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PubMed1994

T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy.

Lymphoproliferation and gamma interferon (IFN-gamma) secretion in response to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculin- and lepromin-positive volunteers and from patients with tuberculosis and leprosy. Peptide recognition was largely promiscuous, with a variety of human leukocyte antigen haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85, and the majority proliferated in response to peptide 6 (amino acids 51 to 70), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 and 21 (amino acids 191 to 220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61 to 80). MTAg85A peptides were also recognized by PBMC from healthy lepromin-positive volunteers and paucibacillary leprosy patients (again in a promiscuous manner), but despite a 90% homology between the 85A proteins of M. leprae and M. tuberculosis, the peptides recognized were different. PBMC from lepromin-positive healthy contacts reacted against peptide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and peptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the MT85A peptides. IFN-gamma production was generally detected simultaneously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicited an IFN-gamma response. In conclusion, regions 41 to 80 and 241 to 295 demonstrated powerful and promiscuous T-cell-stimulatory properties, resulting in proliferative responses and IFN-gamma secretion, respectively, in the majority of reactive subjects tested in this study. These results could be of value in the development of a subunit vaccine for tuberculosis and leprosy.

Amino Acid Sequence↗
PubMed1996

Mapping Epitope Structure and Activity: From One-Dimensional Prediction to Four-Dimensional Description of Antigenic Specificity

Our knowledge of antigenic specificity has greatly increased in recent years mainly through X-ray crystallographic studies of proteins and peptides complexed with monoclonal antibodies. However, our ability to predict the location of antigenic sites in proteins remains limited partly because prediction algorithms reduce the complexity of epitopes to one-dimensional, linear peptide models. Epitopes and paratopes are relational entities definable by their mutual complementarity and adaptation potential as well as by their activity. A complete account of antigenic specificity demands the integration of both structural and binding activity data that can be achieved only through a spatiotemporal four-dimensional analysis. Failure to include the fourth dimension, i.e., time, in the analysis of antigen-antibody complementarity amounts to considering proteins as rigid bodies and ignores the mutual adaptation that occurs when the two partners interact. Reducing four-dimensional protein systems to three-dimensional or two-dimensional representations inevitably distorts our perception of the dynamic nature of epitopes.

Journal Article↗
PubMed1991

Mapping epitopic regions of cholera toxin B-subunit protein.

Continuous overlapping synthetic hexapeptides representing the entire 103 amino acid sequence of the immunodominant B-subunit protein of cholera enterotoxin were used to examine reactivities of a variety of antisera in attempts to detect and define sequence-related (continuous) antigenic regions. The validity of the methods was established by the reactions of polyclonal antisera raised against longer synthetic peptides with appropriate synthetic hexapeptides. An unexpected cross-reaction is attributed to the presence of three identical amino acids (Gln16-Ile17-His18)--although in different order (Gln56-His57-Ile58)--in two parts of the B-subunit chain. Adsorption studies using polyclonal rabbit antisera revealed that, in many instances, denatured B-subunit protein more effectively removed reactivity with hexapeptides than did the native protein. Native holotoxin was more effective than native B-subunit. Sera from human cholera convalescents gave diffuse patterns of reactivity with synthetic hexapeptides--primarily against regions of reactive hexapeptides rather than with clearly defined continuous epitopes. Among many epitopic regions encountered, a strongly reactive tetramer, Ser-Gln-His-Ile (SQHI), was discovered in a highly conserved region, residues 55-58, of the B-subunit amino acid sequence. Adsorption studies revealed that this epitope is apparently exposed on the surface of the native protein. Amino acid substitution revealed the essentiality of Gln and His residues to this epitope. Gly54 was not part of the epitope but substitution of acidic residues Glu and Asp for Gly eliminated reactivity with antibody. The results suggest that continuous epitopes may contribute to the antigenicity of the native toxin protein and may be potentially useful for development of a peptide vaccine.

Amino Acid Sequence↗
PubMed1991

Mapping epitopes on the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum using fusion proteins.

Epitopes for a number of monoclonal antibodies (mAbs) binding (Ca(2+)-Mg2+)-ATPase purified from skeletal muscle sarcoplasmic reticulum have been defined by studying binding to fusion proteins generated from cDNA fragment libraries. Comparison of these results with those of previous studies of binding of mAbs to proteolytic fragments of the ATPase have allowed the definition of the epitopes to within approx. 100 residues and for one (mAb 1/2H7) to within 45 residues. The experiments suggest considerable exposure of the nucleotide binding domain of the ATPase on the top surface of the protein. Those mAbs that were found to inhibit steady-state ATPase activity were found to bind to epitopes in the nucleotide binding domain of the ATPase.

Amino Acid Sequence↗
PubMed1986

Mapping epitope characteristics on carcinoembryonic antigen.

A method of epitope analysis is described in which the binding of one monoclonal antibody (MAb) to radiolabeled carcinoembryonic antigen (CEA) competes with the subsequent binding of an immobilised second MAb. From the degree of blocking obtained, we have identified both structurally related and independent epitopes on CEA. Using this technique to study fifteen MAbs, we have been able to recognise at least 6 unrelated epitopes of the CEA glycoprotein. Further characterisation of these epitopes was accomplished by means of immunohistochemistry. Of the fifteen MAbs, 6 were specific for CEA and reacted with at least 3 unrelated regions of the glycoprotein. Of the remaining 9 MAbs, 2 cross-reacted with erythrocytes, 5 with components of liver and 7 with polymorphonuclear neutrophils. Cross-reactions with liver were varied showing differential antibody specificity for bile canaliculi, Kupffer cells and bile duct epithelium. A high degree of correlation between epitope relatedness and immunohistochemical specificity was found. Two CEA-specific and 4 cross-reactive MAbs were also shown to react with ion-sensitive sites on the CEA glycoprotein.

Adenocarcinoma↗
PubMed1986

Mapping epitopes on a protein antigen by the proteolysis of antigen-antibody complexes.

A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

Animals↗
PubMed1992

Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations.

Five separate monoclonal antibodies (MoAbs) to human thyroid peroxidase (hTPO) were raised by immunising Balb/c mice with hTPO purified from detergent solubilised thyroid microsomes by high performance liquid chromatography (HPLC). The epitope specificities of these MoAbs were determined by assessing their ability to bind to purified recombinant fusion protein fragments of human TPO (TPO(r)) generated in E. coli. A total of seven small overlapping fragments (averaging 104 amino acid residues) of hTPO, encompassing over 90% of the extracellular region of the molecule, were generated as glutathione S-transferase (GST) fusion proteins. The sequential epitopes on TPO(r) recognised by these MoAbs were analysed by both immunoblotting and enzyme linked immunosorbent assay (ELISA). Two different MoAbs (A4 and A5) recognised sequential epitopes within the TPO(r) preparation termed R1a + b (residues 1-160) and more specifically, in the case of MoAb A4, within the subfragment R1b (residues 70-160). The inability of the other MoAbs (A1-A3) to recognise recombinant fragments, suggests they either recognise conformational determinants on the TPO molecule or epitopes that are present on the small regions of the TPO molecule which have not been expressed as recombinant proteins.

Animals↗
PubMed1989

Myelin-associated glycoprotein (MAG) and rat brain-specific 1B236 protein: mapping of epitopes and demonstration of immunological identity.

The myelin-associated glycoprotein (MAG) and the brain 1B236 protein are 100-kDa glycoproteins containing 30% carbohydrate that exist in two developmentally regulated forms and are specific to the nervous system. Recent cDNA cloning experiments in several laboratories using primarily immunological means of identification have determined the complete primary sequence of a rat brain glycoprotein that seems to correspond to both MAG and 1B236, suggesting that these proteins are identical. However, MAG was previously considered to be an oligodendrocyte/myelin specific component in the CNS at all ages, whereas 1B236 was thought to be primarily a neuronal component in adult rats but synthesized by oligodendrocytes at the time of active myelination. The composite term 1B236/MAG was proposed to describe the molecule identified by the cDNAs. In order to explore further the relationship between MAG and 1B236, as well as their developmentally regulated forms, experiments were carried out on rat samples utilizing synthetic peptides corresponding to sequences throughout the 1B236 molecule, antisera raised to synthetic peptides in the C-terminus of 1B236 that distinguish between the two developmentally regulated forms, and well-characterized polyclonal and monoclonal antibodies raised to purified MAG. Epitope mapping demonstrated that reactive sites were distributed throughout the extracellular and intracellular domains of 1B236/MAG. Only antibodies reacting with the smaller of the two forms of 1B236/MAG detected the glycoprotein in the peripheral nervous system. Both anti-MAG and anti-1B236 antibodies revealed a drastic reduction of the level of 1B236/MAG in 25-day-old myelin-deficient rats and in adult quaking mice, and both types of antibodies revealed a slight shift of 1B236/MAG toward higher apparent Mr in quaking mice as had previously been reported for MAG. The results indicate that MAG and 1B236 are almost certainly identical since they cannot be distinguished immunologically by the reagents available and that quantitatively most of the glycoprotein is associated with oligodendrocytes and myelin rather than neurons at all ages.

Animals↗
PubMed2003

A new method for mapping discontinuous antibody epitopes to reveal structural features of proteins.

Antibodies that bind to protein surfaces of interest can be used to report the three-dimensional structure of the protein as follows: Proteins are composed of linear polypeptide chains that fold together in complex spatial patterns to create the native protein structure. These folded structures form binding sites for antibodies. Antibody binding sites are typically "assembled" on the protein surface from segments that are far apart in the primary amino acid sequence of the target proteins. Short amino acid probe sequences that bind to the active region of each antibody can be used as witnesses to the antibody epitope surface and these probes can be efficiently selected from random sequence peptide libraries. This paper presents a new method to align these antibody epitopes to discontinuous regions of the one-dimensional amino acid sequence of a target protein. Such alignments of the epitopes indicate how segments of the protein sequence must be folded together in space and thus provide long-range constraints for solving the 3-D protein structure. This new antibody-based approach is applicable to the large fraction of proteins that are refractory to current approaches for structure determination and has the additional advantage of requiring very small amounts of the target protein. The binding site of an antibody is a surface, not just a continuous linear sequence, so the epitope mapping alignment problem is outside the scope of classical string alignment algorithms, such as Smith-Waterman. We formalize the alignment problem that is at the heart of this new approach, prove that the epitope mapping alignment problem is NP-complete, and give some initial results using a branch-and-bound algorithm to map two real-life cases. Initial results for two validation cases are presented for a graph-based protein surface neighbor mapping procedure that promises to provide additional spatial proximity information for the amino acid residues on the protein surface.

Actins↗
PubMed2006

A novel method for construction of gene fragment library to searching epitopes.

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.

Amino Acid Sequence↗
PubMed1993

Mapping of neutralizing epitopes and the receptor binding site of human interleukin 1 beta.

Antibodies to synthetic peptides of human interleukin 1 beta (IL-1 beta) and to recombinant human IL-1 beta were used to identify epitopes of IL-1 beta associated with the neutralization of its biological activity. Analysis of antisera raised to 17 synthetic peptides derived from the mature IL-1 beta sequence showed that five regions (residues 6-15, 49-80, 58-80, 92-101, and 120-133) were both immunoprecipitating and neutralizing. Using a hexamer epitope mapping method, comparison of the regions recognized by four neutralizing rabbit antisera with those recognized by a rabbit antiserum raised to denatured IL-1 beta suggested two further neutralizing epitopes, residues 39-48 and 83-95. Finally, a neutralizing monoclonal antibody was shown to bind to the peptides 6-11 and 87-95 by peptide binding and mutagenesis. All of these regions appear predominantly on one face of IL-1 beta. The effect of mutations in residues 4-11 and 88-97, which lie within this face, on receptor binding and biological activity was determined. Most of the mutations tested affected both receptor binding and activity, whereas mutations in another face of IL-1 beta (residues 74-80) had no effect. Purification of two of the mutants with reduced bioactivity and receptor binding and analysis by two-dimensional NMR indicated no gross changes in tertiary structure. A third mutant had reduced bioactivity in two different bioassays but no change in receptor binding. Although two-dimensional NMR revealed no gross changes in conformation, small changes did occur at a site distal from that mutated. The data are consistent with other epitope mapping and receptor binding mutagenesis data and suggest that the neutralizing antibodies and receptor recognize different but overlapping regions of IL-1 beta.

Amino Acid Sequence↗
PubMed1995

Mapping of linear epitopes recognized by monoclonal antibodies with gene-fragment phage display libraries.

Epitope mapping with mono- or polyclonal antibodies has so far been done either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizing overlapping short peptides, or by a combination of both methods. Here, we report an alternative method which involves the generation of random gene fragments of approximately 50-200 bp in length and cloning these into the 5' terminus of the protein III gene of fd phages. Selection for phages that bind a given monoclonal antibody and sequencing the DNA inserts of immunopositive phages yields derived amino acid sequences containing the desired epitope. A monoclonal antibody (mAb 215) directed against the largest subunit of Drosophila RNA polymerase II (RPB215) was used to map the corresponding epitope in a fUSE5 phage display library made of random DNA fragments from plasmid DNA containing the entire gene. After a single round of panning with this phage library, bacterial colonies were obtained which produced fd phages displaying the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immunoblots) resulted in overlapping sequences all containing the 15mer epitope determined by mapping with synthetic peptides. Similarly, we have localized the epitopes recognized by a mouse monoclonal antibody directed against the human p53 protein, and by a mouse monoclonal antibody directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning procedure depends on the detection system used (colony immunoblot or ELISA) and there appear to be some restrictions to the use of linker-encoded amino acids for optimal presentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. In general, our phage display method is faster, easier, and cheaper than the construction of overlapping fusion proteins or the use of synthetic peptides, especially in cases where the antigen is a large polypeptide such as the 215 kDa subunit of eukaryotic RNA polymerase II.

Amino Acid Sequence↗
PubMed2003

Mapping T cell epitopes by flow cytometry.

Epitope mapping by flow cytometry is a very modern approach that not only identifies T-cell epitopes but simultaneously allows for detailed analysis of the responding T-cell subsets including lineage, activation marker expression, and other markers of interest. The most frequently used approach is based on the identification of intracellular cytokines in secretion-inhibited activated T cells following stimulation with peptides or peptide pools. A more recently developed assay analyzes T-cell proliferation by measuring the decrease in carboxyfluorescein diacetate succinimidyl ester staining in proliferated cells. This article includes information on peptide configuration, a section on the design and efficient application of peptide pools, and working laboratory protocols for both assays.

Epitope Mapping↗
PubMed2001

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity.

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

Amino Acid Sequence↗
PubMed1996

Location of a protease-hypersensitive region in the multidrug resistance protein (MRP) by mapping of the epitope of MRP-specific monoclonal antibody QCRL-1.

Multidrug resistance protein (MRP) is a Mr 190,000 integral membrane phosphoglycoprotein which has been shown by transfection studies to confer multidrug resistance. We have previously raised and characterized a panel of MRP-specific monoclonal antibodies (MAbs) which detect distinct epitopes in the MRP molecule (D. R. Hipfner et A, Cancer Res., 54. 5788-5792, 1994), and, in the present study, we have identified the epitope of one of these, MAb QCRL-1. Immunoblot analysis of MRP fragments generated by digestion with formic acid or trypsin suggested that the MAb QCRL-1 epitope was located in the region connecting the two halves of MRP. Subsequent analyses of a series of truncated bacterial glutathione S-transferase fusion proteins containing segments of human MRP further localized the MAb QCRL-1 epitope to a region encompassing amino acids 903-956. Similar experiments with an analogous segment of murine MRP demonstrated that MAb QCRL-1 was highly specific for the human protein. The reactivity of MAb QCRL-1 with a series of overlapping hexapeptides and heptapeptides within this region identified the human MRP-specific heptapeptide SSYSGDI (corresponding to amino acids 918-924) as the epitope, and this peptide was shown to specifically inhibit MAb QCRL-1 binding to MRP. The results of these studies confirm that this epitope has a cytoplasmic location consistent with the topology of MRP predicted from hydrophobicity analyses. These experiments also revealed the presence of a number of protease-sensitive sites on either side of the MAb QCRL-1 epitope in the cytoplasmic domain connecting the two halves of MRP. Future epitope-mapping studies with other MRP-specific MAbs win provide additional insights into the topology of MRP, and may help to identify functionally important regions of this protein. Moreover, definition of the epitope recognized by MAb QCRL-1 as well as the other MAbs will facilitate the use of these reagents for immunohistological studies of MRP expression in drug-resistant tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Biochemical nature and mapping of PSMA epitopes recognized by human antibodies induced after immunization with gene-based vaccines.

BACKGROUND: A possible new target for immunotherapy is the prostate-specific membrane antigen (PSMA). The aim of the present study was to define potential PSMA epitopes for antibody binding using sera from patients immunized with gene-based anti-PSMA vaccines. MATERIALS AND METHODS: Sera from prostate cancer patients, immunized repeatedly with plasmid and adenoviral vectors, each encoding for the extracellular portion of human PSMA, were tested for anti-PSMA antibodies by Western blot. PSMA-producing LNCaP cells were used as a control. Recombinant PSMA protein cleaved with different proteinases was used for epitope mapping. Different enzymes were used to cleave the PSMA molecule. RESULTS: Specific anti-PSMA antibodies were detected in the studied patients' sera, mainly against the PSMA protein core. An alignment of the predicted enzyme-cleavage fragments was compared with Western blot results and several antibody epitopes were determined. CONCLUSION: These data demonstrate that multiple gene-based vaccinations induce an anti-PSMA humoral immune response. The antibodies are predominantly specific for the PSMA protein core.

Aged↗
PubMed1998

Functional mapping of CD11b/CD18 epitopes important in neutrophil-epithelial interactions: a central role of the I domain.

In the intestine, lung, and urinary tract, neutrophil (polymorphonuclear leukocyte, PMN) transepithelial migration is dependent on the leukocyte beta2 integrin CD11b/CD18. While the regions of CD11b involved in recognition of several soluble ligands are known, those that mediate PMN-epithelial interactions have not been investigated. In this study, mAbs reactive with four extracellular regions on CD11b, the NH2-terminal region, I (inserted) domain, cation-binding region, and region proximal to the transmembrane domain (C domain), were analyzed for the ability to block CD11b/CD18-mediated interactions with T84 intestinal epithelial cells. In such a manner, epitope mapping was applied to the complex interactions between CD11b/CD18 and a cell-based ligand system. I domain Abs strongly inhibited both adhesion of PMN to epithelial cells and PMN migration across T84 epithelial monolayers. However, the profile of inhibition was distinct from that of other known ligands of CD11b/CD18. CBRM1/32, an Ab to a discontinuous epitope residing within the NH2- and cation-binding domains, strongly inhibited both adhesion and transmigration responses. C domain Abs had minimal effects on adhesion and transmigration. These findings appear applicable to other epithelia, since similar results were obtained in transmigration experiments with CF15 human airway epithelial cells. Finally, Ab inhibition profiles were confirmed with adhesion assays of isolated epithelial cells to purified CD11b/CD18. These findings demonstrate the central role of the I domain and the participation of a discontinuous region shared by the NH2- and cation-binding domains in mediating PMN-adhesive interactions with epithelial cells.

Antibodies, Monoclonal↗