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PubMed1991

Application of recombinant DNA technology in epitope mapping and targeting. Development and characterization of a panel of monoclonal antibodies against the 7B2 neuroendocrine protein.

Three mouse monoclonal antibodies (Moabs) have been obtained with specificity for the 7B2 protein, a proposed member of the granin family of neuroendocrine proteins. Bacterially produced hybrid proteins of 7B2 were used as immunogens. The Moabs were designated MON-100, MON-101, and MON-102. Furthermore, we report the construction of 35 deletion mutants of the glutathione S-transferase-7B2 (GST-7B2) fusion-gene using recombinant DNA technology. The hybrid proteins encoded by eleven of these mutants were used in epitope mapping experiments and the results of these studies strongly suggested that recognition of 7B2 by all three Moabs involved the same 16 amino acid region of 7B2 (from amino acid residue 128-135). This was further substantiated by the observation that MON-101 and MON-102 specifically recognized a conjugate between bovine serum albumin and the synthetic peptide Phe-Glu-Pro-Glu-His-Asp-Tyr-Pro-Gly-Leu-Gly-Lys based upon the deduced amino acid sequence of the predicted epitope region in 7B2. In an approach to generate a series of 7B2-specific Moabs targeted against another epitope region in the 7B2 protein, the hybrid protein encoded by deletion mutant pPV32 was used as the immunogen. This protein lacked the epitope region recognized by the first series of Moabs. A second series of three Moabs, designated MON-142, MON-143, and MON-144, was obtained and, in all three cases, the region of 7B2 from amino acid residue 64-94 appeared to be involved in specific recognition by the Moabs. The whole panel of six anti-7B2 antibodies appeared to be useful in immunoprecipitation and Western blot analysis of the 7B2 protein and specifically stained neuroendocrine cells in immunohistochemical experiments. Using a double determinant sandwich enzyme immunoassay, 7B2 protein levels in rat pituitary were determined as 20 ng/mg tissue.

Amino Acid Sequence↗
PubMed2004

Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display.

Clotting factor VIII (fVIII)-inhibitory antibodies represent a major problem in the treatment of haemophilia A. To understand the inactivation mechanisms and to pave the way towards modifications of recombinant clotting factors that reduce their immunogenicity, the exact localization of immunodominant epitopes is required. Here, a random peptide phage display library was employed to identify epitopes of polyclonal fVIII antibodies isolated from patient's plasma by affinity chromatography. FVIII-binding specificity and inhibitory activity of the isolated fVIII antibodies were confirmed by ELISA and Bethesda assays. Phage selection on the individual samples yielded several phages which were displaced from binding to the respective antibody preparation by fVIII. Their homology with amino acid motifs of human fVIII and immunoprecipitation results with radioactively labelled fVIII fragments suggested putative epitopes in the A1, A2 and C1 domains of fVIII for one and in the C2 domain for another patient. Synthetic peptides corresponding to the A2, C1 and C2 domain epitopes blocked antibody binding to fVIII and partially neutralized the inhibitory activity of the respective plasma in Bethesda assays. These results provide the proof of principle that random peptide libraries can be used for the mapping of epitopes in a polyclonal antibody preparation.

Amino Acid Sequence↗
PubMed2005

Identification and antigenic epitope mapping of immunodominant region amino residues 510 to 672 on the spike protein of the severe acute respiratory syndrome coronavirus.

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510-672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539-559), S1C4 (548-567), S1C7/8 (583-606), and S1C10/11 (607-630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome.

Amino Acid Sequence↗
PubMed1999

Epitope mapping of 53 antibodies against prostate-specific antigen.

A panel of 53 antibodies from the ISOBM TD-3 PSA Workshop were tested for reactivity with iodinated derivatives of free PSA or the alpha1-antichymotrypsin PSA complex using the BIAcore system. Two antibodies (#69 and #83) showed low binding (<8%) for both antigens. One group of antibodies (#25, #26, #33, #54, #68, #73, #77, #78 and #91) had a much lower affinity for the complex (<12%) than for the free antigen (>65%). According to the mapping study, it was possible to categorize the antibodies into 29 different groups. Four antibodies were not classified. The two-dimensional representation of all interactions between the antibodies showed a complex network on the PSA molecule. Antibodies with lower affinity for the complex than for the free PSA appeared to bind epitopes in a common region, and thus it was not possible to perform sandwich assays with antibodies specific for free PSA.

Antibodies↗
PubMed1988

Epitope mapping of HLA-B27 and HLA-B7 antigens by using intradomain recombinants.

To study the HLA-B7 and HLA-B27 antigenic determinants, hybrid genes between these two alleles were constructed by in vivo recombination in Escherichia coli. After transfection of these genes into P815 (high transfection efficiency recipient) murine cells, the bindings of Bw6, HLA-B7, and HLA-B27 allele-specific mAb were studied, as well as that of human anti-HLA-B7 and anti-HLA-B27 monospecific alloantisera. Most of the HLA-B7 antigenic determinants were assigned to the first external domain of the molecule. Four different epitopic areas could be defined: the Bw6 epitope was associated with residues 82 and 83; the BB7.1 epitope to amino acids 63, 67, and 70; the MB40.2 and MB40.3 epitope to amino acid sequence 177-180, and human alloantisera identified as an epitope associated with residue 9. HLA-B27 antigenicity studied by TM-1 mAb was found to involve residues 77 and 80 in the alpha-1 domain. Results obtained with human monospecific alloantisera allowed the definition of an additional allospecific site associated with the NH2 terminal part on the alpha-1 domain of HLA-B27. Epitope mapping fits with data obtained by sequence comparisons and is discussed with reference to the crystallographic three-dimensional structure of the HLA-A2 molecule.

Amino Acid Sequence↗
PubMed1992

Immunoreactivity, epitope mapping and protection studies with anti-conotoxin GI sera and various conotoxins.

Goat or rabbit anti-conotoxin GI sera recognized native or dithiothreitol-reduced alpha-conotoxins GI, MI and SI in an enzyme-linked immunosorbent assay (ELISA). Native mu-conotoxin GIIIA or omega-conotoxin GVIA did not react with either anti-conotoxin GI serum in the assay. The goat anti-conotoxin GI serum neutralized 2.5 LD50S of alpha-conotoxins GI or MI in mouse lethal assays, while the rabbit antiserum had little protective capabilities. Epitope mapping of synthesized conotoxin peptide fragments revealed that both anti-conotoxin GI sera recognized linear sequences from five different alpha-conotoxins: GI, GIA, GII, MI and SI. The CCNPAC sequence was optimally recognized by both antisera.

Amino Acid Sequence↗
PubMed1997

A novel domain of fibronectin revealed by epitope mapping of a monoclonal antibody which inhibits fibroblasts-mediated collagen gel contraction.

The ability of cells to organize collagen fibrils is fundamental to a variety of processes found in embryogenesis, wound healing, fibrosis, and scar formation. We previously isolated a monoclonal antibody (mAb A3A5) which inhibits human fibroblast-mediated collagen gel contraction, an in vitro model producing the process of collagen morphogenesis. Human fibronectin (FN) has been shown to be the antigen of A3A5. The present study aimed at identifying the A3A5 epitope to reveal the mode of binding between collagen, FN, and fibroblasts in the process of gel contraction. The epitope was sought in FN fragments obtained by pepsin digestion and in recombinant FN fragments expressed in Escherichia coli by determining their immunological reactivity with A3A5, and was identified as a short segment consisting of the fourth through the amino half of the fifth FN type III. We propose a new functional domain of FN which plays a crucial role in the binding of fibroblasts to collagen fibrils and is involved in collagen morphogenesis.

Amino Acid Sequence↗
PubMed2005

Human T cell epitope mapping of the Schistosoma mansoni 14-kDa fatty acid-binding protein using cells from patients living in areas endemic for schistosomiasis.

The development of a defined anti-schistosomiasis vaccine would contribute to the current control strategy mainly because immunization provides long-lasting immunity to the disease. Sm14, one of the six Schistosoma mansoni antigens selected by WHO as a candidate to compose a subunit vaccine against schistosomiasis, has been associated with resistance to S. mansoni infection in human beings and is able to induce protection in the murine model. To identify human T cell epitopes in Sm14, we used the TEPITOPE algorithm to select peptides that would most likely bind to several HLA-DR molecules. In this study, three Sm14 epitopes were selected and produced as synthetic peptides. Human T cell responses from schistosomiasis patients living in endemic areas in Brazil were determined by proliferation assay and IL-5 and IFN-gamma measurements. Differential peptide recognition and cytokine production in response to Sm14 epitopes were observed in individuals resistant to S. mansoni infection versus susceptible individuals. Sm14(32-48) and Sm14(53-69) peptides were preferentially recognized by peripheral blood mononuclear cells (PBMCs) of S. mansoni-resistant individuals, and Sm14(53-69) induced significant production of IFN-gamma. Additionally, Sm14(32-48) and Sm14(53-69) were "promiscuous" peptides, since they were able to induce cellular immune responses in individuals carrying 10 and 8, respectively, of the 11 HLA-DR molecules expressed in the studied population. Among Sm14 synthetic peptides tested in this study, we identified Sm14(32-48) and Sm14(53-69) as promising candidates to compose an anti-schistosomiasis vaccine, since they seem to be related to resistance to human schistosomiasis.

Animals↗
PubMed1997

Epitope mapping of horseradish peroxidase (isoenzyme C).

Peptide scanning (PEPSCAN) was used to determine linear antigenic determinants of horseradish peroxidase isoenzyme C (HRPC). For this purpose, we synthesized 303 overlapping hexapeptide fragments (with a step of one amino acid residue) of the protein primary structure and studied their interactions with anti-HRPC polyclonal antisera by ELISA. Experiments with various titers of antisera allowed us to determine linear antigenic determinants of HRPC; several such determinants were spatially located in regular elements of the secondary structure (alpha-helices) found both inside and outside the protein globule. A fraction of epitopes were located in loops and folds of the HRPC peptide chain with irregular shapes. These epitopes contained several functionally important residues: Arg 38, which is part of the active site of the enzyme, as well as Phe 142 and Phe 143, which form a channel allowing aromatic substrates to reach the active site. Amino acid residues that form calcium-binding sites or occur in the vicinity of disulfide bonds are not involved in these epitopes.

Amino Acid Sequence↗
PubMed1988

Human and murine CD4 T cell epitopes map to the same region of the malaria circumsporozoite protein: limited immunogenicity of sporozoites and circumsporozoite protein.

The circumsporozoite (CS) protein is a candidate vaccine antigen for the sporozoite stage in the life cycle of the malaria parasite. Using CS protein purified from recombinant baculovirus-infected cells and a panel of H-2 congenic mice, we are able to demonstrate that this protein is poorly immunogenic in terms of antibody production as a result of Ir gene control. The immune response to the protein is also restricted following immunization with a CS-recombinant vaccinia virus or with sporozoites. Using a panel of overlapping peptides spanning the entire protein, we are able to show that the high responder mice recognize helper T cell epitopes from the same region of the protein as do humans. This region, however, is the polymorphic segment of the protein, which has implications for vaccine development. However, the close overlap of human and murine T cell epitopes demonstrates that murine models may be very useful in epitope mapping and vaccine development for human pathogens. The T cell antigenic regions of this protein fulfil the predictive requirements for the amphipathic helicity algorithm.

Amino Acid Sequence↗
PubMed1996

Epitope mapping of capsid proteins VP2 and VP3 of infectious bursal disease virus.

Twenty hybridoma cell lines producing monoclonal antibodies (MAbs) against serotype 1 infectious bursal disease virus (IBDV) of GBF-1 and the attenuated GBF-1E strains were produced. The MAbs recognized major structural proteins VP2 and VP3. MAb recognition sites were mapped using recombinant Escherichia coli clones which expressed N-terminal and (or) C-terminal truncated virus antigens, and competitive-binding assays. At least 3 conformation-dependent serotype 1 specific virus neutralizing antigenic sites and a linear antigenic site were defined on VP2 and VP3, respectively. Two of the conformational virus neutralizing antigenic sites were localized in the central area of VP2 consisting of 156 amino acid residues, and the linear epitope was localized in C-terminal 105 amino acid residues of VP3. Another conformational virus neutralizing antigenic site recognized with the virus neutralizing MAb GK-5 was not defined because GK-5 did not react with virus antigen expressed in recombinant E. coli. The conformational antigenic site was supposed to be composed of tertiary or quaternary protein structure, which may not be constructed in recombinant E. coli.

Animals↗
PubMed1994

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Amino Acid Sequence↗
PubMed1990

Epitope mapping of recombinant HeLa SmB and B' peptides obtained by the polymerase chain reaction.

SmB and SmB' are the major antigenic proteins contained within small nuclear RNP particles that are recognized by both human SLE and MRL mouse anti-Sm sera. We amplified cDNA obtained from HeLa cells by using the polymerase chain reaction and identified two clones, U2 and L13, that encode SmB and SmB', respectively. The nucleotide sequences of these two clones were identical except for the insertion of a 145-bp sequence in U2 that contained an early in frame termination codon and a potential 3' consensus splice site. The predicted amino acid sequences of HeLa SmB and B' proteins were therefore identical except for the COOH terminal 2 (U2) and 11 (L13) amino acids. U2, L13, and four subclones of U2 (F-B, B-R, F-X, and X-B) were ligated to pATH vectors and expressed as trpE fusion proteins. Epitope mapping with 12 human SLE and 12 MRL/lpr mouse anti-SmB/B' sera revealed that antibodies directed against the X-B peptide accounted for most (65.5 +/- 15.4 and 63.2 +/- 25.3%), B-R intermediate levels (51.5 +/- 30.8 and 18 +/- 19.6%), and F-X none of the anti-SmB activity in human and mouse sera, respectively. Ten human and two mouse sera contained antibodies that cross-reacted with epitopes located within the proline-rich, COOH-terminal, 27-residue peptide encoded by B-R and the NH2-proximal F-B peptide. These observations suggest that a) the polymerase chain reaction is a powerful ancillary method to synthesize autoantigens, b) SmB and B' in HeLa cells are derived from alternative splicing of a common RNA transcript, and c) both SLE and MRL anti-SmB/B' sera recognize multiple epitopes (some shared and some unique) on these proteins.

Amino Acid Sequence↗
PubMed1995

Peroxisomal membrane protein PMP68 of mouse liver: cloning of a cDNA encompassing the nucleotide binding fold and epitope mapping of monoclonal antibodies to the expressed protein.

We have isolated and sequenced a cDNA which encodes 376 amino acids toward the carboxy-terminus, and encompassing the putative nucleotide binding fold, of PMP68 (mouse liver peroxisomal integral membrane protein of 68 kDa) the major integral membrane protein of mouse liver peroxisomes. The protein sequence predicted from this cDNA shows 97.9% amino acid identity to this same region of rat liver PMP70, a member of the ATP-binding cassette protein superfamily (K. Kamijo, S. Taketani, S. Yokota, T. Osumi, and T. Hashimoto, 1990, J. Biol. Chem. 265, 4534-4540). The section of the cDNA encoding the hydrophilic and putative cytoplasmic domain of PMP68 was expressed as a recombinant fusion protein in bacteria. Two monoclonal antibodies raised against this protein have been epitope-mapped to peptides generated by cyanogen bromide cleavage of the fusion protein. Antibody 1A4 recognizes a peptide whose sequence contains the first motif of the putative nucleotide binding fold of PMP68, and antibody 8F11 recognizes a carboxy-terminal peptide which includes the second motif of this nucleotide binding fold. These antibodies are expected to be useful in the elucidation of the biological function of this putative membrane transporter.

ATP-Binding Cassette Transporters↗
PubMed2001

Human retinal S-antigen: T cell epitope mapping in posterior uveitis patients.

Uveitis, an intraocular inflammatory disease which affects the uveal tract and the retina of the eye in humans, is one of the major causes of visual impairment. Posterior uveitis is often associated with inflammation of the retina and vitreous. Unfortunately, etiological diagnosis of the disease is not possible in the majority of patients. It is generally felt that an autoimmune mechanism may be involved in so-called idiopathic cases. The role of retinal S-antigen, its 20 linear peptides spanning the entire sequence, and 2 additional peptides, known to be uveitopathogenic in experimental animals, was studied in 26 patients with uveitis. Lymphocyte proliferative response was tested in vitro to identify the epitopes of S-antigen involved and to establish their role in the pathogenesis of uveitis. Of 26 uveitis patients tested, 11 showed a significant T cell proliferative response in vitro to at least 1 antigen used. None among the controls showed any response to the peptides or native S-antigen used in this study. We have found that uveitis patients respond most frequently to peptide 4 (61--80), peptide 5 (81--100), peptide 8 (141--160), peptide 9 (161--180), peptide 12 (221--240), and peptide 13 (241--260) of the human S-antigen. These results further confirm that autoimmunity to retinal S-antigen may play a role in the etiopathogenesis of a subset of patients with idiopathic uveitis.

Adolescent↗
PubMed1996

Epitope mapping on intact, heated and reduced molecular variants of human chorionic gonadotrophin.

Monoclonal antibodies (MAbs) raised against purified human chorionic gonadotrophin (hCG) (n = 30) and synthetic peptides derived from hCG (n = 3) were able to recognize by Western blotting several hCG dimers (57-47 and 42 kDa), free beta-subunits (35-32, 26 and 16 kDa) and free alpha-subunit (21 kDa) which coexist in commercially available hCG preparations. According to differences in the immunoreactivity of hCG-related molecular forms observed under native or denaturing conditions such as boiling or reducing hCG samples before or after gel electrophoresis, nine classes of MAbs able to recognize different immunoreactive domains were determined. Three domains corresponded to continuous epitopes recognized by MAbs raised against hCG-related peptides. The six remaining domains, recognized by the other MAbs, contained discontinuous epitopes from which three were surface-oriented and three disguised in the holo-hormone. This solid-phase approach, combining native and denaturing conditions, represents a simple and powerful tool to screen the specificity of MAbs from varying sources and to investigate molecular variants of proteic hormone.

Antibodies, Monoclonal↗
PubMed1990

B cell responses to paramyosin. Isotypic analysis and epitope mapping of filarial paramyosin in patients with onchocerciasis.

To examine the fine specificity of the human immune response to filarial paramyosin, the antigenicity of an expressed rcDNA (2.55 kb) of Dirofilaria immitis paramyosin was detailed by ELISA. Using sera from patients infected with Onchocerca volvulus, we analyzed both the entire paramyosin molecule and six subcloned fragments for their IgG, IgG subclasses, and IgE responses. Patients from both Guatemala (64% positive) and Ghana (100% positive) reacted to paramyosin with specific IgG levels above normal controls. Although there was no anti-paramyosin subclass restriction common to all patients, the IgG3 response in the Ghananians was significantly greater than that of Guatemalans (p less than 0.001). IgE anti-paramyosin responses showed positive correlations with IgG2 (p less than 0.001), IgG4 (p less than 0.002), and IgG1 (p less than 0.04) responses. Epitope mapping using the IgG response to the six subclones demonstrated preferential recognition of the amino terminal end of the molecule (nucleotides 1 to 360). IgG2 reactivity was clearly localized to the most amino-terminal 120 amino acids, and the IgG4 antibodies recognized amino acids immediately adjacent to this fragment. These studies examining the fine specificity of anti-filarial immune reactions should provide a method for understanding how parasites either evade or induce host immune responses.

Adolescent↗