Effects of double mutation at two distant IgE-binding sites in the three-dimensional structure of the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity

Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.     

Expression of hypoallergenic Der f 2 derivatives with altered intramolecular disulphide bonds induces the formation of novel ER-derived protein bodies in transgenic rice seeds

House dust mites (HDM) are the most common source of indoor allergens and are associated with allergic diseases worldwide. To benefit allergic patients, safer and non-invasive mucosal routes of oral administration are considered to be the best alternative to conventional allergen-specific immunotherapy. In this study, transgenic rice was developed expressing derivatives of the major HDM allergen Der f 2 with reduced Der f 2-specific IgE reactivity by disrupting intramolecular disulphide bonds in Der f 2. These derivatives were produced specifically as secretory proteins in the endosperm tissue of seeds under the control of the endosperm-specific glutelin GluB-1 promoter. Notably, modified Der f 2 derivatives aggregated in the endoplasmic reticulum (ER) lumen and were deposited in a unique protein body (PB)-like structure tentatively called the Der f 2 body. Der f 2 bodies were characterized by their intracellular localization and physico-chemical properties, and were distinct from ER-derived PBs (PB-Is) and protein storage vacuoles (PB-IIs). Unlike ER-derived organelles such as PB-Is, Der f 2 bodies were rapidly digested in simulated gastric fluid in a manner similar to that of PB-IIs. Oral administration in mice of transgenic rice seeds containing Der f 2 derivatives encapsulated in Der f 2 bodies suppressed Der f 2-specific IgE and IgG production compared with that in mice fed non-transgenic rice seeds, and the effect was dependent on the type of Der f 2 derivative expressed. These results suggest that engineered hypoallergenic Der f 2 derivatives expressed in the rice seed endosperm could serve as a basis for the development of viable strategies for the oral delivery of vaccines against HDM allergy.     

Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.     

Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity

The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 A resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel beta-sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.     

The binding of mouse hybridoma and human IgE antibodies to the major fecal allergen, Der p I, of Dermatophagoides pteronyssinus. Relative binding site location and species specificity studied by solid-phase inhibition assays with radiolabeled antigen

The relative binding site location and species specificity of 19 mouse hybridoma antibodies, produced in four laboratories, to Dermatophagoides pteronyssinus major fecal allergen, Der p I, was studied by using immobilized mAb and inhibitions of radiolabeled Ag binding. Four mAb groups were defined, within which 4, 6, 8, and 5 mAb, respectively, cross-inhibited each other. Five mAb were members of both group 2 and 3, demonstrating a considerable overlap of epitopes between the corresponding antibody-binding regions. The degree of mAb species specificity, as assessed by inhibition with cold Der p I and Ag Der m I and Der f I from the related species, Dermatophagoides microceras and Dermatophagoides farinae, was highly variable even for mAb binding to the same region on the Ag. Five cases of cross-reactivity between Der p I and Der m I and one case of cross-reactivity between Der p I and Der f I were found. The N-terminal 30 amino acids of the three species showed 7 substitutions between Der p I and Der m I/Der f I and 2 between Der f I and Der p I/Der m I. Single mAb inhibited up to 65% of labeled Der p I binding to immobilized human IgE from allergic patients' sera and up to 24% of labeled Der p I binding to immobilized rabbit antibodies. The spectrum of species specificities in human IgE sera, as assessed by inhibitions with cold Ag, was similar to that of the mAb. No evidence for the presence of strictly sequential epitopes, reactive with either mAb or human IgE was found, as judged from the weak inhibitory activity of acid-denatured Der p I.     

Crystal Structure of Der f 7, a Dust Mite Allergen from Dermatophagoides farinae

Background

Der f 7 is the group 7 allergen from the dust mite Dermatophagoides farinae, homologous to the major allergen Der p 7 from D. pteronyssinus. Monoclonal antibody that bind to residues Leu48 and Phe50 was found to inhibit IgE binding to residue Asp159, which is important for the cross-reactivity between Der f 7 and Der p 7.

Methodology/Principal Findings

Here, we report the crystal structure of Der f 7 that shows an elongated and curved molecule consisting of two anti-parallel β-sheets – one 4-stranded and the other 5-stranded – that wrap around a long C-terminal helix. The overall fold of Der f 7 is similar to Der p 7 but key difference was found in the β1–β2 loop region. In Der f 7, Leu48 and Phe50 are in close proximity to Asp159, explaining why monoclonal antibody binding to Leu48 and Phe50 can inhibit IgE binding to Asp159. Both Der f 7 and Der p 7 bind weakly to polymyxin B via a similar binding site that is formed by the N-terminal helix, the 4-stranded β-sheet and the C-terminal helix. The thermal stability of Der f 7 is significantly lower than that of Der p 7, and the stabilities of both allergens are highly depend on pH.

Conclusion/Significance

Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and overall 3D structure but with significant differences in the region proximal to the IgE epitope and in thermal stability. The crystal structure of Der f 7 provides a basis for studying the function and allergenicity of this group of allergens.     

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.     

Antigen Der f I from the dust mite Dermatophagoides farinae: structural comparison with Der p I from Dermatophagoides pteronyssinus and epitope specificity of murine IgG and human IgE antibodies

The physicochemical and antigenic properties of an allergen purified from Dermatophagoides farinae, Der f I, were compared with Der p I from Dermatophagoides pteronyssinus. On SDS-PAGE, Der f I migrated as a single polypeptide chain with the same m.w. as Der p I (24,000). Two isoallergenic peaks of Der f I were identified on preparative isoelectric focusing (pI 5.7 to 6.3 and pI 6.6 to 6.95). Fractions from each peak were shown to have an identical amino acid composition (which was similar but not identical to Der p I) and the same N-terminal amino acid sequence. There was a good correlation between quantitative intradermal skin tests to both purified allergens and to D. farinae extract in mite-allergic patients, with positive results when using as little as 10(-5) micrograms/ml of Der f I. The majority of sera with detectable IgE antibody to D. farinae also had IgE antibody to Der f I both among children (29/42 = 69%) and adults (55/63 = 87%). By RAST, there was an excellent correlation between IgE antibody to Der f I and Der p I in sera from 42 mite-allergic children (n = 0.94, p less than 0.001). Polyclonal IgG antibodies from six mice immunized with Der f I showed preferential binding to that allergen, and most monoclonal antibodies (16 of 18) raised against Der f I did not bind Der p I. However, two monoclonal antibodies from this fusion showed cross-reactive binding to both allergens. Immunoabsorption experiments, using D. pteronyssinus and D. farinae extracts coupled to Sepharose, showed that a large proportion of murine antibodies (74% to Der p I and 60 to 93% to Der f I) could not be absorbed by the heterologous extract on the immunosorbent. In contrast, in sera from seven mite-allergic patients, most of the specific IgE and IgG antibody (i.e., greater than or equal to 82%) was removed by either immunosorbent. Thus, Der f I and Der p I represent a homologous pair of major allergens which possess both cross-reacting and species-specific epitopes. The antibody response in mice immunized with either allergen in complete Freund's adjuvant was largely directed against species-specific epitopes, whereas in allergic humans, IgE- and IgG-specific antibodies bound predominately to cross-reacting epitopes.     

Molecular determinants for antibody binding on group 1 house dust mite allergens

House dust mites produce potent allergens, Der p 1 and Der f 1, that cause allergic sensitization and asthma. Der p 1 and Der f 1 are cysteine proteases that elicit IgE responses in 80% of mite-allergic subjects and have proinflammatory properties. Their antigenic structure is unknown. Here, we present crystal structures of natural Der p 1 and Der f 1 in complex with a monoclonal antibody, 4C1, which binds to a unique cross-reactive epitope on both allergens associated with IgE recognition. The 4C1 epitope is formed by almost identical amino acid sequences and contact residues. Mutations of the contact residues abrogate mAb 4C1 binding and reduce IgE antibody binding. These surface-exposed residues are molecular targets that can be exploited for development of recombinant allergen vaccines.     

Relationship between the magnitude of IgE production in mice and conformational stability of the house dust mite allergen,Der p 2

BackgroundProtein antigens are degraded by endosomal protease in antigen presentation cell. T cells recognize peptides derived from antigen proteins bound to class II major histocompatibility complex molecules. We previously reported that an increase in the conformational stability of an antigen depressed its immunogenicity. However, there is little information on antigens with differences in molecular properties such as net charges and molecular weight.MethodsDenaturation experiments against guanidine hydrochloride. The serum IgE levels to protein antigens at 35 days after the first immunization analyzed using ELISA.ResultsThe Der p 2 mutations in which Ile13 is mutated to Ala (I13A) and Ala122 is mutated to Ile (A122I) were shown to have lower and higher conformational stability than the wild-type, respectively, by denaturation experiments. The amount of IgE production by the less stable I13A mutant was higher and that of the stable A122I mutant was lower than that of the wild-type.ConclusionOur results suggest that the increased conformational stability of Der p 2 depressed the IgE production in mice.General significanceThese findings should provide a milestone for the engineering of allergen vaccines.     

Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.     

Expression of house dust mite allergens Der f 1 and Der f 2 in leaves of Nicotiana benthamiana

In test systems for diagnostics of type I allergy, recombinant allergens in native conformation are used, because immunoglobulins E (IgE), responsible for the development of this type of allergy, recognize conformational epitopes of protein allergens. To obtain recombinant allergens of the Dermatophagoides farinae house dust mites (HDM), Der f 1 and Der f 2, two systems of heterological expression were used: Escherichia coli and Nicotiana benthamiana plants. IgE from sera of children allergic to HDM were shown to recognize the recombinant Der f 2 protein synthesized in both E. coli and N. benthamiana plants, as well as the recombinant Der f 1 protein produced in N. benthamiana plants, while mature form of Der f 1 produced in E. coli did not interact with IgE.     

Cloning and expression of cDNA encoding the complete prepro-form of an isoform of Der f 1, the major group 1 allergen from house dust mite Dermatophagoides farinae

cDNA clones encoding a major house dust mite allergen, Der f 1, were isolated from a Dermatophagoides farinae cDNA library by plaque immunoscreening using rabbit anti-Der f 1 serum. The sequences cover the complete open reading frame encoding the prepro-form. The sequence is different from previously reported cDNA of Der f 1 in six bases and the encoded amino acid sequence is different in two residues. Pro-forms of Der f 1 and its mutant, in which the N-glycosylation motif was disrupted, expressed in Pichia pastoris were converted to the mature forms by an in vitro activation process and they showed significant IgE-binding. The biologically active rDer f 1 molecules would be useful for diagnostic testing and allergen-specific immunotherapy. In contrast, Der f 1 directly expressed in Escherichia coli without the prosequence had very low IgE binding. The hypoallergenic Der f 1 polypeptide could be useful for safer and more effective immunotherapy.     

Alpha-Actinin Is a New Type of House Dust Mite Allergen

Main indoor allergens for humans are from house dust mites. There are more than 30 allergens in Dermatophagoides farinae but only fourteen allergens have been identified from this mite including Der f 1–3, 6, 7, 10, 11, 13–18, and 22. A native allergen protein (Der f 24, 90 kDa) was purified from D. farinae by gel filtration and anionic exchange liquid chromatography combined with IgE immunodetection. Its primary structure was determined by Edman degradation, mass spectrometry analysis and cDNA cloning. Enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, basophil activation test (BAT) and skin prick test (SPT) were performed to evaluate the allergenicity. It was identified as an alpha (α)-actinin containing a CaM-like domain with EF-hand motifs. Der f 24 reacted to sera from 85.4% (35/41) of patients on western blot analysis. It reduced ∼20% sera IgE reactivity to D. farinae extracts on a competitive ELISA. Eighty percent (8/10) of patients with D. farinae allergy showed positive reactions to Der f 24 in skin prick test. The expression of CD63 on basophils from patients was up-regulated by Der f 24 by ∼5.4-fold. Alpha-actinin was identified as a new type of house dust mite allergen. To the best of our knowledge, this is the first report of α-actinin as an allergen.     

Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10-100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2-3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.     

Block Copolymer/DNA Vaccination Induces a Strong Allergen-Specific Local Response in a Mouse Model of House Dust Mite Asthma

Background

Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity.

Objective

The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice.

Methods

Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements.

Results

Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted.

Conclusions & Clinical Relevance

DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.     

Population growth and allergen accumulation of Dermatophagoides farinae cultured at 20 and 25 °C

House dust mites are cultured to obtain mite allergen material to produce allergen extracts (vaccines) for diagnostic tests, immunotherapy, and research purposes. Research laboratories and manufacturers have their own culturing protocols to grow these mites and these may vary between manufacturers and between research laboratories. The temperature at which mites are cultured may influence the allergen composition, allergen ratio of Der 1: Der 2 and endotoxin levels in the extracts produced from these cultured mites. In order to produce standardized and uniform extracts, across the industry and in various research laboratories, the influence of culture conditions must be understood. Here we determined how temperature affects mite population growth rates, dynamics of allergen production, Der f 1: Der f 2 ratio and endotoxin levels in extracts made from Dermatophagoides farinae mites cultured at 20 and 25 °C. We found that Der f 1 and Der f 2 accumulated exponentially in the cultures with Der f 1 accumulating faster than Der f 2. When the live mite populations peaked, the ratios for Der f 1: Der f 2 were 4.1 and 4.7 for cultures reared at 20 and 25 °C, respectively. Most of the Der f 1 and Der f 2 allergen in whole cultures is not in mite bodies and is lost when the mite material is washed. Thus, if the ratio of Der f 1 and Der f 2 is an important consideration for commercial and research extracts, then the temperature at which the mites are cultured and the collection procedure are important considerations.     

深圳地区粉尘螨Ⅱ类变应原基因的多态性分析及其表达蛋白的变应原性鉴定

粉尘螨Ⅱ类变应原(Der fⅡ)是粉尘螨Dermatophagoides farinae主要变应原之一,可引发Ⅰ型变态反应。从深圳地区挑取活粉尘螨,经形态鉴定后纯培养,提取其总RNA,RT-PCR扩增出Der fⅡ 基因,克隆到pMD18-T载体后测序。通过计算机软件分析该基因的多态性。将该目的基因克隆到pET-His表达载体上得到重组质粒pET-Der fⅡ。工程菌经IPTG诱导培养,表达Der fⅡ目的蛋白,该蛋白主要以包涵体形式存在。重组Der fⅡ蛋白通过6His-tag蛋白纯化系统进行分离、纯化,并进行Western blot检测该重组蛋白与对粉尘螨过敏患者血清中IgE的反应性。结果表明,克隆的5株深圳地区的Der fⅡ 基因与GenBank公布的Der fⅡ(AB195580.1)核苷酸序列同源性为96.8%～99.3%,推导的氨基酸序列同源性为93.8%～98.6%。表达纯化的5种重组Der fⅡ蛋白经Western blot检测,表明都具有良好的变应原性。     

Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (?=16nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.     

Mapping of conformational IgE epitopes with peptide-specific monoclonal antibodies reveals simultaneous binding of different IgE antibodies to a surface patch on the major birch pollen allergen, Bet v 1

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients' IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients' IgE binding to Bet v 1 (52-75%) were obtained with mAbs specific for two peptides comprising aa 29-58 (P2) and aa 73-103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.