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PubMed2002

[Epitope mapping of hepatitis C virus non-structure protein 5 from a 7 peptide phage library by using immobilized specific monoclonal antibody].

OBJECTIVE: To screen HCV NS5 mimotopes by using monoclonal antibody and phage peptide library. METHODS: By using HCV NS5 monoclonal antibody as selective molecule, a 7 peptide phage library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing. RESULTS: Twelve positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was confirmed as the mimotope of HCV NS5. CONCLUSIONS: HCV mimotope is obtained by phage peptide library screening. The result provides a new approach for HCV therapy and vaccine development.

Amino Acid Sequence↗
PubMed2004

Epitope mapping of anti-PR3 antibodies using chimeric human/mouse PR3 recombinant proteins.

Autoantibodies against proteinase 3 (PR3) and myeloperoxidase (MPO) (ANCA = anti-neutrophil cytoplasmic antibodies) are used as diagnostic tools for patients with small vessel vasculitis. ANCA are detected by different assays, but the correlation between the results of these assays is generally poor. The overall aim of the study was to provide a framework for the future development of new assays with an increased diagnostic yield. In order to express discrete epitopes of human PR3 (hPR3), the nonantigenic molecules murine PR3 (mPR3) and human leucocyte elastase (HLE) were used as a framework. We constructed recombinant chimeric vectors and were able to produce 6 hPR3/mPR3 proteins and 3 hPR3/HLE proteins. Anti-PR3 monoclonal antibodies differed in their binding pattern to the chimeras, but no distinct binding region could be identified for any monoclonal antibody. The recombinant hPR3/mPR3 were also tested in ELISA with sera from patients with Wegener's granulomatosis with renal involvement. The results show that patients have antibodies to different constructs, indicating that the patients vary in their antibody repertoire from the beginning of the disease, and that patients may have antibodies from a broad range of clones early in the course of the disease. Recombinant hPR3/mPR3 chimeric proteins have a potential to be used as antigens in future ANCA assays.

Amino Acid Sequence↗
PubMed1989

Murine monoclonal antibodies which recognize active sites of Escherichia coli NusA protein and epitope mapping by gene fusion.

Seven hybridomas producing murine monoclonal antibodies reactive against NusA protein of Escherichia coli were prepared. Antigenic determinants of these monoclonal antibodies have been mapped by immunoblotting analyses using fusion proteins containing parts of NusA. The epitope of the N14 antibody maps in a hydrophobic amino acid (aa) cluster and consists of at least Ala-181 and Ser-183 residues. nusA1 and nusA11 mutations, which cause aa changes of these residues, abolish the antigenic reactivity to the N14 antibody. These antibodies react with intact NusA protein, indicating that the epitopes are exposed on the surface of NusA. Most of these epitopes cluster around the nusA1 and nusA11 mutation loci.

Amino Acid Sequence↗
PubMed2000

Characterization and epitope mapping of neutralizing monoclonal antibodies produced by immunization with oligomeric simian immunodeficiency virus envelope protein.

In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmDeltaB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmDeltaB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.

Amino Acid Sequence↗
PubMed2003

Epitope mapping and specificity of the anti-alpha-synuclein monoclonal antibody Syn-1 in mouse brain and cultured cell lines.

While alpha- and beta-synuclein largely overlap in their expression in the vertebrate brain, only alpha-synuclein accumulates in the fibrillar aggregates typical of Parkinson's disease. It is thus critical to have immunological reagents that distinguish between these two protein isoforms. The monoclonal antibody Syn-1 (Transduction Labs) has been frequently used for the specific detection of alpha-synuclein. In this report, the epitope for Syn-1 is localized within residues 91-99 of human alpha-synuclein. Sequence differences exist in this domain that account for the specificity of Syn-1 for alpha- versus beta-synuclein. However, Syn-1 also displays reactivity with additional species (approximately 45 kDa) in brain homogenates from both wild-type and alpha-synuclein null mice, indicating a potential for cross-reactivity with a protein species that is unrelated to alpha-synuclein in brain tissue or extracts.

Animals↗
PubMed2006

Preparation, characterization and epitope mapping of monoclonal antibodies specific to human mast cell carboxypeptidase.

Human mast cell carboxypeptidase (hMC-CP) is a unique product of mast cells. Unlike tryptase and chymase, its potential function and expression in diseased conditions remain largely unknown. To develop an assay for hMC-CP, the recombinant fusion protein of hMC-CP and purified native skin hMC-CP was prepared, and two novel monoclonal antibodies against hMC-CP named CCP1 (IgG1 isotype) and CCP2 (IgM isotype) were raised in the present study. Epitope analysis shows that CCP1 and CCP2 antibodies recognize epitopes located in the region of amino acids 112-202 of hMC-CP, and hydrophilicity analysis implies that epitopes might be located in the amino acid residues 123-134 and 165-177. Furthermore, using a competition enzyme-linked immunosorbent assay, it was shown that the epitope recognized by CCP1 is close to that recognized by CCP2 or the two antibodies partially share the same epitope. Flow cytometry analysis shows that basophilic leukemia cell line KU812 reacts with both CCP1 and CCP2 antibodies, suggesting that this cell line expresses hMC-CP. In conclusion, although the two antibodies possess different isotypes, they may partially share the same epitope. These two antibodies will be valuable tools for the development of an assay to detect the levels of hMC-CP in the biological fluids in man.

Animals↗
PubMed2002

Molecular and immunological characterization and IgE epitope mapping of Pen n 18, a major allergen of Penicillium notatum.

The mould genus, Penicillium, is a significant source of environmental aero-allergens. A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization-time-of-flight MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni(2+)-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified. Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.

Allergens↗
PubMed1989

Epitope mapping of foot-and-mouth disease virus with neutralizing monoclonal antibodies.

Epitopes of strain A22 Iraq 24/64 of foot-and-mouth disease virus have been mapped with monoclonal antibodies (MAbs). Three methods were used: (i) an indirect ELISA using an overlapping set of peptides, (ii) production of neutralization escape variants against each MAb and (iii) sequencing of neutralization escape variants. The study has shown that the virus has at least three overlapping liner neutralizing epitopes within a major antigenic site on VP1. The presence of a second, conformational site was demonstrated but its position on the virus particle was not located. Synthetic peptides with sequences representing the major site elicit antibodies which have similar broad cross-neutralizing activity to polyclonal serum or neutralizing MAbs produced with the virus against a range of field isolates.

Amino Acid Sequence↗
PubMed2003

Epitope map for a growth hormone receptor agonist monoclonal antibody, MAb 263.

Monoclonal antibody (MAb) 263 is a widely used monoclonal antibody that recognizes the extracellular domain (ECD) of the GH receptor. It has been shown to act as a GH agonist both in vitro and in vivo, and we report here that it must be divalent to exert its effect on the full-length receptor. To understand the mechanism of its agonist action, we have determined the precise epitope for this antibody using a novel random PCR mutagenesis approach together with expression screening in yeast. A library of 5200 clones of rabbit GH receptor ECD mutants were screened both with MAb 263 and with an anticarboxy-tag antibody to verify complete ECD expression. Sequencing for clones that expressed complete ECD but were not MAb 263 positive identified 20 epitope residues distributed in a discontinuous manner throughout the ECD. The major part of the epitope, as revealed after mapping onto the crystal structure model of the ECD molecule, was located on the side and upper portion of domain 1, particularly within the D-E strand disulfide loop 79-96. Molecular dynamics docking of an antibody of the same isotype as MAb 263 was used to dock the bivalent antibody to the 1528-A2 epitope and to visualize the likely consequences of MAb binding. The minimized model enables the antibody to grasp two receptors in a pincer-like movement from opposite sides, facilitating alignment of the receptor dimerization domains in a manner similar to, but not identical with, GH.

Amino Acid Sequence↗
PubMed1995

Epitope mapping and functional properties of anti-intercellular adhesion molecule-3 (CD50) monoclonal antibodies.

Intercellular adhesion molecule-3 (ICAM-3, CD50), a member of the immunoglobulin gene superfamily, is a major ligand for the lymphocyte function-associated antigen 1 (LFA-1, CD18/CD11a) in the resting immune system and plays a role as a signaling and costimulatory molecule on T lymphocytes. In this study we have generated a large panel of anti-ICAM-3 monoclonal antibodies (mAb) and show that the biological effects of these antibodies are critically dependent on the epitope recognized. By using an adhesion assay employing COS cells expressing LFA-1 binding to recombinant chimeric ICAM-3-Fc proteins (which overcomes the confounding effects of interleukocyte LFA-1/ICAM binding events), we have been able to examine the effects of these antibodies in blocking LFA-1/ICAM-3 adhesion. Our data suggests that only a small minority of ICAM-3 mAb, recognizing a distinct epitope, are able to mimic the effects of LFA-1 binding to ICAM-3. Moreover these antibodies are functionally distinct as defined by their costimulatory activity and ability to elicit interleukin-2 production and cell proliferation in T lymphocytes.

Animals↗
PubMed1995

Epitope mapping of the V3 domain of feline immunodeficiency virus envelope glycoprotein by monoclonal antibodies.

A panel of six IgG monoclonal antibodies (MAbs) was produced by immunizing mice with a 22 amino acid synthetic peptide, designated V3.3, of the third variable region of feline immunodeficiency virus (FIV) envelope glycoprotein. This peptide is known to induce neutralizing antibodies in cats. In ELISA all MAbs reacted with purified SDS-disrupted FIV and in flow cytometry all MAbs stained permeated, persistently infected FL4 cells but not unfixed FL4 cells; this indicated that the MAbs recognize essentially cryptic epitopes of the gp100 V3 loop. By direct ELISA using partially overlapping synthetic peptides and by competition binding studies, the anti-V3.3 MAbs were shown to detect at least four distinct epitopes, two located in the amino-terminal half and two in the carboxy-terminal half of the sequence. When tested for neutralizing activity by the syncytium inhibition assay in Crandell feline kidney cells, all anti-V3.3 MAbs neutralized FIV at high dilution. However, at low dilution two MAbs exhibited much less neutralizing activity. These results indicate that the V3 region of FIV contains multiple epitopes involved in neutralization.

Amino Acid Sequence↗
PubMed1998

Epitope mapping of monoclonal antibodies by mass spectrometry: identification of protein antigens in complex biological systems.

We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an extract of the lysate of cells overexpressing the Ty1 Gag protein. MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope-containing peptide fragment. Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells. Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS. To our knowledge, this constitutes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system.

Antibodies, Monoclonal↗
PubMed2005

Structure, epitope mapping, and docking simulation of a gibberellin mimic peptide as a peptidyl mimotope for a hydrophobic ligand.

Using NMR spectroscopy and simulated annealing calculations, we determined the solution structure of the disulfide-linked cyclized decapeptide ACLPWSDGPC (SD), which is bound to an anti-(gibberellin A(4)) mAb 4-B8(8)/E9 and was found to be the first peptidyl mimotope for a hydrophobic ligand. The resulting structure of the peptide showed a beta-turn-like conformation in residues three to seven and the region converges well (average rmsd 0.54 A). The binding activity and the epitopes of the peptide to the antibody were assessed using saturation transfer difference (STD)-NMR experiments. We also conducted docking simulations between the peptide and the mAb to determine how the peptide is bound to the mAb. Resonances around the beta-turn-like conformation of peptide SD (residues 3-5) showed strong STD enhancement, which agreed well with results from docking simulation between peptide SD and the mAb. Together with the commonality of amino acid residues of the mAb involved in interactions with gibberellin A(4) (GA(4)) and peptide SD, we concluded that peptide SD is bound to the antigen-binding site of mAb 4-B8(8)/E9 as a GA(4) mimic, confirming evidence for the existence of peptide mimics even for hydrophobic ligands.

Amino Acid Sequence↗
PubMed2006

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action.

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.

Amino Acid Sequence↗
PubMed2004

Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments.

Individual domains from extracellular proteins are potential reagents for biochemical characterization of ligand/receptor interactions and antibody binding sites. Here, we describe an approach for the identification and characterization of stable protein domains with cell surface display in Saccharomyces cerevesiae, using the epidermal growth factor receptor (EGFR) as a model system. Fragments of the EGFR were successfully expressed on the yeast cell surface. The yeast-displayed EGFR fragments were properly folded, as assayed with conformationally specific EGFR antibodies. Heat denaturation of yeast-displayed EGFR proteins distinguished between linear and conformational antibody epitopes. In addition, EGFR-specific antibodies were categorized based on their ability to compete ligand binding, which has been shown to have therapeutic implications. Overlapping EGFR antibody epitopes were determined based on a fluorescent competitive binding assay. Yeast surface display is a useful method for identifying stable folded protein domains from multidomain extracellular receptors, as well as characterizing antibody binding epitopes, without the need for soluble protein expression and purification.

Animals↗
PubMed1987

A mouse monoclonal antibody to human alpha 2-macroglobulin (alpha 2M) crossreacts with alpha 2M from mouse: epitope mapping and characterization of subunit structure of murine alpha 2M.

Mice were immunized with the isolated C-terminal heat-induced fragment of human alpha 2-macroglobulin (alpha 2M) and the spleens were used to prepare hybridomas. A monoclonal antibody (Mab) designated F17D5 was selected for further characterization. The epitope defined by Mab F17D5 was not expressed on alpha 2M, on alpha 2M-methylamine, or on alpha 2M-proteinase complexes. On the other hand, the antibody reacted avidly with denatured human alpha 2M and with some types of alpha 2M from other species, including mouse, on nitrocellulose-immunoblotting. The epitope of Mab F17D5 was mapped to less than 250 residues C-terminal of the internal thiolester of human alpha 2M. This was based on CNBr fragmentation of the 60 kDa C-terminal heat-fragment and on peptide mapping of alpha 2M, derivatized at the internal thiolester GLX-residue with 125I-labeled histamine. Murine alpha 2M was confirmed to contain two types of subunit: a 185 kDa subunit and a combination of 165 kDa and 35 kDa polypeptides. By partial disulfide bond reduction, heat-fragmentation and immunoblotting with Mab F17D5, the structure of murine alpha 2M was compared to that of human alpha 2M. The F17D5-epitope was mapped to a 30 kDa heat-induced fragment, which was released by denaturation without reduction. This fragment contained an intrachain disulfide bridge. By analogy with human alpha 2M, the 35 kDa subunit would be located at the C-terminal end of murine alpha 2M, disulfide-bonded to the major 165 kDa subunit.

Animals↗
PubMed1998

Sequence diversity, predicted two-dimensional protein structure, and epitope mapping of neisserial Opa proteins.

The sequence diversity of 45 Opa outer membrane proteins from Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria sicca, and Neisseria flava indicates that horizontal genetic exchange of opa alleles has been rare between these species. A two-dimensional structural model containing four surface-exposed loops was constructed based on rules derived from porin crystal structure and on conservation of sequence homology within transmembrane beta-strands. The minimal continuous epitopes recognized by 23 monoclonal antibodies were mapped to loops 2 and 3. Some of these epitopes are localized on the bacterial cell surface, in support of the model.

Amino Acid Sequence↗
PubMed2006

Epitope mapping of human aromatic L-amino acid decarboxylase.

Autoimmune polyendocrine syndrome type I (APS I) is a rare hereditary condition considered a model disease for organ specific autoimmunity. A wide range of autoantibodies targeting antigens present in the affected organs have been identified. Autoantibodies against aromatic L-amino acid decarboxylase (AADC) are present in about 50% of APS I patients. In order to increase our understanding of autoantibody specificity in APS I, the aim of the present study was to localize target regions on AADC recognized by sera from APS I patients. Using several complementing strategies, we have shown that autoantibodies against AADC mainly recognize conformational epitopes. The major antigenic determinants were detected N-terminally to amino acid residue 237. Replacement of amino acids 227-230 (ERDK) with alanine residues reduced the reactivity towards AADC by >80% in all patient sera tested, suggesting that amino acids 227-230 are an important part of an immunodominant epitope.

Aromatic-L-Amino-Acid Decarboxylases↗