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PubMed1992

Epitope analysis of the allergen ovalbumin (Gal d II) with monoclonal antibodies and patients' IgE.

Ovalbumin (OVA) is a major allergen (Gal d II) of hen egg white and is often the cause of hypersensitivity reactions to food. Further knowledge of the antigenic and allergenic epitopes of allergens will provide better treatment of this disease. To analyse these epitopes we produced a panel of monoclonal antibodies (mAbs) against native OVA. The initial information about the epitopes was obtained with the binding patterns of these mAbs in IEF-immunoprints and western blots of OVA under reducing and non-reducing conditions. It was possible to demonstrate that the different conformations of OVA exhibit different epitopes, and that there are other epitopes which are shared by each conformation. Seven different, although sometimes overlapping epitopes, could be determined on native OVA; four different epitopes on denaturated non-reduced OVA by means of immunoblots of the intact molecule. The number of epitopes which could be differentiated by the mAbs was increased by the use of peptide blots after CNBr fragmentation of the molecule. IgE binding to different OVA conformations and to CNBr-fragments of OVA was also detectable and appears in the same regions as the reactivity of some mAbs. Western blots of OVA and CNBr-peptides demonstrate that some antigenic/allergenic binding sites seem at least partly to be continuous epitopes. The identification of the CNBr-fragments was performed by a microsequence analysis of blotted CNBr-fragments after a 2-dimensional electrophoresis. IgE was found to bind the two largest CNBr-fragments (residues 41-172 and 301-385), but not the fragment corresponding to residues 173-196. A number of monoclonal antibodies also reacted with the two large fragments, especially with fragment 301-385, and some bind also to shorter peptides, such as fragment 173-196, which were not reactive to patients' IgE. Most of the monoclonal antibodies and patients' IgE bind to the fragments 41-172 and 301-385 in 2D-PAGE blots suggesting that these fragments are involved in an immunogenic structure.

Amino Acid Sequence↗
PubMed1994

Immunogenic combinations of HIV-1 B- and heterologous T-cell epitopes.

Peptides were synthesized which combined HIV-1 B-epitopes from gp41, p34pol and heterologous T-cell epitopes from hepatitis B virus (HBV) core or tetanus toxoid. Mixtures of these composite peptides and peptides representing single HIV-1 B-epitopes were used to immunize rabbits in an adjuvant-free immunization regimen. Fusion to T-cell epitopes made the HIV-1 B-epitopes immunogenic and high titers of anti-HIV-1 antibodies were reached. Efficient antibody response against an immunorecessive HIV-1 B-epitope from p34 pol introduced as a B+T-composite also developed in rabbits pre-immunized by composites of the same T-cell epitopes but with a B-epitope from gp41. Fusion changed the fine antigenicity of the epitopes, but at least part of the antibodies against gp41-containing B+T composites recognized whole viral gp160. Composite peptides stimulated T-cells in the majority of the immunized animals.

Amino Acid Sequence↗
PubMed1996

Longitudinal study of an epitope-biased serum haemagglutination-inhibition antibody response in rabbits immunized with type A influenza virions.

This paper describes a longitudinal study of the antibody specificities generated to the haemagglutinin (HA), the major envelope glycoprotein of type A influenza virus particles, during primary and secondary antibody responses in rabbits. Two New Zealand White rabbits (no. 191, no. 192) and an English Half-Lop rabbit (no. 193) were immunized intravenously with beta-propiolactone-inactivated virus at 0, 28 and 56 days. Haemagglutination-inhibition (HI) antibody specificities were measured using neutralizing antibody double escape mutants selected with monoclonal antibodies (mabs) specific for an epitope in antigenic site A, site B and site D. In this regard the epitope reactivity to these mabs was represented as A+B-D-, A-B+D- and A-B-D+, where "+" and "-" represent the non-mutated and mutated epitopes respectively. The HI response was well developed at 7 days after primary immunization and at this time the response was equally divided between all three epitopes. Two rabbits (no. 191 and no. 193), showed a bias to the site B epitope from 14 days onwards, such that about half of the total HI activity was to this epitope and the other half was made up of HI antibody to the other two epitopes in approximately equal proportions. In the other New Zealand White rabbit (no. 192) a non-biased response extended throughout the primary response and for one week after the second immunization. Apart from this, a bias to a single epitope was clearly evident in all rabbits after the second immunization, and this constituted up to 70% of the total HI antibody response. The antibody response did not broaden and remained essentially unchanged even after a third immunizing injection. The observed bias to the site B epitope during the secondary response of HA-specific antibody is in accord with a previous cross-sectional study of nine other rabbits.

Animals↗
PubMed1990

Lipooligosaccharide epitopes shared among gram-negative non-enteric mucosal pathogens.

The non-enteric Gram-negative human pathogens, B. catarrhalis, H. ducreyi, H. influenzae, N. gonorrhoeae and N. meningitidis, do not have repeating O-antigens as part of their principle surface glycolipid, the lipooligosaccharide (LOS). Because they have similar LOS structures, we studied the conservation of LOS oligosaccharide epitopes among these organisms. Twenty-one monoclonal antibodies (mAbs) generated by immunizing mice with H. influenzae, N. gonorrhoeae and N. meningitidis were studied for cross reactivity. Five mAbs generated against non-typable H. influenzae were the only strain-specific antibodies. Ten mAbs reacted to LOS epitope(s) common to a genera or species, and six mAbs bound to epitope(s) on the LOS of strains from different genera. Some cross reactive mAbs bound to LOS bands of similar molecular weights, while others bound to bands of varying molecular weights. mAb 3F11, whose epitope mimics a human blood-group antigen, bound to a 4.8 kDa LOS band in N. gonorrhoeae and H. ducreyi, two pathogens that infect genital epithelium. mAb 3D9, whose epitope consists of 2-keto-3-deoxyoctulosonic acid (KDO), reacted with different LOS bands in N. gonorrhoeae, H. influenzae and some R mutants of S. minnesota. A 14 kb restriction fragment containing lipooligosaccharide synthesis genes responsible for the assembly of the 3D9 epitope in H. influenzae hybridized to all H. influenzae strains tested but did not hybridize to gonococcal and S. minnesota strains that expressed this epitope. These studies demonstrate that conserved LOS epitope(s) exist among different species and genera of non-enteric human pathogens and that different genetic mechanisms may have evolved in these pathogens to assemble some of these conserved epitopes.

Antibodies, Monoclonal↗
PubMed1996

Engineering of doubly antigenized immunoglobulins expressing T and B viral epitopes.

BACKGROUND: Concomitant with the advent of molecular biology techniques and the ability of immunoglobulins (Ig) to recognize proteins, carbohydrates, lipopeptides and nucleic acids, vaccinologists have taken advantage to develop a variety of prophylactic and therapeutic vaccine prototypes. Presentation of epitopes to the immune system by Ig molecules as a carrier platform offers several advantages: (i) long exposure of the antigen to antigen processing cells (APCs) by virtue of their long half life, (ii) lack of the immune response to self Ig, focusing the immune response to protective epitopes rather than irrelevant epitopes, (iii) it takes advantage of the properties of Fc fragment of various isotypes like crossing the placenta (IgG) or homing in epithelia (IgA), and (iv) targeting various antigens by virtue of their binding specificity. OBJECTIVES: This study was aimed to genetically and enzymatically engineer immunoglobulins (Igs) able to express and to deliver concomitantly immunodominant T and B viral epitopes. STUDY DESIGN: Using a genetic engineering approach we replaced the complementary determining region 3 (CDR3) and complementary determining region 2 (CDR2) of an anti-arsonate 91A3 mAb with the immunodominant HA110-120 T cell epitope and HA150-159 B cell epitope of hemagglutinin (HA) of influenza A/PR8 virus, respectively. The second doubly antigenized Ig (Ig-HA-Gal-B) was constructed on an Ig in which CDR3 was replaced with HA110-120 T cell epitope while the HA150-159 B cell epitope was enzymatically assembled through an imidic bond on the galactose (Gal) residues of the carbohydrate moiety. RESULTS AND CONCLUSIONS: Both genetically and genetically/enzymatically doubly antigenized Ig constructs (dAIg) were properly folded and they were able to activate peptide-specific T cells and to elicit anti-viral antibody response in mice. This demonstrates that the CDR loops as well as carbohydrate moieties of immunoglobulins represent permissive sites for grafting foreign epitopes without altering the structural integrity of immunoglobulins and the immunogenicity of the viral peptides.

Animals↗
PubMed2004

Microarray immunoassay: association of clinical history, in vitro IgE function, and heterogeneity of allergenic peanut epitopes.

BACKGROUND: IgE epitope mapping of food allergens is a prerequisite for engineering hypoallergenic immunotherapeutic agents and might reveal basic information regarding a patient's immune response. Mapping of large numbers of epitopes by using individual patient sera has been impractical with current techniques. OBJECTIVE: We sought to develop a peptide microarray-based immunoassay to map peanut epitopes by using microliter quantities of serum. METHODS: A set of 213 overlapping 20-residue peptides was synthesized corresponding to the primary sequences of Ara h 1, Ara h 2, and Ara h 3. These were arrayed in triplicate along with the corresponding recombinant proteins onto glass slides and used for immunolabeling. RESULTS: Seventy-seven patient and 15 control sera were analyzed. The majority of patients (97%) had specific IgE to at least one of the recombinant allergens, and 87% had detectable IgE to sequential epitopes. Microarray mapping correlated well with previous studies. However, the analysis of individual patients revealed remarkable heterogeneity in the number and patterns of epitope recognition. High epitope diversity was found in patients with a history of more severe allergic reactions. Also, sensitization of effector cells with more diverse IgE antibodies conferred greater reactivity to specific allergen. CONCLUSIONS: The protein microarray immunoassay confirmed that Ara h 1, Ara h 2, and Ara h 3 are major peanut allergens and allows for parallel epitope analysis. This has led to the discovery of an additional important epitope of Ara h 1 and the recognition of a high degree of patient heterogeneity. This qualitative difference in epitope diversity might provide prognostic information about the patient.

2S Albumins, Plant↗
PubMed2004

Epitope down-modulation as a mechanism for the coexistence of competing T-cells.

Efficient immune responses against pathogens are frequently characterized by the simultaneous targeting of multiple epitopes. However, it remains unclear how the targeting of multiple epitopes is maintained in the face of competition for antigenic stimulation. Here, we investigate this question by using mathematical models of the population dynamics of a viral pathogen, antigen presentation sites and T-cells. We first show that direct competition for access to antigen presenting sites and indirect competition through killing of the pathogen select for dominance of the T-cell response with the highest affinity for its epitope. We then incorporate in our model that epitopes can become down-modulated following interaction with epitope specific T-cells. We demonstrate that epitope down-modulation leads to differentiation of epitope presentation on antigen presenting sites. This differentiation promotes the coexistence of multiple epitope specific responses. Hence, we propose that the functional relevance of epitope down-modulation may be to enable the persistence of a broad immune response despite competition for antigenic stimulation.

Animals↗
PubMed2005

Identification of dominant epitopes of synthetic immunocontraceptive vaccines that induce antibodies in dogs.

The specificities of immunoglobulin G antibodies obtained from the sera of dogs inoculated with totally synthetic immunocontraceptive vaccine candidates based on luteinising hormone releasing hormone (LHRH: amino acid sequence HWSYGLRPG) were examined using peptides expressed in a phage display library. The three vaccine candidates each contained a different T helper-cell epitope chemically linked with the same LHRH amino acid sequence HWSYGLRPG and all of them elicited high antibody titres against the hormone. Delineation of epitopes recognised by sera from vaccinated dogs using a phage display library indicated that two of the three vaccine candidates induced antibody directed to the consensus sequence xHWSxxLxxx whereas the third vaccine candidate induced antibody against the consensus sequence xxxxxxxRPx. Two of the three vaccine candidates elicited antibodies against B cell epitopes present within the helper T-cell epitope component of the vaccine whereas the third vaccine did not. The occurrence of anti-T helper cell epitope antibodies appeared to have little or no effect on the generation of the anti-LHRH responses indicating that carrier-induced epitope suppression was not operating here. Our results also demonstrated that with animal sera of high quality, it is possible to delineate immunodominant epitopes recognised by polyclonal antibodies with high efficiency using phage display library. The approach has utility in the definition of immunodominant epitopes, which may "decoy" antibody responses away from other epitopes, which may be more useful in prophylaxis or therapy.

Animals↗
PubMed2005

Uneven distribution of MHC class II epitopes within the influenza virus.

The identification of T cell epitopes is crucial for the understanding of the host immune response during infection. While much is known about the MHC class I-restricted response following influenza virus infection of C57BL/6 mice, with over 16 CD8 epitopes identified to date, less is known about the MHC class II-restricted response. Currently, only a few I-A(b)-restricted T helper epitopes have been identified. Therefore, several important questions remain about how many class II epitopes exist in this system and whether these epitopes are evenly distributed within the most abundant viral proteins. In order to address these questions, we analyzed the repertoire of epitopes that drive the CD4+ approximately 20-30 epitopes drive the CD4 T cell response and that the majority of these peptides are derived from the NP and HA proteins. We were also able to demonstrate that vaccination with one of the newly identified epitopes, HA(211-225)/A(b), resulted in increased epitope-specific T cell numbers and a significant reduction in viral titers following influenza virus challenge.

Animals↗
PubMed2006

Adjuvanticity of stealth liposomes on the immunogenicity of synthetic gp41 epitope of HIV-1.

Present study aims to enhance the efficacy of liposomes as an adjuvant by steric protection and strengthen the path of vaccine research. PEG grafted liposomes carrying epitopes on their surface showed enhanced adjuvanticity than liposomes carrying epitopes for elicitation and prolongation of immune response to an antigenic epitope of gp41, a transmembrane protein of HIV-1. The multiples of epitope were incorporated onto the surface of liposomes by conjugating them with phosphatidylethanolamine that was used in the formulation of liposomes at an optimized ratio. Furthermore, the liposomes carrying epitopes on their surface were sterically protected by shielding with methoxy-poly(ethylene glycol), mass 20 kDa. Methoxy-poly(ethylene glycol) was activated to its electrophilic N-succinimide carbonate derivative, methoxy-poly(ethylene glycol)-N-succinimide carbonate, that formed a urethane linkage with the amino group of phosphatidylethanolamine. The epitope was covalently coupled to phosphatidylethanolamine through an amide bond between the -COOH group of the epitope and -NH2 group of phosphatidylethanolamine under the catalysis of 1-ethyl-3-(3-dimethylaminopropy-1)-carbodiimide. PEG grafted epitopes carrying liposomes showed about two times higher immune response and prolonged persistence of antibodies than that of liposomes carrying epitopes without PEG moieties.

AIDS Vaccines↗
PubMed1998

Immunohistochemical localization of different epitopes of advanced glycation end products in human atherosclerotic lesions.

To better understand the role of advanced glycation end products (AGEs) in atherogenesis, we developed specific antibodies against different immunological epitopes of AGE structures, including Nepsilon-(carboxymethyl)lysine-protein adduct (CML) and a structure(s) other than CML (nonCML), and demonstrated the immunohistochemical localization of CML- and nonCML-epitopes in atherosclerotic lesions of human aorta, which were obtained at autopsy from 20 nondiabetic patients (12 males and eight females; mean age, 60.8+/-16.7 years). Monoclonal anti-CML antibody (6D12) recognized not only AGE-modified proteins, but also CML-modified proteins. On the other hand, polyclonal anti-nonCML antibody reacted to AGE-modified proteins, but not to CML-modified proteins. Both antibodies were unreactive to the early-stage products of glycation, including fructose-modified butyloxycarbonyl-lysine and fructose-epsilon-aminocaproic acid. Atherosclerotic lesions included diffuse intimal thickening (DIT), fatty streaks (FS), atherosclerotic plaques (AP) and complicated lesions. An immunohistochemical analysis showed both CML- and nonCML-epitopes to be found along the collagen fibers in DIT in subjects more than 40 years old, but not in subjects less than 40 years old. CML-epitopes accumulated mainly in the cytoplasm of macrophage/foam cells, while nonCML-epitopes accumulated exclusively in the extracellular spaces in FS. APs showed the CML-epitope stored macrophage/foam cells, and the accumulation of both CML- and nonCML-epitopes in the lipid-rich fibrous area. An immunohistochemical analysis with a monoclonal antibody against oxidized low density lipoprotein (FOH1a/DLH3) showed the presence of this antigen within the cytoplasm of the macrophage/foam cells in atherosclerotic lesions, which were also positive for the CML-epitopes. These findings thus suggest that the heterogeneous localization of AGEs in atherosclerotic lesions depends on their different epitopes, and that a close link, therefore, exists between the peroxidation of LDL and the formation of AGEs in atherosclerotic lesions.

Adult↗
PubMed2003

Characterization of polysaccharide conformational epitopes by surface plasmon resonance.

SPR techniques can provide a wealth of insight into the nature of protein-carbohydrate interactions. Information not obtained readily by other methodologies can be gathered relatively quickly in a label-free manner with low sample consumption. This chapter focused on two applications in which SPR has been used to map conformational epitopes on bacterial polysaccharides recognized by protective antibodies. In one example, methods for demonstrating the conformational nature of the epitope recognized by an anti-GBS antibody were described. Dramatic epitopic stabilization at 2 repeating units with further significant stabilization between 7 and 20 repeating units was demonstrated. In a second example, SPR methods were employed in characterization of the epitope recognized by a protective antibody against the group B meningococcus. It was shown that the antibody bound a long epitope, in excess of eight monosaccharides and probably helical, on NPr-GBMP but did not bind to GBMP. The binding of the protective antibody to GBMP only when GBMP is cell associated, or with an attached lipid, indicated that the protective GBM epitope consists of more than GBMP. NPr-GBMP mimics a cell surface complex consisting of extended helical portions of the GBMP in association with a second molecule, possibly a phosphoglycerolipid. SPR experiments indicated that the protective nature of certain antibodies induced by the NPr-GBMP vaccine is attributable to their recognition of an abundant internal epitope on NPr-GBMP and cell-associated GBMP. A nonprotective antibody, specific for NPr-GBMP, recognized a terminal and consequently minor epitope on the polysaccharide. Low levels of this nonprotective antibody binding to cells and unpurified polysaccharide confirmed recognition of a minor epitope on the natural antigen as well. In contrast, a protective antibody exhibited a high level of binding to the cell-associated antigen.

Animals↗
PubMed2001

Contrasting Epstein-Barr virus-specific cytotoxic T cell responses to HLA A2-restricted epitopes in humans and HLA transgenic mice: implications for vaccine design.

This study investigates the hierarchy of cytotoxic T cell (CTL) responses to twelve HLA A2-restricted epitopes from the latent, lytic and structural proteins of Epstein-Barr virus (EBV) in acute infectious mononucleosis and in healthy seropositive donors and the relative immunogenecity of these epitopes in transgenic mice. Responses to the lytic epitope were uniformly strong in all healthy seropositive individuals and acute infectious mononucleosis donors while moderate or low responses were observed to the latent and structural epitopes, respectively in both groups studied. In contrast, when HLA A2/Kb transgenic mice were immunised with these peptide epitopes, CTL responses were observed to all epitopes with a maximal response to the epitopes within the structural proteins and low to moderate responses to the latent epitopes. This hierarchy of CTL responses in mice was also reflected in an MHC stabilisation analysis. These contrasting CTL responses in humans following natural infection compared to the immunogenicity of these epitopes and their ability to stabilise MHC may need to be considered when designing an EBV vaccine.

Acute Disease↗
PubMed2003

Neutralization epitopes of the HIV-1 primary isolate DH012.

To study HIV-1 primary isolate neutralization, we have used DH012 as a model to study the immunogenicity of several DH012 immunogens and determine the potential neutralization epitopes in the virus envelope glycoprotein. Previously, we identified that DH012 infected animals mount potent neutralizing activity against a conformational epitope (CEV) that involves multiple variable regions. In this study, we show that the conformational epitope can be reconstituted with one gp120 recombinant fragment containing sequences from the V1/V2 loop and the bridging sheet of gp120 and a V3 peptide. In contrast to DH012 infection, we previously demonstrated that animals immunized with DH012 gp120 induced potent neutralizing antibodies directed at the V3 domain of gp120. In this study, a second neutralizing activity against the V1/V2 region of gp120 was identified from the same guinea pig sera. In summary, several neutralization epitopes are identified on DH012, including the CEV, V1/V2, V3, 17b, IgG1b12, and 2G12 epitopes. Infectious DH012 virus carrying oligomeric envelope appears to raise primarily neutralizing antibodies that recognize a discontinuous conformationally dependent epitope whereas the monomeric gp120 induces antibodies that are primarily directed at epitopes in the V3 and V1/V2 domains. The DH012 neutralizing epitopes, such as V1/V2 and V3, are either cryptic or poorly immunogenic in chimpanzees. However, immunogens, such as gp120, could be designed to induce neutralizing activity against epitopes that are poorly immunogenic, such as V1/V2 of DH012, in the native envelope glycoproteins.

Amino Acid Sequence↗
PubMed2003

Ii-Key/MHC class II epitope hybrid peptide vaccines for HIV.

The Ii-Key/MHC class II epitope hybrid acts on MHC class II molecules to facilitate replacement of antigenic peptides with the epitope tethered to the Ii-Key motif. Hybrid peptides linking an immunoregulatory segment of the Ii protein (Ii-Key peptide) through a polymethylene bridge to MHC class II epitopes of HIV gp160 or gag are potent vaccines to elicit CD4(+) T cell responses. More potent responses to two MHC class II epitopes, HIV gp160(843-852) or HIV gag(279-292), occurred in mice immunized with Ii-Key hybrid peptides than with epitope-only peptides, as measured in IL-4 and IFN-gamma ELISPOT assays of splenic lymphocytes stimulated in vitro by epitope-only peptides. Both the number of responding cells and cytokine output per cell were increased. The Ii-Key/MHC class II epitope hybrid acts on MHC class II molecules to facilitate replacement of antigenic peptides with the epitope tethered to the Ii-Key motif. Such antigenic peptide constructs create opportunities to enhance greatly Th1 or Th2 responses to MHC class II epitopes for therapeutic purposes.

AIDS Vaccines↗
PubMed1999

Epitope insertion into variable loops of HIV-1 gp120 as a potential means to improve immunogenicity of viral envelope protein.

We report on the properties of a set of HIV-1 IIIB Env mutants carrying a linear gp41 epitope insertion (LLELDKWASL) in the V1, V2, V3 or V4 variable loop. Insertion of the epitope, which is defined by the HIV-1 neutralizing MAb 2F5, was well tolerated in the V1, V2 and V4 loops, as these mutants were properly expressed, retained reactivity to conformation-dependent monoclonal antibodies and exhibited patterns similar to the parental Env molecule. However, insertion of this epitope in the V3 loop was associated with drastically reduced protein expression. Relative to parental Env molecule, the V1, V2 and V4 insertion mutants demonstrated significantly increased binding to mAb 2F5 in vitro. To evaluate immunogenicity, mice and guinea pigs were immunized with plasmid expression vectors for the mutant proteins. For both mice and guinea pigs, all four mutants elicited anti-gp120 antibody responses. In mice the V1 and V3 insertion mutants, but neither the V2 or V4 insertion mutant nor the parental env, elicited significant titers against the epitope peptide, whereas in guinea pigs, V2 insertion mutant was most effective in eliciting anti-2F5 peptide antibody responses. While original V2 2F5 insertion mutant failed to elicit anti-2F5 peptide responses in mice, studies with 14 additional V2 insertion mutants revealed several insertion sites at which the epitope was able to induce epitope-specific antibody responses. This indicates that the precise position at which the epitope insertion takes place dictates the ability of the mutant to induce the epitope-specific antibody responses. When tested for virus neutralization activity, the guinea pig sera that contain high titers of anti-2F5 peptide antibody failed to enhance the virus neutralizing activity, suggesting that the configuration of 2F5 epitope plays a critical role in inducing neutralizing antibody responses. The results from this study may have potential implications with respect to modification of the HIV-1 Env molecule for the purpose of improving HIV-1 Env immunogenicity.

AIDS Vaccines↗
PubMed1998

Serological cross-reactivity between Brucella abortus and yersinia enterocolitica 0:9: IV. Evaluation of the M- and C-epitope antibody response for the specific detection of B. abortus infections.

Smooth lipopolysaccharides (SLPS) from Brucella abortus contain A-epitopes against which the majority of serum antibodies are directed during infections. SLPS from Yersinia enterocolitica 0:9 possesses identical epitopes, which are the cause for serological cross-reactivity. All Brucella spp. possess M- and C-epitopes which are not present in Y. enterocolitica 0:9. In order to examine the usefulness of these M- and C-epitopes for discriminatory serological testing, a panel of sera were used in this study, comprising sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected cattle of comparable strength in the serological brucellosis tests to the sera from Y. enterocolitica 0:9-infected heifers, sera from B. abortus-infected bovines with strong serological reactions and sera from animals free from B. abortus or Y. enterocolitica infections. These sera were tested in blocking ELISAs with seven M- and one C-epitope-specific monoclonal antibodies in combination with SLPS from B. melitensis M16 high in M-epitopes as antigen. Strong B. abortus sera inhibited most strongly, while negative sera showed no or little inhibition. Sera with weak or intermediate titres blocked to a lower extent. Unexpectedly, the sera from Y. enterocolitica 0:9-infected heifers showed inhibition behaviour virtually identical to the comparable sera from B. abortus infected animals. Absorbing out of the A-epitope specific serum antibodies with either Y. enterocolitica 0:9 SLPS or with Y. enterocolitica 0:9 bacteria, indicated the presence of M- or C-epitope-specific serum antibodies in some sera from B. abortus-infected cattle but not in the sera from Y. enterocolitica 0:9-infected animals. These results demonstrate that the M- or C-epitope-specific antibody response in sera from B. abortus infected cattle is only of limited value for the serological discrimination between B. abortus and Y. enterocolitica 0:9 infections.

Animals↗
PubMed2001

Characterization and identification of allergen epitopes: recombinant peptide libraries and synthetic, overlapping peptides.

For the understanding of the relationship between protein structure and allergenicity, it is important to identify allergenic epitopes. Two methods to characterize primarily linear epitopes are compared using the major allergen from brown shrimp (Penaeus aztecus), Pen a 1, as an example. A recombinant peptide library was constructed and synthetic, overlapping peptides, spanning the entire Pen a 1 molecule, were synthesized and tested for specific IgE reactivity. Both methods identified IgE-binding of Pen a 1, however, the SPOTs procedure resulted in the identification of more epitopes of the major shrimp allergen Pen a 1 than the usage of the recombinant peptide library. For detection of specific IgE antibodies, the usage of 125I-labeled detection antibody seems to be superior over enzyme-labeled anti IgE antibodies. The regeneration of SPOTs membranes is possible, but it is prudent to test regenerated membranes for residual activity. If a given food allergen contains significant linear epitopes, which seems to be true for stable major allergens such as those of peanut and shrimp the SPOTs system may be more advantageous than the use of recombinant peptides libraries. However, if allergens are studied that contain more conformational epitopes, recombinant peptide libraries may help to identify the relevant epitopes. It has to be emphasized that no system for epitope identification will detect all epitopes and that the relevance of identified epitopes has to be confirmed with other methods such as inhibition studies, crystallographic analysis or the immunological evaluation of modified whole allergens.

Allergens↗