Reduction of human melanoma tumor growth in severe combined immunodeficient mice by passive transfer of antibodies induced by a high molecular weight melanoma-associated antigen mimotope vaccine.

PURPOSE: The high molecular weight melanoma-associated antigen (HMW-MAA) is an attractive target for immunotherapy of malignant melanoma. We have recently generated a vaccine based on the HMW-MAA mimotope 225D9.2+ that was able to induce anti-HMW-MAA antibodies with antitumor activity in vitro. Here, we investigated the antitumor activity of these antibodies in a human melanoma xenotransplant severe combined immunodeficient (SCID) mouse model. EXPERIMENTAL DESIGN: Tumors were established by injecting the human melanoma 518A2 cells into C.B.17 SCID/SCID mice. In tumor prevention experiments, 200 microg purified total IgG antibodies were injected intravenously the same day or on day 5 in therapeutic experiments. Antibody administration was repeated every fourth day and tumor volumes were measured. Antibody specificity and tumor infiltration by macrophages were investigated by immunohistochemistry. RESULTS: Within 35 days after cell inoculation, antibody treatment reduced tumor growth up to 40% in the therapeutic and up to 62% in the tumor prevention experiments compared with the control mice. In tumors of all groups, a similar distribution of the HMW-MAA and no differences in infiltration of macrophages were detected by immunohistochemistry. CONCLUSIONS: Here, we showed that antibodies induced by the 225D9.2+ mimotope effectively inhibited melanoma tumor growth. Additional mechanisms besides antibody-dependent cell cytotoxicity like disruption of interactions of melanoma cells mediated by extracellular matrix components seem to be involved in tumor growth inhibition. Based on our findings, we suggest that active immunization with this mimotope might be a promising strategy for treatment of melanoma.

Naturally occurring hypoallergenic Bet v 1 isoforms fail to induce IgE responses in individuals with birch pollen allergy.

BACKGROUND: Engineered hypoallergens are currently being investigated for specific immunotherapy of allergic diseases in preclinical and clinical studies. Naturally occurring hypoallergens have by and large not been considered as a source of vaccine candidates. OBJECTIVE: Evaluation of the antibody response in atopic individuals induced by birch pollen containing isoforms of the major birch pollen allergen Bet v 1. METHODS: Isoform-specific antibody isotype responses for Bet v 1.0101, Bet v 1.0401, and Bet v 1.1001 were determined for 35 sera of individuals with birch pollen allergy. Isoform structures were compared and related to IgE-binding inhibitory capacities and induction of mediator release in human Fcvarepsilon receptor transformed rat basophilic leukemia cells. RESULTS: Bet v 1.0101 induced a predominant IgE response, whereas the significant highest levels of IgG(4) antibodies were directed against Bet v 1.0401. Bet v 1.1001 induced only a minimal antibody response. Structural comparisons revealed that most of the amino acid differences between the isoforms were located on the protein surfaces. IgE induced by Bet v 1.0101 only partly cross-reacted with the 2 other isoforms and bound to them with notably lower affinity. Bet v 1.0401 and Bet v 1.1001 also were poor inducers of mediator release. CONCLUSION: Bet v 1 isoforms possess highly variant immunogenic and allergenic properties. Bet v 1.0101 acts as the sensitizing agent, whereas Bet v 1.0401 and Bet v 1.1001 can induce only a minimal IgE response.

Mimotopes identify conformational B-cell epitopes on the two major house dust mite allergens Der p 1 and Der p 2.

House dust mite allergy occurs in 10-20% of the population. Improvement of the present immunotherapy requires detailed knowledge about the structure of the allergens. Mimotopes selected from phage peptide libraries imitate the conformational epitopes of a natural allergen. The aim of our study was to generate epitope mimics for the two major allergens of house dust mite. When the monoclonal anti-Der p 1 and anti-Der p 2 antibodies were used for biopannings, mimotopes were selected which bound also specific IgE from human allergic patients' sera. The conformational matching of these mimotopes on the 3D structure of the natural allergens determined discontinuous epitopes in both cases, representing conformational B-cell epitopes relevant for binding of human IgE. Therefore, these mimotopes are potential candidates for the directed induction of blocking antibodies and epitope-specific immunotherapy of mite allergy.

Active induction of tumor-specific IgE antibodies by oral mimotope vaccination.

A role of IgE antibodies in cancer surveillance has been implicated for a long time. Studies dealing with IgE antibodies directly targeted to tumor antigens have shown marked anticancer effects mediated by this antibody class. Thus, the basic function of IgE antibodies may be to control tumor growth. Thus far, cancer-specific IgE has only been applied passively. Consequently, the aim of this study was to establish an active vaccination protocol to induce tumor antigen-specific IgE antibodies, and to evaluate functional properties. We previously generated epitope mimics, so-called mimotopes, for the epitope recognized by the anti-HER-2 antibody trastuzumab. Upon i.p. immunizations, IgG antibodies with trastuzumab-like properties could be elicited. In the present study, we immunized BALB/c mice via the oral route with these trastuzumab mimotopes, under simultaneous neutralization and suppression of gastric acid. As shown in preceding experiments, this feeding regimen effectively induces Th2 immune responses. Oral immunizations with trastuzumab mimotopes under hypoacidic conditions indeed resulted in the formation of IgE antibodies towards the HER-2 antigen. Moreover, anti-HER-2 IgE-sensitized effector cells mediated SK-BR-3 target cell lysis in an antibody-dependent cytotoxicity assay. We conclude that directed and epitope-specific induction of IgE against tumor antigens is feasible with an oral mimotope vaccination regimen, and that these antibodies mediate anticancer effects.

Characterization of intrinsic and extrinsic risk factors for celery allergy in immunosenescence.

Recent studies indicated an underestimation of allergies in elderly. In our experimental food allergy model of protein feeding under acid-suppression we aimed to assess whether food allergy can be induced in immunosenescent mice. Furthermore, the impact of gastric digestion on celery allergenicity was evaluated in aged patients. Measurements of serum zinc and iron levels in senescent and adult BALB/c mice for definition of the nutritional status indicated a possible alteration of the immune response in the aged animals due to reduced zinc and iron levels. Feedings of mice with digestion-sensitive celery proteins under physiological gastric conditions induced IgG1 and IgG2a in the aged and preferentially IgG1 in the adult animals. In contrast, incomplete digestion due to acid-suppression rendered celery-specific IgE, positive skin tests and elevated IL-5 levels in both age groups. Also in aged celery allergic patients (mean age 72 years) properly digested celery showed decreased capacity to bind and crosslink IgE as evaluated by skin tests and IgE immunoblot. Thus, in the geriatric murine model, celery allergy was induced only if gastric digestion was hindered. Accordingly, gastric proteolysis decreased in vitro and in vivo IgE-reactivity against celery proteins in aged allergic patients.

Exercise with latex sport bands represents a risk for latex allergic patients.

Based on two clinical observations of adverse reactions during exercise with latex sport bands, we aimed to assess the possible risk for allergic patients posed by this equipment by investigating allergen content and IgE binding potential. Protein extracts of three different latex sport bands were characterized with sera of latex allergic patients. The IgE recognition profile of the allergic patients was identified by component resolved diagnosis and the allergen composition of the extracts was characterized by inhibition assays with the recombinant latex allergens Hev b 1, 3, 5, 6.02, and 8. The sera showed pronounced IgE binding to all three blotted extracts, however with diverse patterns. Inhibition assays revealed the presence of Hev b 1, 3, 5, and 8 in latex sport band extracts. The clinical relevance of contained allergens was demonstrated by strong skin reactions when testing with latex sport bands. From our results we conclude that latex sport bands contain clinically relevant allergens and may cause latex allergic individuals to experience allergic symptoms, potentially amplified by exercise-induced mechanisms. Even though latex is labeled on products, it is important that patients as well as athletic trainers and physical therapists recognize the risk of adverse reactions with these bands.

Anti-ulcer treatment during pregnancy induces food allergy in mouse mothers and a Th2-bias in their offspring.

The treatment of dyspeptic disorders with anti-acids leads to an increased risk of sensitization against food allergens. As these drugs are taken by 30-50% of pregnant women due to reflux and heartburn, we aimed here to investigate the impact of maternal therapy with anti-acids on the immune response in the offspring in a murine model. Codfish extract as model allergen was fed with or without sucralfate, an anti-acid drug, to pregnant BALB/c mice during pregnancy and lactation. These mothers developed a codfish-specific allergic response shown as high IgG1 and IgE antibody levels and positive skin tests. In the next step we analyzed whether this maternal sensitization impacts a subsequent sensitization in the offspring. Indeed, in stimulated splenocytes of these offspring we found a relative Th2-dominance, because the Th1- and T-regulatory cytokines were significantly suppressed. Our data provide evidence that the anti-acid drug sucralfate supports sensitization against food in pregnant mice and favors a Th2-milieu in their offspring. From these results we propose that anti-acid treatment during pregnancy could be responsible for the increasing number of sensitizations against food allergens in young infants.

Immunization with mimotopes prevents growth of carcinoembryonic antigen positive tumors in BALB/c mice.

PURPOSE: The carcinoembryonic antigen (CEA) is a glycoprotein that is overexpressed in nearly 50% of all human and veterinarian tumors. At present, anti-CEA antibodies are being tested in clinical studies as passive immunotherapeutics. This study aims to establish an active immunotherapy for the poorly immunogenic CEA glycoprotein by generating antigen surrogates. EXPERIMENTAL DESIGN: We used the monoclonal anti-CEA antibody Col-1 and the biopanning method to generate peptide mimics (mimotopes) of the Col-1 epitope. The peptide showing the highest specificity and mimicry was synthesized as an octameric multiple antigenic mimotope (MAM). Subsequently, immunogenicity of the selected mimotope was examined in BALB/c mice. We assessed antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity mediated by the induced antibodies on CEA-expressing HT29 tumor cells. Furthermore, after immunization, the BALB/c mice were transplanted s.c. with Meth-A/CEA tumor cells. RESULTS: When BALB/c mice were immunized with this MAM, they generated a specific humoral immune response against CEA. The mimotope-induced polyclonal and poly-isotypic antibodies induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro. Furthermore, when MAM-immunized mice were transplanted s.c. with Meth-A/CEA cells expressing human CEA, a suppressed tumor growth was observed. CONCLUSION: From our results, we can conclude that the Col-1 epitope of the glycoprotein CEA can be translated into an immunogenic peptide mimic. The mimotope-induced antibodies recognize CEA and do effectively inhibit growth of CEA-positive tumors. Based on these finding, we suggest that the generated mimotopes are candidates for active immunotherapy of CEA-expressing tumors.

Internal images: human anti-idiotypic Fab antibodies mimic the IgE epitopes of grass pollen allergen Phl p 5a.

BACKGROUND: The role of anti-idiotypic antibodies in allergic disease is still poorly understood. According to Jerne, anti-idiotypic antibodies to IgE should represent internal images of an allergen. Our aim was to ultimately prove whether this hypothesis holds true in allergy. Here, we describe the selection of anti-idiotypic antibodies against Phl p 5a-specific IgE directly from the B-cell repertoire of a grass pollen allergic individual. METHODS: Taking Phleum pratense grass pollen allergen Phl p 5 as a model, we selected anti-idiotypic antibodies against allergen-specific IgE directly from the B-cell repertoire of an allergic individual. We screened a combinatorial phage display library of human monovalent antibody heavy and light chain fragments (Fabs) with anti-Phl p 5a-IgE to identify and characterize Fabs with anti-idiotypic specificity. RESULTS: Five different Fab clones with anti-idiotypic specificity for anti-Phl p 5a-IgE were identified. Their hypervariable regions revealed partial sequence homology with solvent accessible antigenic sites of Phl p 5a, which have been identified by our previous mimotope approach. Phagemid DNA derived from the phage clones was used to produce two soluble recombinant anti-idiotypic Fab clones in E. coli. As a proof of molecular mimicry, both Fabs induced anti-Phl p 5a-specific antibodies in immunized BALB/c mice. Molecular modeling of the heavy and light chain hypervariable loops of the anti-idiotypic Fabs illustrated structural similarity with dominant IgE epitopes of Phl p 5a. CONCLUSION: In this straightforward phage technology approach, antibodies with anti-idiotypic specificities could be isolated from a human allergic's repertoire. As predicted by the immune network hypothesis, their hypervariable domains mimic IgE epitopes like internal images and, more importantly, induce allergen-specific immune responses in the absence of the allergen.

Mimotopes identify conformational epitopes on parvalbumin, the major fish allergen.

Parvalbumin, the major fish allergen, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. A detailed knowledge of allergenic structures is crucial for developing a vaccine inducing blocking antibodies specifically directed towards the IgE binding epitopes. In the present study we aimed to use the phage display technique to generate mimotopes, which mimic epitopes on parvalbumin. Parvalbumin-specific IgE was purified from sera of fish-allergic patients and used for screening of a constrained decamer phage library. After four rounds of biopanning using parvalbumin-specific IgE, five phage clones were selected which were specifically recognized by parvalbumin-specific IgE as well as IgG. DNA sequencing and peptide alignment revealed a high degree of sequence similarities between the mimotopes. Interestingly, on the surface of natural parvalbumin three regions could be defined by computational mimotope matching. In accordance, previously defined allergenic peptides of cod parvalbumin highlighted areas in close proximity or overlapping with the mimotope matching sites. From the presented data we conclude that our approach identified conformational epitopes of parvalbumin relevant for IgE and IgG binding. We suggest that these mimotopes are suitable candidates for an epitope-specific immunotherapy of fish-allergic patients.

Anti-ulcer drugs promote IgE formation toward dietary antigens in adult patients.

Recently, we have demonstrated that anti-ulcer drugs, such as H2-receptor blockers and proton pump inhibitors, promote the development of immediate type food allergy toward digestion-labile proteins in mice. The aim of this study was to examine the allergological relevance of these findings in humans. In an observational cohort study, we screened 152 adult patients from a gastroenterological outpatient clinic with negative case histories for atopy or allergy, who were medicated with H2-receptor blockers or proton pump inhibitors for 3 months. IgE reactivities to food allergens before and after 3 months of anti-acid treatment were compared serologically. Ten percent of the patients showed a boost of preexisting IgE antibodies and 15% de novo IgE formation toward numerous digestion-labile dietary compounds, like milk, potato, celery, carrots, apple, orange, wheat, and rye flour. Thus, the relative risk to develop food-specific IgE after anti-acid therapy was 10.5 (95% confidence interval: 1.44-76.48). The long-term effect was evaluated 5 months after therapy. Food-specific IgE could still be measured in 6% of the patients, as well as significantly elevated serum concentrations of ST2, a Th2-specific marker. An unspecific boost during the pollen season could be excluded, as 50 untreated control patients revealed no changes in their IgE pattern. In line with our previous animal experiments, our data strongly suggest that anti-ulcer treatment primes the development of IgE toward dietary compounds in long-term acid-suppressed patients.

Matching of trastuzumab (Herceptin) epitope mimics onto the surface of Her-2/neu--a new method of epitope definition.

As seen with the proto-oncogene Her-2/neu, antibodies targeting different parts of a receptor can have opposing effects. Depending on epitope specificity, in this case, tumor growth can be inhibited--but also enhanced. Therefore, the definition of molecular binding sites is of increasing importance in modern medicine. We here introduce a novel approach for binding site localization, utilizing information obtained by the phage display technique. This is a high throughput screening method for identification of peptide mimics, so called mimotopes, of any binding structure of interest. All target molecules whose structure is available in the RCSB Protein Data Bank can be scanned for mimotope matches on their surface. In this study, we present the matching results of five mimotopes defined for the epitope recognized by trastuzumab (Herceptin), a humanized monoclonal antibody inhibiting tumor growth, on Her-2/neu. The localization thus obtained corresponds to the known trastuzumab epitope. We therefore suggest the algorithm as a novel way of binding site definition, circumventing co-crystallization experiments.

Isolation and structural analysis of peptide mimotopes for the disialoganglioside GD2, a neuroblastoma tumor antigen.

The disialoganglioside GalAcbeta1-4(NeuAcalpha2-8NeuAcalpha2-3)Galbeta1-4Glcbeta1-1Cer (GD2) is expressed on various tumors, including neuroblastoma, and was defined as a relevant tumor antigen. The monoclonal anti-GD2 antibody 14.18 is widely used for diagnostic purposes in neuroblastoma, and in its mouse/human chimeric form (ch14.18) now enters passive immunotherapeutic regimens in phase II clinical trials. This study aimed to generate structural mimics of the 14.18 epitope of GD2. Therefore, we used the ch14.18 antibody for selecting immunoreactive GD2 peptide mimotopes from a decamer phage display library. In all, 13 GD2 peptide mimics could be determined by biopanning and their specificity was demonstrated by exclusive recognition by the ch14.18 antibody. Furthermore, their nature of being GD2 mimics and their degree of mimicry was confirmed by competition with the natural antigen. When performing a comparative visualization of the GD2 epitope and selected mimotopes using a three-dimensional computer modeling system (BALLView), we demonstrated fitting of the GD2 molecule and the mimotopes in the antigen-binding pouch of a GD2 specific antibody. Moreover, the computer modeling argued for optimal affinity of the GD2 mimotopes. We thus provide evidence that the generation of GD2 peptide mimotopes is successful when using the neuroblastoma antibody ch14.18 for selection, and that this approach might offer a tool to develop a vaccination strategy against this malignant pediatric tumor.

Epitope-specific antibody response to Mel-CAM induced by mimotope immunization.

Peptide mimotopes of tumor antigen epitopes have been proposed as components of tumor vaccines. In this study, we determined the immunogenicity of melcam mim1 and melcam mim2, peptide mimics of an epitope of the melanoma cell-adhesion molecule (Mel-CAM). BALB/c mice were vaccinated either with mimotopes or mimotopes coupled to tetanus toxoid (TT). The antibody responses of mice to melcam mim1, melcam mim2, and recombinant Mel-CAM were analyzed by an ELISA and immunoblot analyses. TT-coupled mimotopes led to high titers of IgG mainly of the IgG2a subclass to melcam mim1 and melcam mim2. Immunization with each of the mimotope formulations induced antibodies that cross-reacted with recombinant Mel-CAM. Uncoupled mimotopes induced lymphocyte proliferation and cytokine production in spleen cell cultures indicating that both peptide mimotopes also contained T cell epitopes. TT-coupled mimotopes induced T helper (Th)1 (interleukin (IL)-2, interferon-gamma) and Th2 (IL-4, IL-5) cytokines, whereas uncoupled mimotopes induced a Th1-biased T cell response. Our results suggest that mimotopes potentially represent a novel vaccine approach to induce a tumor antigen-specific humoral and cellular response.

Antiulcer drugs promote oral sensitization and hypersensitivity to hazelnut allergens in BALB/c mice and humans.

BACKGROUND: Hazelnut allergy can be a consequence of sensitization to cross-reactive pollen, especially from the Fagales family. However, severe allergic reactions after ingestion of hazelnuts without associated pollen allergy have been reported. In these cases, oral sensitization by hazelnut ingestion is plausible. OBJECTIVE: We have reported that antiulcer drugs promote oral sensitization to digestion-labile food allergens. Because hazelnut proteins were sensitive to gastric digestion in our in vitro assay, we aimed to analyze the effect of antiulcer treatment on oral sensitization to hazelnut proteins. DESIGN: BALB/c mice were fed hazelnut extract with or without antiulcer drugs. In parallel, gastroenterologic patients (n = 153) were screened during antiulcer treatment for specific immunoglobulin (Ig) E to hazelnut and inhalative allergens in vitro and in vivo. RESULTS: Mice fed hazelnut extract in combination with antiulcer drugs formed anaphylactogenic IgG1 toward hazelnut and developed type I skin reactivity to hazelnut extract. In the human study population, 5 of 153 (3.3%) patients developed hazelnut-specific IgE, 4 of 5 developed specific skin reactivity, 3 of 5 had a positive result to oral provocation, and 2 of 5 manifested a food allergy to hazelnut after a 3-mo course of antiulcer treatment. Immunoblot testing with recombinant allergens showed that hazelnut, but not Fagales pollen, was the genuine elicitor in mice and humans. CONCLUSION: Our experimental and epidemiologic data suggest that the intake of antiulcer drugs may lead to the induction of immediate-type food hypersensitivity toward hazelnut.

Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier.

Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9A using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development.

Plant-based heterologous expression of Mal d 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein.

Mal d 2 is a thaumatin-like protein and important allergen of apple fruits that is associated with IgE-mediated symptoms in apple allergic individuals. We obtained a full-length cDNA clone of Mal d 2 from RNA isolated from ripe apple (Malus domestica cv. Golden Delicious). The cDNA's open reading frame encodes a protein of 246 amino acid residues including a signal peptide of 24 residues and two putative glycosylation sites. The deduced amino acid sequence of the mature Mal d 2 protein results in a predicted molecular mass of 23,210.9Da and a calculated pI of 4.55. Sequence comparisons and molecular modeling place Mal d 2 among those pathogenesis-related thaumatin-like proteins that contain a conserved acidic cleft. In order to ensure the correct formation of the protein's eight conserved disulfide bridges we expressed Mal d 2 in Nicotiana benthamiana plants by the use of a tobacco mosaic viral vector. Transfected N.benthamiana plants accumulated Mal d 2 to levels of at least 2% of total soluble protein. MALDI-TOF mass spectrometric analyses of the recombinant Mal d 2 and its proteolytic fragments showed that the apple-specific leader peptide was correctly cleaved off by the host plant and that the mature recombinant protein was intact and not glycosylated. Purified recombinant Mal d 2 displayed the ability to bind IgE from apple-allergic individuals equivalent to natural Mal d 2. In addition, the recombinant thaumatin-like Mal d 2 exhibited antifungal activity against Fusarium oxysporum and Penicillium expansum, implying a function in plant defense against fungal pathogens.

Targeting antigens to murine and human M-cells with Aleuria aurantia lectin-functionalized microparticles.

Neuraminidases act as a virulence factors for several pathogens that invade the human body through Peyer's patch M-cells. Because of the structural similarity of Aleuria aurantia lectin (AAL) to neuraminidases, we hypothesized that AAL might also target human M-cells. In an in vitro human M-cell co-culture model significantly more particles were transported across the epithelium when microparticles were functionalized with AAL versus those that were not. Moreover, high concentrations of AAL induced no detectable cytotoxic effects on the related intestinal epithelial cell cultures, epithelial Caco2- and HT29-MTX-E12-cells. Upon incubation with AAL, PBMCs of allergic volunteers proliferated in response to AAL and secreted the cytokines, IL-2, IFN-gamma, IL-10 and IL-5 in a concentration-dependent manner, indicating immune-stimulatory properties of the lectin. We conclude that AAL-coated microparticles may have the potential to target entrapped antigens to human M-cells for oral vaccination.

Cross-reactive N-glycans of Api g 5, a high molecular weight glycoprotein allergen from celery, are required for immunoglobulin E binding and activation of effector cells from allergic patients.

Allergy diagnosis relying on the determination of specific IgE is frequently complicated by the presence of cross-reacting IgE of unclear clinical relevance. Particularly, the anaphylactogenic activity of IgE directed to cross-reactive carbohydrate moieties of glycoproteins from plants and invertebrates has been a matter of debate. In this study, we present the biochemical and immunological characterization of Api g 5, a glycoprotein allergen from celery with homology to FAD containing oxidases. Carbohydrate analysis of the allergen revealed the presence of glycans carrying fucosyl and xylosyl residues, structures previously shown to bind IgE. Chemical deglycosylation of the protein completely abolished binding of serum IgE from all 14 patients tested. Likewise, basophils from a patient allergic to mugwort pollen and celery were stimulated only by native Api g 5, whereas the deglycosylated allergen did not trigger release of histamine. IgE inhibition immunoblots showed that native Api g 5 other than the deglycosylated protein completely inhibited IgE binding to high molecular weight allergens in protein extracts from birch pollen, mugwort pollen, and celery. A similar inhibition was accomplished using the IgE binding oligosaccharide, MUXF, coupled to bovine serum albumin. All these observations taken together confer convincing evidence that IgE directed to cross-reactive carbohydrates is capable of eliciting allergic reactions in vivo.

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment.

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.