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PubMed2005

Epitope mapping in multiple sclerosis using the ELISPOT assay.

Multiple sclerosis (MS) is thought to be an autoimmune disease in which an unknown trigger initiates an immune response against brain proteins. This autoaggressive response causes the breakdown of the myelin sheaths that protect nerve axons, leading to impaired nerve conduction and subsequent neurodegeneration that are characteristic of MS. Many studies have attempted to determine the exact target within the brain. However, there appear to be multiple targets, which may change over time. No single study has examined all targets nor looked at how they can change over the course of the disease and whether these changes are related to the course of disease. We have approached this by using the single-cell resolution capability of the enzyme-linked immunospot assay to examine cytokine reactivity in MS patients in response to a very large set of overlapping peptides that span the two major proteins of myelin: myelin basic protein and proteolipid protein. Our goal was to use the enzyme-linked immunospot assay to perform comprehensive epitope mapping in relapsing-remitting MS patients in a longitudinal study to help define the role of myelin responses in disease progression.

Amino Acid Sequence↗
PubMed2001

Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor.

A protocol based on saturation transfer difference (STD) NMR spectra was developed to characterize the binding interactions at an atom level, termed group epitope mapping (GEM). As an example we chose the well-studied system of galactose binding to the 120-kDa lectin Ricinus communis agglutinin I (RCA(120)). As ligands we used methyl beta-D-galactoside and a biantennary decasaccharide. Analysis of the saturation transfer effects of methyl beta-D-galactoside showed that the H2, H3, and H4 protons are saturated to the highest degree, giving evidence of their close proximity to protons of the RCA(120) lectin. The direct interaction of the lectin with this region of the galactose is in excellent agreement with results obtained from the analysis of the binding specificities of many chemically modified galactose derivatives (Bhattacharyya, L.; Brewer, C. F. Eur. J. Biochem. 1988, 176, 207-212). This new NMR technique can identify the binding epitope of even complex ligands very quickly, which is a great improvement over time-consuming chemical modifications. Efficient GEM benefits from a relatively high off rate of the ligand and a large excess of the ligand over the receptor. Even for a ligand like the biantennary decasaccharide with micromolar binding affinity, the binding epitopes could easily be mapped to the terminal beta-D-Gal-(1-4)-beta-D-GlcNAc (beta-D-GlcNAc = N-acetyl-D-glucosamine) residues located at the nonreducing end of the two carbohydrate chains. The binding contribution of the terminal galactose residue is stronger than those of the penultimate GlcNAc residues. We could show that the GlcNAc residues bind "edge-on" with the region from H2 to H4, making contact with the protein. Analysis of STD NMR experiments performed under competitive conditions proved that the two saccharides studied bind at the same receptor site, thereby ruling out unspecific binding.

Acetylglucosamine↗
PubMed1990

Identification of functional regions on the tail of Acanthamoeba myosin-II using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites.

We used a series of COOH-terminally deleted recombinant myosin molecules to map precisely the binding sites of 22 monoclonal antibodies along the tail of Acanthamoeba myosin-II. These antibodies bind to 14 distinguishable epitopes, some separated by less than 10 amino acids. The positions of the binding sites visualized by electron microscopy agree only approximately with the physical positions of these sites on the alpha-helical coiled-coil tail. On the other hand, the epitope map agrees precisely with competitive binding studies: all antibodies that share an epitope compete with each other for binding to myosin. Antibodies with adjacent epitopes can compete with each other at linear distances up to 5 or 6 nm, and many antibodies that bind 3-7-nm apart can enhance the binding of each other to myosin. Most of the antibodies that bind to the distal 37 nm of the tail disrupt assembly of octameric minifilaments and, depending upon the exact location of the binding site, stop assembly at specific steps yielding, for example, monomers, antiparallel dimers, parallel dimers or antiparallel tetramers. The effects of these antibodies on assembly identify sites on the tail that are required for individual steps in minifilament assembly. Experiments on the assembly of truncated myosin-II tails have revealed a complementary group of sites that participate in the assembly reactions (Sinard, J.H., D.L. Rimm, and T.D. Pollard. 1990. J. Cell Biol. 111:2417-2426). Antibodies that bind to the distal tail but do not affect assembly appear to have a low affinity for myosin-II. Antibodies that bind to the proximal 50 nm of the tail do not inhibit the assembly of minifilaments. Many antibodies that bind to the tail of myosin-II, even some that have no obvious effect on minifilament assembly, can inhibit the actomyosin ATPase activity and the contraction of an actin gel formed in crude extracts. An antibody that binds between amino acids 1447 and 1467 inhibits the phosphorylation of serine residues distal to residue 1483.

Acanthamoeba↗
PubMed2005

Epitope mapping of Ly-49G and G-like receptors: CK-1 antibody defines a polymorphic site of functional interaction with class I ligand.

Ly-49 receptors regulate mouse natural killer cell functions. Members of the polymorphic Ly-49 multigene family recognize specific alleles of major histocompatibility complex class I (MHC I) or MHC I-like proteins. Previous studies have provided insight into the nature of Ly-49A and -C interaction with their high-affinity MHC I ligands, H-2Dd and Kb, respectively. Unlike Ly-49C, recognition of MHC I by Ly-49A is regulated in part by residues within the beta4-beta5 loop of its ectodomain. Ly-49A and -G are within the same Ly-49 subfamily, and both receptors recognize Dd. However, there have been no studies that define specific sites on Ly-49G that mediate class I MHC recognition. The Ly-49G receptors of different inbred mouse strains can differ as a result of amino acid polymorphisms within their ectodomains. In this report, we have generated a novel antibody, CK-1, which recognizes Ly-49G(B6) and a Ly-49G(B6)-like receptor, Ly-49M(nonobese diabetic), but not Ly-49G(BALB/c). By exploiting the differences within ectodomains of C57BL/6 and BALB/c Ly-49G allele products, we identified epitopes recognized by the Ly-49G-specific antibodies CK-1 and Cwy-3, whose epitopes mapped within the beta4-beta5 loop and the beta1 strand, respectively, and were nonoverlapping. Although both antibodies specifically recognized the Ly-49G(B6) ectodomain, Cwy-3 was unable to block its interaction with MHC I, and CK-1 significantly inhibited it. The importance of residues within the beta4-beta5 loop in Ly-49G recognition demonstrates that its interaction with MHC I is similar to that of Ly-49A but not Ly-49C.

Amino Acid Sequence↗
PubMed2005

Epitope mapping of monoclonal antibodies specific to serovar of Leptospira, using phage display technique.

Random heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of five monoclonal antibodies that were specific to L. australis, L. bangkok, and L. bratislava. Phages selected by biopanning were cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Almost all of the peptide epitopes were continuous or linear. Interestingly, in phages reacting with the monoclonal antibody (MAb) clones F11, F20, 2C3D4, and 8C6C4A12, the deduced amino acid sequence of the displayed peptides corresponded to a segment of hypothetical protein of the Leptospira genome (L. interrogans serovar Lai and Copenhageni). Considering the deduced amino acid sequences of phages reacting with the MAb clones F11, F20, 2C3D4, and 8C6C4A12, the consensus motif -SKSSRC-, -TLINIF-, -SSKSYR- and -CTPKKSGRC- appeared respectively. No similarity was observed among phage reacting with the MAb clone F21. The results demonstrate that T7 phage display technique has potential for epitope mapping of leptospiral MAbs, and for rapid analysis of the interactions between phage display peptides with the MAb. The finding of a phage peptide that binds to MAb with protective activity can be further tested as a candidate for leptospirosis vaccine in the future.

Antibodies, Monoclonal↗
PubMed1994

Alzheimer's paired helical filaments: amyloid precursor protein epitope mapping.

Paired helical filaments (PHF) were electrophoretically purified and solubilized from Alzheimer's neurofibrillary tangles and consisted of a primary 66 kDa protein on SDS-PAGE analysis. A panel of antibodies raised against restricted regions of the beta-amyloid precursor protein (APP) were employed for epitope mapping studies of this 66 kDa PHF protein. Western blot studies revealed that C-terminal APP antibodies were immunoreactive with the 66 kDa PHF protein. Further analysis revealed that only antisera raised against peptides that include the beta/A4-amyloid region within the C-terminal portion of APP were immunoreactive with PHF proteins. These data complement previous immunocytochemical studies which indicated that C-terminal APP antibodies preferentially label PHF-containing neurofibrillary tangles in Alzheimer's brain. The present data suggest a similarity of secondary or tertiary structure between beta/A4-amyloid and PHF which accounts for the cross-reactivity of beta/A4-amyloid antibodies with PHF proteins.

Alzheimer Disease↗
PubMed1985

Monoclonal antibodies against rat brain hexokinase. Utilization in epitope mapping studies and establishment of structure-function relationships.

The effects of seven monoclonal antibodies on various functions of rat brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) have been assessed. Specifically, effects on catalytic properties (Km values for substrates, glucose and ATP X Mg2+; Ki for inhibition by glucose 6-phosphate), binding to the outer mitochondrial membrane, and glucose 6-phosphate-induced solubilization of mitochondrially bound hexokinase were examined. Epitope mapping studies with the native enzyme provided information about the relative spatial distribution of the epitopes on the surface of the native molecule. Binding of nucleotides (ATP or ATP X Mg2+) was shown to perturb the epitopes recognized by two of these antibodies. Neither nucleotides nor other ligands (glucose, glucose 6-phosphate, Pi) had detectable effect on epitopes recognized by the other five antibodies. Peptide mapping techniques in conjunction with immunoblotting permitted assignment of the epitopes recognized by several of the antibodies to specific segments within the overall primary structure. These results, together with previous work relating to the organization of structural domains within the molecule, permitted development of a three-dimensional model which provides a useful representation of major structural and immunological features of the enzyme, and depicts the association of those features with specific functions.

Adenosine Triphosphate↗
PubMed1992

Epitope mapping of the nonspecific cross-reacting antigen using various related recombinant proteins expressed in Chinese hamster ovary cells and eight distinct monoclonal antibodies.

Antigenic epitopes of nonspecific cross-reacting antigen (NCA) recognized by 8 different monoclonal antibodies (MAbs) were analyzed in relation to the domain structures of NCA [domains N, I (A1-B1) and M] and CEA [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M]. We reconstructed cDNAs for NCA-N, NCA-N-I-M, CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M in a eukaryotic expression vector, pdKCR-dhfr, and expressed them in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested against eight different MAbs reactive with NCA. All 8 MAbs had been shown to recognize the protein epitopes of the NCA molecule and classified into two groups in terms of the reactivity with NCA and CEA; Group X, 5 clones reactive with both NCA and CEA; and Group Y, 3 clones reactive only with NCA. The epitopes recognized by two of five Group X MAbs were found to be present on the domain N of the NCA molecule as well as of the CEA molecule, and those of the three others were on the domain I (A1-B1) of both molecules, respectively. All three epitopes of Group Y MAbs, which were unique to NCA, were present on the domain I (A1-B1) but not on the domain N of the NCA molecule. The epitope mapping reported here helps form the basis for understanding the relation between the chemical structure and antigenic activities of the NCA molecule and may be useful to study the functions of the NCA molecule, especially those of the respective domains.

Animals↗
PubMed2006

Mapping epitopes and antigenicity by site-directed masking.

Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (beta-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to beta-lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with beta-lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops.

Animals↗
PubMed1991

Fusion protein based epitope mapping of the MPB57 protein from Mycobacterium bovis BCG and its epitope insertion into the native protein.

The gene coding for the 12-kDa protein (MPB57) of Mycobacterium bovis BCG has recently been cloned and sequenced (R. Yamaguchi, K. Matsuo, A. Yamazaki, S. Nagai, K. Terasaka, and T. Yamada. 1988. FEBS Lett. 240: 115-117). To map linear B-cell epitopes by beta-galactosidase fusion proteins, we have constructed convenient vectors (pUR278S, pUR288S, and pUR289S) with the SmaI site. Based on recognition by polyclonal antibodies, two epitope regions on the MPB57 protein were identified, both of which corresponded to the amino acid sequences Glu20 to Val45 (26 residues, epitope I region) and Ile78 to Leu86 (9 residues, epitope II). Complementary oligonucleotides encoding epitope II were synthesized, polymerized by a ligase reaction, inserted into the native MPB57 protein gene, and expressed in Escherichia coli, giving rise to epitope-inserted proteins. Their stability and potential uses are described.

Amino Acid Sequence↗
PubMed1990

Epitope map of the HLA-B7 CREG using affinity-purified human alloantibody probes.

Monoclonal antibodies (mAb) recognizing the B7 CREG have been used to construct an epitopic map of HLA-B7. Similar studies with human HLA alloantisera have been lacking due to the polyclonal nature of the alloantibodies (aAb). Detergent-solubilized HLA Class I antigens were purified and coupled to activated CH-Sepharose 4B. Sequential affinity isolation of aAb populations using a series of HLA antigen columns enabled us to produce a battery of aAb eluates against both the private B7, B13, B27, and B omega 60 determinants and the public B7-42, B7-60, B7-60-61, B7-27-13-60, B7-42-22-27, B7-8-42-60-41, and B omega 6 epitopes. The topographic relationship of the B7 family of determinants recognized by the Ab probes was derived using crosscompetition Ab blocking assays with quantitation by indirect immunofluorescence and FACS analysis. We have found that aAb and mAb of similar specificity crossblock; Ab of different specificity give complex patterns including both overlapping blocking between the alpha domains and Ab-induced conformational change of the molecule. From these investigations, we conclude that HLA Class I alloantigens bear both multiple, topographically distinct public epitopes and separate private determinants that can be distinguished using human aAb probes. At least four discrete epitopes are expressed by each molecule of the HLA-B7 CREG and can be ascribed to unique aa substitutions on the hydrophilic beta loops of the distal heavy chain domains and also on several exposed areas of the alpha helices. These findings are extremely similar to those of the HLA-A2 CREG and suggest that possibly all Class I molecules possess a comparable, complex degree of serologic polymorphism.

Antibodies, Monoclonal↗
PubMed1995

Epitope mapping of anti-HIV and anti-HCV monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library.

A large filamentous phage library (1 x 10(9) clones) displaying random 30-amino-acid (aa) sequences on the N terminus of the pIII coat protein was constructed and characterized. Clones in the library were affinity selected for binding to monoclonal antibodies (mAb) against two viral antigens, the HIV gp120 protein and the HCV core protein. The obtained aa sequences precisely identified the epitopes recognized by the mAb. Binding of peptide-carrying phages to the Ab was demonstrated by ELISA, Western blot and the surface plasmon resonance (SPR) method. The mAb-specific peptides were transferred via genetic techniques onto the N terminus of Escherichia coli alkaline phosphatase (AP). When fused to the enzyme, the peptides maintained their ability to bind their respective mAb, indicating that the peptides contained the necessary contact residues for binding. The affinity of the peptides was estimated to be 100 nM by SPR. A comparison is presented of the relative affinities of phage-derived peptides to the native viral epitopes also displayed on the AP scaffold. The approach of transferring epitopes from phage to AP for further evaluation should be applicable to many other mAb or receptors.

Acid Phosphatase↗
PubMed2004

Epitope mapping of Porphyromonas gingivalis heat-shock protein and human heat-shock protein in human atherosclerosis.

To identify T- and/or cross-reactive B-cell epitopes of P. gingivalis and human heat-shock protein (HSP)60 in atherosclerosis patients, we synthesized 104 overlapping synthetic peptides spanning whole molecules of P. gingivalis HSP60 and human HSP60, respectively. T-cell epitopes of P. gingivalis HSP were identified with the use of previously established P. gingivalis HSP-reactive T-cell lines. B-cell epitopes of P. gingivalis HSP60 and human HSP60 were identified by the use of patients' sera. Anti-P. gingivalis, anti-P. gingivalis HSP60, or anti-human HSP60 IgG antibody titers were higher in the atherosclerosis patients compared with the healthy subjects. Five immunodominant peptides of P. gingivalis HSP60, identified as T-cell epitopes, were also found to be B-cell epitopes. Moreover, 6 cross-reactive B-cell epitopes of human HSP60 were identified. It was concluded that P. gingivalis HSP60 might be involved in the immunoregulatory process of atherosclerosis, with common T- and/or B-cell epitope specificities and with cross-reactivity with human HSP60.

Aged↗
PubMed1993

Protein P4 of double-stranded RNA bacteriophage phi 6 is accessible on the nucleocapsid surface: epitope mapping and orientation of the protein.

Protein P4, an early protein of double-stranded RNA bacteriophage phi 6, is a component of the virion-associated RNA polymerase complex and possesses a nucleoside triphosphate (NTP) phosphohydrolase activity. We have produced and characterized a panel of 20 P4-specific monoclonal antibodies. Epitope mapping using truncated molecules of recombinant P4 revealed seven linear epitopes. The accessibility of the epitopes on the phi 6 nucleocapsid (NC) surface showed that at least the C terminus and an internal domain, containing the consensus sequence for NTP binding, protrude the NC shell. Four of the NC-binding antibodies distorted the integrity of the NC by releasing protein P4 and the major NC surface protein P8. This finding suggests a close contact between these two proteins. The dissociation of the NC led to the activation of the virion-associated RNA polymerase. The multimeric status of the recombinant P4 was similar to that of the virion-associated P4, indicating that no accessory virus proteins are needed for its multimerization.

Amino Acid Sequence↗
PubMed1987

Epitope mapping of monoclonal antibodies to gag protein p19 of avian sarcoma and leukaemia viruses.

We have characterized a set of 15 monoclonal antibodies to p19gag, one of the internal proteins of avian sarcoma and leukaemia viruses. All the antibodies work in immune precipitations as well as in immunoblotting, though with different efficiencies. We have developed a simple epitope mapping technique, which uses partial chemical cleavages at methionine or tryptophan residues followed by immunoblotting from SDS-polyacrylamide gels, to localize the epitopes of nine of these antibodies. The epitopes fall into at least four classes. The mapping procedure should also be useful for other antigens of known primary structure.

Antibodies, Monoclonal↗