, blocked cofactor activity for C3b. Knowledge of the repeats bearing the epitopes for these MoAbs should facilitate the further characterization of CR1.

Animals↗
PubMed2001

Epitope mapping of human alpha-fetoprotein.

The epitope structure of human alpha-fetoprotein (AFP) was studied using more than 50 monoclonal antibodies (MAB) to human AFP. These MAB obtained from various world laboratories of the TD-2 AFP Workshops of the International Society for Oncodevelopmental Biology and Medicine (ISOBM-1996-1998-2000) were analyzed by competitive immunoaffinity electrochromatography (IAE) on nitrocellulose membranes (NCM). Five types of interaction of the AFP-MAB complex with the MAB fixed on NCM were found: 1) complete neutralization; 2) partial neutralization; 3) unidirectional neutralization; 4) enhanced binding; 5) lack of interaction. By IAE, 51 MAB were found to recognize 23 different epitopes in the AFP molecule. Based on these findings, an epitope map of AFP was designed which consists of eight epitope clusters and eight individual epitopes. The epitope location is considered with respect to the conformational state of the AFP molecule. Possible causes of the five types of interaction found on neutralization are discussed.

Antibodies, Monoclonal↗
PubMed1999

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.

Epitope Mapping↗
PubMed1992

Epitope mapping of polyclonal and monoclonal antibodies against two alpha-bungarotoxin-binding alpha subunits from neuronal nicotinic receptors.

Recently, cDNAs for alpha subunits of two different neuronal alpha-bungarotoxin-binding proteins (alpha BgtBP) were isolated from chick brain, designated alpha BgtBP alpha 1 and alpha BgtBP alpha 2. These are now also referred to as subunits alpha 7 and alpha 8, respectively. Expression studies in Xenopus oocytes have indicated that alpha 7 subunits are able to form cation channels that are sensitive to nicotinic ligands, and therefore represent bona fide nicotinic acetylcholine receptor subunits. Polyclonal and monoclonal antibodies (mAbs) have been produced against: (i) affinity-purified chick brain alpha BgtBP; and (ii) fusion proteins containing the unique cytoplasmic sequences alpha 7(327-412) and alpha 8(293-435). Here, synthetic overlapping peptides corresponding to their deduced amino acid sequences are used to map the epitopes recognized by the different antibodies. The polyclonal response to affinity-purified alpha BgtBPs and the fusion proteins indicates that sequence segments 290-420 of both subunits contain several major and minor epitopes. mAbs selected for their ability to bind both native and denatured alpha BgtBPs isolated from chick brain also recognize subunit-specific sequential epitopes within the sequence segment 290-420. The epitopes recognized by the mAbs correspond to the minor epitopes defined using antisera. The mAbs characterized in these studies will provide useful probes for further studies of alpha BgtBP structure and histological localization.

Amino Acid Sequence↗
PubMed1996

Epitope Mapping by Label-Free Biomolecular Interaction Analysis

The diversity of B-cell response to a large immunogen gives rise to a series of antibodies that can be used for epitope mapping of an antigen. This is based on the relative reaction pattern for all antibodies in relation to each other and other ligands to the studied protein. With the introduction of an instrument system, BIAcore, label-free real-time biomolecular interaction analysis (BIA) was made possible. It is based on biosensor technology, with a carboxymethyl-dextran-coated gold surface and an integrated fluidics for transport of liquid. The basic idea is to measure label-free binding of an analyte from a continuous flow to an immobilized ligand in real time. With an automatic approach, quantitative analysis and sequential injection characteristic biospecific binding parameters such as affinity and kinetic constants can be measured. The instrument system was adopted at an early stage for epitope mapping. With label-free detection, antibodies from tissue culture media can be analyzed without purification. Binding of both antigen and a series of antibodies can be individually determined in molar ratio by sequential injections. The quantitative aspects of BIA offer the possibility of further refined epitope mapping. The relative binding pattern for 30 monoclonal antibodies against HIV-1 p24 core protein has been analyzed. Multideterminant analysis and peptide identification of binding sites were performed. Verification of the binding pattern has also been performed in relation to mapping with ELISA as well as the binding to peptides derived from the antigen sequence. Functional domains of proteins in relation to an epitope map have been identified for Taq polymerase.

Journal Article↗
PubMed1996

The application of epitope mapping in the development of a new serological test for Helicobacter pylori infection.

Epitope mapping was applied to the derived amino acid sequences of the urease A and urease B genes of Helicobacter pylori. This identified 15 epitopes of which five were the most immunodominant. These were LTPKELD (Ure A), FISP, QIPTAF, EVGKVA and SIP (Ure B). Peptide 1 representing LTPKELD and peptide 2 representing EVGKVA were used to develop ELISA procedures for detecting antibody specific to H. pylori infection. The sensitivity, specificity and efficiency values for peptide 1 reactive IgM were 31.6, 92.8 and 52.5% and for peptide 1 IgG were 52.6, 35.7 and 45.4%. The corresponding values for peptide 2 IgM were 31.6, 100 and 60.6% and for peptide 2 IgG were 63.2, 71.4 and 66.6% respectively. When the tests were combined so that a positive for either peptide was counted as a positive overall the figures for IgM were 52.6, 92.8 and 69.6%. Thus epitope mapping delineated peptides against which specific IgM was produced in active H. pylori infection.

Antibodies, Bacterial↗
PubMed1994

Epitope mapping of cytochrome P450 2B4 by peptide scanning.

Overlapping hexapeptides covering the whole sequence of the cytochrome P450 2B4 have been synthesized on the solid supports and tested by ELISA using the polyclonal antiserum against cytochrome 2B4. 70 hexapeptide fragments have been found to interact specifically with the antiserum, i.e. to possess antigenic activity. The mapped linear epitopes occupy about 43% of the whole sequence of 2B4. They presumably form clusters in the regions of No. 60-150, 210-300, 390-430 and 465-486 amino acid residues. The use of cytochrome P450 DataBase has allowed to classify the revealed antigenic determinants into absolutely specific for 2B4, specific only for 2B4 and 2B5, characteristic for 2B subfamily and widely distributed in family 2.

Amino Acid Sequence↗
PubMed1998

Epitope mapping of a function-blocking beta 1 integrin antibody by phage display.

Integrins are a major class of cell surface receptors involved in cell-cell and cell-matrix adhesion and communication. Ha2/11 is a function-blocking anti-rat beta 1 integrin hamster IgM that should be a useful reagent for understanding beta 1 integrin function. We demonstrate that Ha2/11 cross reacts with human, Xenopus, and Drosophila beta 1 integrins, and use phage display to map the epitope for Ha2/11 to residues within the sequence LRSGEPQTF which lies 18 amino acids proximal to the putative I domain in beta 1 integrins. Monoclonal antibody mapping experiments, mutational analyses, and direct binding assays have implicated integrin I domains in both cation and ligand binding. Our data therefore suggest that Ha2/11 blocks beta 1 integrin function by interfering with I domain-mediated ligand binding.

Amino Acid Sequence↗
PubMed1993

Topology of P-glycoprotein as determined by epitope mapping of MRK-16 monoclonal antibody.

There is growing evidence for the direct role of P-glycoprotein mediating multidrug resistance in tumor cells. P-glycoprotein is thought to function as an energy-dependent drug efflux pump. The monoclonal antibody MRK-16 binds to an external domain of P-glycoprotein and partially inhibits drug efflux in multidrug-resistant cells. As an approach toward elucidating the mechanism by which MRK-16 affects drug transport, we undertook the definition of the precise binding site of this antibody. In this study we have mapped the epitope of MRK-16 monoclonal antibody to a resolution of a single amino acid using a series of overlapping synthetic peptides. We demonstrate that MRK-16 recognizes only the class I isoform (MDR1) of human P-glycoprotein and that its epitope encompasses at least two (first and fourth) of the six predicted extracellular peptide loops. These results suggest that the epitope of MRK-16 is discontinuous and that the sequences involved which are separated by about 625 amino acids in the linear sequence must be spatially situated in close proximity in the native protein. Based on these results, we present a model for transmembrane alpha-helical packing of P-glycoprotein in the lipid bilayer. This may have implications for understanding the function of P-glycoprotein in drug transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Epitope mapping on N-terminal region of taenia solium paramyosin.

Epitope mapping of the amino-terminal 20aa sequence from Taenia solium paramyosin (TPmy), an immunodominant protein involved in the complex host-parasite relationship in human and porcine cysticercosis is reported. A 12-mer random peptide phage display library was screened with antibodies raised against a synthetic peptide corresponding to the amino-terminal 20aa sequence of TPmy, its highly immunodominant region. In total, 57 clones isolated in two panning conditions were analyzed, of which a single group of 14 sequences found in 25 clones shared a consensus motif showing structural similarity with the antigen Arg10-Thr16 region.

Amino Acid Sequence↗
PubMed2003

Epitope mapping for the monoclonal antibody that inhibits intramolecular electron transfer in flavocytochrome b2.

Flavocytochrome b(2) (yeast L-lactate dehydrogenase) carries one FMN and one protohaem IX on each of its four subunits. The prosthetic groups are bound to separate domains, the haem domain (residues 1-99) and the flavin domain (residues 100-485), which interact for electron transfer between lactate-reduced FMN and haem b(2); in vivo, the latter reduces cytochrome c. In the crystal structure, one haem domain out of two is mobile. Previously we have described a monoclonal antibody, raised against the tetramer, that only recognizes the native haem domain and prevents electron transfer between flavin and haem, while having no effect on flavin reduction by the substrate [Miles, Lederer and Lê (1998) Biochemistry 37, 3440-3448]. In order to understand the structural basis of the uncoupling between the domains, we proceeded to site-directed mutagenesis, so as to map the epitope on the surface of the haem domain. We analysed the effects of 14 mutations at 12 different positions, located mostly in the domain interface or at its edge; we also analysed the effect of replacing protohaem IX with its dimethyl ester. We used as criteria the antibody-mediated inhibition of cytochrome c reduction by flavocytochrome b(2), competitive ELISA tests and surface plasmon resonance. We have thus defined a minimal epitope surface on the haem domain; it encompasses positions 63, 64, 65, 67, 69 and 70 and one or both haem propionates. When the haem and flavin domains are docked for electron transfer, the 65, 67 and 70 side chains, as well as the haem propionates, are excluded from solvent. The present results thus indicate that, when bound, the antibody acts as a wedge between the domains and constitutes a physical barrier to electron transfer.

Amino Acid Substitution↗
PubMed2005

IgE and IgG4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2.

BACKGROUND: Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping. OBJECTIVE: We designed a redundant peptide microarray for IgE and IgG4 epitope mapping of the previously characterized peanut allergen, Ara h 2. METHODS: Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies. RESULTS: By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG(4) signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length-7 of which (78%) agreed with previous mapping. CONCLUSION: Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map.

2S Albumins, Plant↗
PubMed1996

Epitope mapping of homologous and cross-reactive antigens by monoclonal antibodies to streptococcal cell membrane (mAb to SCM).

An approach to epitope mapping of a series of anti-streptococcal cell membrane (SCM) mAbs is described. Evaluations by enzyme-linked immunosorbent assay (ELISA) of one control mAb HB-35 and 13 different anti-SCM mAbs were made on homologous SCM antigen and human basement membrane antigens isolated from glomeruli (GBM) and lung (LBM). These anti-SCM mAbs were previously shown to be cross-reactive in a variety of systems with both GBM and LBM. The binding capacities were measured for all 14 mAbs on ELISA plates sensitized with SCM antigen or the cross-reactive GBM or LBM antigens, at 5 micrograms/ml or approximately 20 pM/well. From the 50% binding capacity dilution the pM of mAb bound/pM antigen-well was calculated which translated into an estimate of the ratio-number of epitopes bound. Observations with the homologous and cross-reactive antigens showed multiple reactive epitope ratios to eight mAbs whereas the other five yielded a ratio value of one or two on the tested antigens. Plates blocked with a specific dilution of one mAb evaluated the binding by a second mAb providing both binding and specificity data. One mAb (I-F-3) blocked all the other anti-SCM mAbs on all three antigen plates. An additive effect was noted by three mAbs, I-G-8, II-C-4 and II-D-8 with most of the other mAbs. The order of placement, however, made distinctive differences; II-F-4 showed an additive or enhancement effect on I-B-5 but no reciprocal effect was seen. A similar effect was made with I-G-8 and II-C-4 or I-F-7. Possible interpretations are that each mAb is binding different epitopes each fully exposed, and the order of placement of the mAbs makes no difference. Where an enhanced effect was observed it is suggested that the binding of the first mAb changed the conformation of the antigen, thereby opening and exposing additional epitope(s) to the second antibody. Or, in contrast, where the second mAb was blocked by the first, a fixing of the protein conformation is suggested thereby occluding the other epitope, as seen with I-F-3 and II-C-4. These epitope mapping procedures confirmed that all 13 anti-SCM mAbs were binding at different epitopes. The nature of basement membrane collagens and how this relates to post-streptococcal sequelae will be discussed.

Antibodies, Bacterial↗
PubMed1992

Epitope mapping of two isoforms of a trans Golgi network specific integral membrane protein TGN38/41.

TGN38/41 is an integral membrane protein predominantly located in the trans Golgi network (TGN) of rat (NRK) cells. We have used a cDNA expression system to map the epitopes recognised by a panel of antibodies raised to TGN38/41 as a preliminary step in the accurate identification of the region(s) of the molecule responsible for its correct intracellular location. These studies have confirmed the predicted topology of the molecule, and have identified a region in the cytoplasmic domain which is immunologically (and hence potentially functionally) conserved between species.

Amino Acid Sequence↗