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PubMed1995

Epitope mapping of monoclonal antibodies to keratin 19 using keratin fragments, synthetic peptides and phage peptide libraries.

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a full-length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.

Amino Acid Sequence↗
PubMed1990

Comparative epitope mapping of murine monoclonal and human autoantibodies to human PDH-E2, the major mitochondrial autoantigen of primary biliary cirrhosis.

Immunization with recombinant human pyruvate dehydrogenase (PDH)-E2, the major autoantigen of primary biliary cirrhosis, readily induces a vigorous murine antibody response but does not generate hepatic disease. To determine the fine specificity of this response, 18 mAb were generated from three strains of mice and the reactive epitopes mapped. An initial examination of mAb suggested that they behaved similarly to the antimitochondrial autoantibodies in primary biliary cirrhosis (PBC) because i) all polyclonal antisera and 2 of 18 mAb reacted with all species of mammalian PDH-E2 examined including mouse PDH-E2, ii) 15 of 18 mAb inhibited PDH enzyme function, and iii) the reactivity of mAb toward rPDH-E2 were blocked by PBC sera. However, fine examination of the reactive sequences of the PDH-E2 complex revealed that antibodies identical to those in PBC patients were not produced by experimental immunization. In contrast to PBC, none of the mAb or murine polyclonal sera were able to react with protein X, a lipoic acid-containing component of the PDH complex previously shown to cross-react with PDH-E2 when probed with PBC sera. Although the epitopes for 12 mAb were localized within the inner lipoyl domain, none reacted with mouse PDH-E2 or cross-reacted with the outer lipoyl domain as observed in PBC. In addition, the epitopes of the two mAb which did react with all mammalian species of mitochondria were not localized within the PBC epitope. These findings indicate the highly immunogenic nature of the inner lipoyl domain of PDH-E2. The inability to elicit antibodies of the same specificity in mice, considered together with the highly localized autoantibody response in humans, suggests that antimitochondrial autoantibodies are most likely the result of specific breakdown of tolerance to a unique autoepitope.

Acetyltransferases↗
PubMedDate not supplied

Epitope mapping of autoantigens using recombinant proteins and synthetic peptides.

In order to evaluate mechanisms of autoantibody production, three hypotheses (random polyclonal B cell activation, molecular mimicry and immune response against restricted self antigens) were considered in relation to autoantibody recognition. Polyclonal B cell activation was considered unlikely because lupus sera recognized a relatively small number of eukaryotic protein antigens on immunoblots and failed to show enhanced reactivity against prokaryotic epitopes. Although two interesting examples of molecular mimicry between autoantigens and two different viruses have been suggested, it is yet to be shown that lupus antibodies bind to the shared epitopes on the viruses and that the viruses induce such antibodies. Finally, epitope mapping of several different autoantigens suggest that the antigens are themselves the focus of the immune response and initiate and/or maintain autoantibody production.

Amino Acid Sequence↗
PubMed1987

Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides.

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.

Allergens↗
PubMed2006

Epitope mapping of the domains of human angiotensin converting enzyme.

Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.

Amino Acid Sequence↗
PubMed1994

[Molecular cloning, expression and epitope mapping of nuclear antigen].

The antinuclear antibody is a major characteristic phenomenon in systemic type of autoimmune diseases. There is a good correlation between symptoms or diseases and the types of antinuclear antibodies in these patients. Therefore, the study of the mechanisms of antinuclear antibody production is thought to be important in the elucidation of the pathogenesis of such autoimmune diseases. The conventional methods to detect antinuclear antibodies have been useful to date in clinical studies. However, these methods are not sufficient for more precise investigations. Thus, we have applied the recently developed molecular biology to this field. cDNAs which encode nuclear antigens have been cloned and their protein products were expressed as recombinant fusion protein in E. coli. By this procedure, we could establish simple and reproducible method to detects antinuclear antibodies. Furthermore, epitope mapping of nuclear antigens could be performed using deletion mutants generated by enzymatic manipulations of the cDNAs. These epitope studies are facilitating a more precise understanding of the mechanisms of antinuclear antibody production.

Antibodies, Antinuclear↗
PubMed1990

HIV-1 envelope protein gp120 expression by secretion in E. coli: assessment of CD4 binding and use in epitope mapping.

A non-glycosylated form of the HIV-1 envelope protein gp120 and four truncated derivatives have been expressed as non-fused secreted products in the periplasmic space of E. coli. We show that the full length molecule, whilst folded and soluble, fails to bind to CD4 consistent with other work that suggests an essential role for carbohydrate in gp120 function. In addition, when used in conjunction with the truncated derivatives, rapid epitope mapping of anti-gp120 monoclonal antibodies is achieved using both Western-blot and ELISA formats.

Antibodies, Monoclonal↗
PubMed1991

Epitope mapping by mutagenesis distinguishes between the two tertiary structures of the histidine-containing protein HPr.

Thirty-four of the 85 residues of the histidine-containing protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system have been changed by site-directed mutagenesis. Many of the mutations have wild-type activity suggesting an unaltered tertiary structure but have altered binding to three monoclonal antibodies: Jel42, Jel44, and Jel323. This altered binding defines the residues that are involved in the epitopes of HPr. At present, two different three-dimensional structures have been determined for HPr, one from two-dimensional nuclear magnetic resonance spectra and the other from x-ray diffraction of HPr crystals. The epitope mapping for Jel42 does not distinguish between the tertiary structures. However, only the HPr structure derived from two-dimensional nuclear magnetic resonance spectra is consistent with a contiguous surface binding site that can be defined as the epitope for Jel44. Thus the x-ray structure may represent a partially unfolded HPr.

Amino Acid Sequence↗
PubMed1996

Epitope mapping studies with human anti-cytochrome P450 3A antibodies.

A subset of patients with hypersensitivity reactions to the aromatic anticonvulsants phenytoin, carbamazepine, and phenobarbital have circulating antibodies that recognize members of the rat cytochrome P450 (CYP) 3A subfamily. These antibodies do not recognize related human CYP3A proteins despite the high degree of structural similarity. To investigate the relationship between P450-mediated drug metabolism and the development of anti-P450 antibodies, we initiated epitope mapping studies by screening a library of fusion proteins constructed from rat CYP3A1 with an anti-CYP3A1-positive patient serum sample. Positive signals from colony lifts were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblotting, and a 26-amino acid sequence corresponding to amino acids 342-367 of the CYP3A1 protein (NKAPPTY-DTVMEMEYLDMVLNETLRL) was identified as containing the epitope recognized by IgG3 antibodies in this serum sample. By subjecting inserts from two clones into a second round of library construction and screening by immunoblot analysis, we further defined the epitope to EYLDMVLNETLRL. Single amino acid deletions identified DMVLNETLRL as the minimum amino acid sequence required for antibody binding. The corresponding sequence in the four human CYP3A proteins differs by only one amino acid (DMVVNETLRL) This amino acid is critical to antibody recognition as immunoreactivity of the L361V mutant is markedly reduced. Anti-CYP3A antibodies in nine of nine additional sera also recognized the 13-amino acid epitope; for five of these sera, the minimum antibody binding sequence was DMVLNETLRL. The proximity of this epitope to a region determining substrate specificity may provide the link among reactive metabolite production, hapten formation, and the production of anti-P450 antibodies in anticonvulsant-induced idiosyncratic reactions.

Amino Acid Sequence↗
PubMed1992

Human T lymphotropic virus types I- and II-specific antibody-dependent cellular cytotoxicity: strain specificity and epitope mapping.

Antibody-dependent cellular cytotoxicity (ADCC) against human T lymphotropic virus (HTLV) types I and II was investigated using sera obtained from infected individuals or from rabbits immunized with HTLV-I or -II envelope peptides. Target cells included an HTLV-I-transformed cell line (C91/PL), an HTLV-II-transformed cell line (729pH6neo), and Epstein Barr virus (EBV)-transformed B lymphocytes expressing HTLV-I or -II env or gag gene products after infection with vaccinia/HTLV recombinants. ADCC activity was directed at HTLV-I and -II envelope glycoproteins but not against core (gag) components. In contrast to the human immunodeficiency virus system, significant cross-reactivity between HTLV-I- and -II-directed ADCC activity was observed. Epitope mapping studies using sera from rabbits that had been immunized with HTLV-I or -II envelope peptides suggested that the critical epitopes for ADCC activity are located primarily in hydrophilic regions of the exterior (gp46) part of the envelope glycoprotein.

Animals↗
PubMed1986

Epitopic mapping of structural and nonstructural Aleutian disease virus proteins.

Common antigenic properties for p85 and p75 but a different antigenic character for p71 Aleutian disease virus (ADV) proteins were demonstrated by Western blot analysis with monoclonal antibodies. It was shown that four hybridomas (ADV-Hy 47, 66, 77 and 84) with specific reactivity for structural proteins p85 and p75 also recognized p25 but not the p71, nonstructural, protein. In turn, the monoclonal antibody ADV-Hy 2 recognized the p71 protein only. For further studies of their antigenic properties, the ADV proteins were subjected to enzymatic or chemical cleavage. The derived peptide fragments were analyzed by epitopic mapping. Depending on the cleavage reagent and monoclonal antibody applied, specific peptide maps were revealed. The maps of p85 and p75 were very similar, indicating that both proteins shared an extensive antigenic relationship. After cleavage with alpha-chymotrypsin and N-chlorosuccinimide and by using the ADV-Hy 84 monoclonal antibody, unique peptide fragments were identified with p85 which had no counterparts in p75 fragments.

Aleutian Mink Disease Virus↗
PubMed2005

Validation of in silico prediction by in vitro immunoserological results of fine epitope mapping on citrate synthase specific autoantibodies.

In silico antibody-antigen binding predictions are generally employed in research to rationalize epitope development. These techniques are widely spread despite their technical limitations. To validate the results of these bioinformatic calculations evidence based comparative in vitro studies are necessary. We have used a well-conserved mitochondrial inner membrane antigen-citrate synthase to develop a model for comparative analysis of the predicted and the immunoserologically verified epitopes of circulating autoantibodies. Epitopes were predicted using accepted tools: the GCG Wisconsin package and TEPITOPE 2000. An overlapping multipin ELISA assay--covering 49% of the citrate synthase molecule--was developed to map autoantibody epitopes of individuals (healthy, systemic autoimmune, and heart transplanted) in different immunopathological conditions. From the 40 synthesized decapeptides 34 were predicted in silico and 27 were validated in vitro. Thirty-two percent of epitopes were recognized by majority of sera 47% by at least one sera. False positive predictions were 21%. There was major difference in the recognized epitope pattern under different immunopathological conditions. Our results suggest that special databases are needed for training and weighing prediction methods by clinically well-characterized samples, due to the differences in the immune response under different health status. The development of these special algorithms needs a new approach. A high number of samples under these special immunological conditions are to be mapped and then used for the "fine tuning" of different prediction algorithms.

Amino Acid Sequence↗
PubMed1999

Epitope mapping for the anti-rabbit cholesteryl ester transfer protein monoclonal antibody that selectively inhibits triglyceride transfer.

Among the monoclonal antibodies (Mab) against rabbit plasma cholesteryl ester transfer protein (CETP), Mab 14-8F cross-reacted with human CETP and selectively inhibited triglyceride transfer but not cholesteryl ester transfer (Ko, K. W. S., T. Ohnishi, and S. Yokoyama. 1994. J. Biol. Chem. 269: 28206;-28213). The epitope of this antibody was studied by using synthetic fragment peptides of rabbit and human CETP. Mab 14-8F reacted with the peptide R451-Q473 of human CETP near the carboxyl-terminal and not with the peptides representing any other regions, and inhibited the binding of human CETP to the goat antibody against its carboxyl-terminal peptide R451-S476. The experiments with a series of the fragment peptides in this region revealed that the epitope requires the segment 465-473 (EHLLVDFLQ) of human CETP or 485-493 (KHLLVDFLQ) of rabbit CETP (core epitope) though neither peptide by itself binds to the antibody. Both peptides needed extension at least by one residue beyond either amino- or carboxyl-end in order to show the reactivity to the antibody, but the effect was not highly residue-specific at least at the amino-end. Circular dichroism analysis demonstrated the increase of helical conformation by the extension of the "core epitope" peptides to either direction. Thus, the epitope is dependent on conformation of the core epitope induced by the presence of an additional residue(s) in either end. The core epitope occupies the central 64% of the reported linear epitope of Mab TP2, a widely used anti-human CETP monoclonal antibody that inhibits both cholesteryl ester and triglyceride transfer.Therefore, we conclude that the limited interaction of Mab with a common lipid interaction site causes selective inhibition of the transfer of triglyceride that has presumably lower priority than cholesteryl ester for the CETP reaction.

Amino Acid Sequence↗
PubMed1996

Antigenic determinants at the carboxy terminus of chicken egg white riboflavin carrier protein (RCP): epitope mapping and antibody-mediated pregnancy curtailment in rodents.

The epitopic core sequences recognized by three monoclonal antibodies raised to chicken riboflavin carrier protein (RCP) were mapped to the C-terminal tail-end of the protein using the pepscan method. A 21-residue synthetic peptide corresponding to residues 200-219 of the protein and comprising the regions corresponding to the antibodies was synthesized. Administration of polyclonal antibodies specific to this peptide led to termination of early pregnancy in mice. Also, active immunization of rats with the peptide-purified protein derivative conjugate inhibited establishment of pregnancy. These results demonstrate the functional importance of the C-terminal 200-219 region of chicken RCP.

Amino Acid Sequence↗
PubMed1993

Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I.

The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide-linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty-one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope-mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I. Two monoclonal antibodies directed against reduced/alkylated chain 1 bound to the overlapping peptides 53-66 and 60-70 of chain 1. The monoclonal antibody directed against reduced/alkylated chain 2 bound to the overlapping peptides 36-49 and 43-56 of chain 2. Binding specificity was demonstrated by inhibition by reduced/alkylated Fel d I for all three monoclonal antibodies. Another monoclonal antibody against reduced/alkylated Fel d I had been found to bind predominantly to reduced/alkylated chain 2 on immunoblot in previous studies. It bound to peptides 1-16 and 60-70 of chain 1 and peptides 1-14 and 50-63 of chain 2; it is therefore probably directed against a conformational epitope formed by these four regions. Possibly because of low affinity of this monoclonal antibody, specificity of its binding could not be verified by inhibition studies. A panel of monoclonal antibodies directed against native Fel d I bound to peptides 1-16 and 60-70 of chain 1 and peptides 1-14 and 43-56 of chain 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗