The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50–60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13

BACKGROUND: More than 70% of the patients allergic to grass pollen exhibit IgE-reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level. OBJECTIVE: The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capacity. METHODS: Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE-binding frequency to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELISA measurements. Pre-adsorption of sera with purified allergens and subsequent incubation of nitrocellulose-blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu-C. RESULTS: More than 50% of 300 patients displayed IgE-binding with both allergens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a 'background smear'. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 was degraded rapidly. CONCLUSION: Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.     

Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11

BACKGROUND: Allergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. OBJECTIVE: To clone and characterize the 20 kDa timothy grass pollen allergen Phl p 11. METHODS: Phl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. RESULTS: Phl p 11 cDNA defined an acidic polypeptide of 15.8 kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n = 184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. CONCLUSION: As the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.     

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen Phl p 13 from timothy grass pollen (Phleum pratense)

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.     

Design of an optimally-diagnostic skin test solution for diagnosis of sensitivity to timothy grass (Phleum pratense) pollen

Background Although most of the common allergen extracts that are used for diagnosis of type 1 hypersensitivity are now well standardized, this gives no assurance that they are within the concentration range that gives the best chance of a true diagnosis. Objective The objective of this study was to identify the most appropriate concentration range of timothy grass pollen Phleum pratense extract to diagnose sensitivity to this pollen correctly through skin-testing. Methods Dilutions of a well-standardized extract were made and used to skin test ‘true’ positive and ‘true’ negative populations of subjects as identified by case history, challenge tests and radioallergosorbent test (RAST). Weal diameters were measured and the data were submitted to receiver operating characteristics (ROC) analysis. For any particular weal size cut–off, the optimal diagnostic concentration (ODC) range was thus calculated. Results A 3 mm weal diameter cut-off was chosen as an appropriate size for routine diagnosis. Therefore the ODC range at this diameter was used to establish a product target concentration and specification for formulation of the diagnostic reagent. This method of allergen extract standardization can lead to a true-biological unitage that can be used for labelling purposes. Conclusion The optimun concentration range at which to formulate an allergen extract, in terms of an in vitro immunologically based assay, can be determined by carrying out ROC analysis of the results of clinical studies as described in this communication. Diagnostic units (DU), are now used by us for labelling of such final formulations which conveys the information that the product is at the most appropriate concentration for diagnosis.     

Phleum pratense alone is sufficient for allergen-specific immunotherapy against allergy to Pooideae grass pollens

Background Specific immunotherapy is the only causal treatment of allergy available today. Traditionally, therapeutic products based on either a single grass species or a mix of such extracts are used for grass pollen immunotherapy. Investigations comparing the immunological response to these allergen preparations are needed to ensure optimal treatment. The objective of this study was to investigate patterns of T and B cell cross-reactivity to Pooideae single-species extracts and to extract mixes.
Methods IgG4 induced by immunotherapy with Phleum pratense extract was investigated for cross-reactivity using nine single-species extracts and four mixes. For the mixes, studies of IgE cross-reactivity were also performed. T cell cross-reactivity was investigated in lines specific to nPhl p 1 or nPhl p 5 allergens, and the amounts of group 1 and 5 allergens in the extracts were quantified by a single radial immunodiffusion.
Results The levels of treatment-induced IgG4 detected by all the extracts displayed a clear correlation to that detected by the P. pratense pollen extract. The IgE studies confirmed the cross-reactivity of P. pratense -specific B cells towards the allergens contained in the mixes, and the T cell studies demonstrated cross-reactivity towards group 1 and 5 major allergens in extracts of six temperate grass species.
Conclusion Extensive T and B cell cross-reactivity was observed towards the allergens of the Pooideae grasses, and the degree of B cell cross-reactivity was independent of the number of species included in the extract mixes. This implies that treatment with pollen extract of just one Pooideae species will affect the allergic responses caused by any of the temperate grasses in this subfamily.     

Heterogeneity of commercial timothy grass pollen extracts

Background The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources.
Objective To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity.
Methods Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients.
Results The allergen extracts showed broad variations in protein compositions and amounts (24.1–197.7 μg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32–384 ng/mL; Phl p 2: 1128–6530 ng/mL, Phl p 5: 40–793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients.
Conclusions Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.     

Measurement of IgE antibodies against purified grass-pollen allergens (Phl p 1, 2, 3, 4, 5, 6, 7, 11, and 12) in sera of patients allergic to grass pollen

BACKGROUND: Current allergy diagnosis is performed with allergen extracts which contain a variety of allergenic and nonallergenic components. The availability of highly purified and well-characterized allergen molecules seems to be an advantage of component-based diagnosis. METHODS: With the immunoenzymatic CAP FEIA System, we measured specific IgE levels to the recombinant allergens rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, and native Phl p 4 in 77 sera of patients allergic to grass pollen, in order to evaluate the IgE-binding frequency to these purified grass-pollen allergens and their relationship to rBet v 4, rBet v 2, and other allergens. RESULTS: The frequency of sensitization was as follows: rPhl p 1=93.5%; rPhl p 2=67.5%; rPhl p 5=72.7%; rPhl p 6=68.8%; rPhl p 7=7.8%; rPhl p 11=53.2%; rPhl p 12=35.1%; and native Phl p 4=88.3%. As expected, rPhl p 7 and rPhl p 12 had a very good correlation (Spearman's r) with Bet v 4 (r=0.95%, P<0.05) and rBet v 2 (r=0.99, P<0.05), respectively. Good correlations of rPhl p 12 with papain (r=0.93, P<0.05), latex (r=0.92, P<0.05), and bromelain (r=0.86, P<0.05) were found. Highly variable individual sensitization patterns were observed. CONCLUSIONS: A new clinical approach has allowed the determination of specific allergograms for the different patients and may therefore be of great importance for more specific diagnosis. The use of component-resolved diagnostics may be useful to evaluate the allergen content of an extract for immunotherapy by monitoring patient's IgE and IgG directed to relevant allergens.     

Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens.

In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen-allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen-allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta-galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofed to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy.     

Allergen content of grass pollen preparations for skin prick testing and sublingual immunotherapy

Background:  The allergen content of diagnostics and immunotherapeutics is crucial for effective diagnosis and treatment. The aim of this study was to quantify and compare the allergen content of different grass pollen preparations for skin prick testing and sublingual immunotherapy (SLIT).
Methods:  Five skin prick test (SPT) solutions and 10 sublingual immunotherapeutics were analysed for protein and allergen concentration by Bradford assay, inhibition of IgE-binding to Phleum pratense ImmunoCAPs and content of the main allergen Phl p 5 by two-site enzyme immunoassay. In addition, the grass pollen preparations were compared by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses.
Results:  Protein concentrations of SPT solutions ranged from 15 to 427 μg/ml, and Phl p 5 concentrations ranged from 0.15 to 18.3 μg/ml. The ranking of SPT solutions concerning Phl p 5 content and IgE inhibition capacity was the same, and the ranking of protein and allergen content was closely correlated ( r  = 0.9). Protein content of the maintenance doses of the immunotheurapeutics ranged from 5 to 153 μg, Phl p 5 content ranged from 0.2 to 21.6 μg. IgE inhibition capacity of the maintenance doses was closely correlated to their Phl p 5 and protein content. SDS-PAGE and immunoblots confirmed the differences in protein and allergen content.
Conclusions:  Grass pollen preparations for SPT and SLIT varied greatly concerning protein and allergen content. Whereas this result corresponds to previous analyses results of SPT solutions, it was the first comparison of grass pollen immunotherapeutics. For diagnosis and therapy, these differences should be taken into account.     

Phl p 3: Structural and immunological characterization of a major allergen of timothy grass pollen

BACKGROUND: The relevant importance of individual allergens for allergic sensitization is only partially understood. More detailed information on allergen structure and how it influences immunological responses can lead to better diagnosis of disease and improved preparations for allergen-specific immunotherapy. Grass pollen contains several different allergens, and although the group 3 allergens have been classified long ago, their structure and allergenicity have been poorly investigated. OBJECTIVE: To characterize Phl p 3 from timothy grass pollen and compare it with Phl p 2 with respect to biochemical structure and allergenicity. METHODS: Natural Phl p 2 and Phl p 3 were separated from a pollen extract by chromatography and characterized by 2D electrophoresis and protein sequencing. The complete sequences were determined by DNA cloning and detected in natural pollen extracts by mass spectrometry. Further comparisons of the allergens were made for IgE-binding and cross-reactivity, allergenicity was determined by basophil CD203c activation and skin prick test and 3D structures were compared by molecular modelling. RESULTS: Phl p 3 reveals molecular masses of 10.958 and 10.973 kDa and pIs of 8.9 and 9.3, respectively, Phl p 2 a molecular mass of 10.816 kDa and a pI of 4.6. The sequence identity is 58%. In spite of these differences in the primary structures, both allergens reveal similar conformational structures, resulting in similar immunological and allergological moieties. CONCLUSIONS: The group 3 and group 2 allergens are major allergens with similar 3D structures. Although they differ considerably in their protein sequences and their pIs, they show only a slightly higher immunological reactivity for Phl p 3 on the B-cell level (conformational epitopes). But distinct differences between the sequences may influence reactivity at the T cell level.     

Food proteins from different allergen families sensitize Balb/c mice to family-specific immune responses

Abstract

The classification of food allergens based on the structure and function of proteins contributes to the study of the relationship between bioinformatics and potential allergenicity of allergens. Food allergens always share sequence similarity with the allergens in the same allergen families. For that reason, food proteins from different allergen families may induce different patterns of immune responses in animal models. Female Balb/c mice (3–4-weeks-old) were sensitized with food proteins once per week for 4 weeks, and then challenged 2 weeks later (on Day 42 of study). Blood was collected (to obtain serum levels of histamine and protein-specific IgG₁ and IgE antibodies) and measures of vascular permeability were performed 20?min after the challenge. Five food proteins (11S globulin, OVA [ovalbumin], HAS [human serum albumin] and LRP [lysine-responsive storage protein] of different allergen families, and Cry 1Ab/Ac [crystal protein]) were used to assess patterns of immune responses for each allergen family and then bioinformatics and digestive stability in simulated gastric fluid were employed to assess the overall utility of the Balb/c. The assay results indicated that, in this model, histamine and protein-specific IgE antibody levels and vascular permeability could be used to identify allergenicity of 11S globulin, OVA, and PAP (potato acid phosphatase) only. However, the results of the protein-specific IgG₁ measures could only distinguish allergic food proteins with negative control. Based on bioinformatic analyses, the five different food proteins clearly induced distinct patterns of immune responses in the Balb/c model.     

The international collaborative study establishing the first international standard for timothy (Phleum pratense) grass pollen allergenic extract

A selected candidate international reference preparation of timothy grass (Phleum pratense)-pollen extract was studied together with two other freeze-dried timothy pollen allergenic extracts in a multinational study. The collaborators used RAST inhibition, histamine release, quantitative immunoelectrophoresis (crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis and rockets), isoelectric focusing, and other methods. The total allergenic potencies measured in RAST inhibition were evaluated for validity of linearity and parallel-line response. The relative concentrations of some important individual allergenic components were measured. The relative potencies for the total allergenic activity and the timothy components studied in each preparation were expressed relative to the selected candidate. This preparation was established in 1983 by the World Health Organization expert committee on biologic standardization as the international standard for timothy grass-pollen extracts with assigned units of 100,000 IU per ampule.     

Generation of a low Immunoglobulin E-binding mutant of the timothy grass pollen major allergen Phl p 5a

BACKGROUND: Immunotherapy of grass pollen allergy is currently based on the administration of pollen extracts containing natural allergens. Specifically designed recombinant allergens with reduced IgE reactivity could be used in safer and more efficacious future therapy concepts. OBJECTIVES: This study aimed to generate hypoallergenic variants of the timothy grass major allergen Phl p 5a as candidates for allergen-specific immunotherapy. METHODS: Three deletion mutants were produced in Escherichia coli and subsequently purified. The overall IgE-binding capacity of the mutants was compared with the recombinant wild-type allergen by membrane blot and IgE-inhibition assays. The capacity for effector cell activation was determined in basophil activation assays. T cell proliferation assays with allergen-specific T cell lines were performed to confirm the retention of T cell reactivity. Structural properties were characterized by circular dichroism analysis and homogeneity by native isoelectric focusing. The deletion sites were mapped on homology models comprising the N- and C-terminal halves of Phl p 5a, respectively. RESULTS: The double-deletion mutant rPhl p 5a Delta(94-113, 175-198) showed strongly diminished IgE binding in membrane blot and IgE-inhibition assays. Both deletions affect predominantly alpha-helical regions located in the N- and C-terminal halves of Phl p 5a, respectively. Whereas deletion of Delta175-198 alone was sufficient to cause a large reduction of the IgE reactivity in a subgroup of allergic sera, only the combination of both deletions was highly effective for all the sera tested. rPhl p 5a Delta(94-113, 175-198) consistently showed at least an 11.5-fold reduced capacity to activate basophils compared with the recombinant wild-type molecule, and the T cell proliferation assays demonstrated retention of T cell reactivity. CONCLUSION: The mutant rPhl p 5a Delta(94-113, 175-198) fulfils the basic requirements for a hypoallergenic molecule suitable for a future immunotherapy of grass pollen allergy; it offers substantially reduced IgE binding and maintained T cell reactivity.     

Concentrations of major grass group 5 allergens in pollen grains and atmospheric particles: implications for hay fever and allergic asthma sufferers sensitized to grass pollen allergens

BACKGROUND: Grass pollen allergens are the most important cause of hay fever and allergic asthma during summer in cool temperate climates. Pollen counts provide a guide to hay fever sufferers. However, grass pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet, grass pollen allergens are known to be associated with atmospheric respirable particles. OBJECTIVE: We aimed (1) to determine the concentration of group 5 major allergens in (a) pollen grains of clinically important grass species and (b) atmospheric particles (respirable and nonrespirable) and (2) to compare the atmospheric allergen load with clinical data to assess different risk factors for asthma and hay fever. METHODS: We have performed a continuous 24 h sampling of atmospheric particles greater and lower than 7.2 microm in diameter during the grass pollen season of 1996 and 1997 (17 October 1996-16 January 1997) by means of a high volume cascade impactor at a height of about 15 m above ground in Melbourne. Using Western analysis, we assessed the reactivity of major timothy grass allergen Phl p 5 specific monoclonal antibody (MoAb) against selected pollen extracts. A MoAb-based ELISA was then employed to quantify Phl p 5 and cross-reactive allergens in pollen extracts and atmospheric particles larger and smaller than 7.2 microm. RESULTS: Phl p 5-specific MoAb detected group 5 allergens in tested grass pollen extracts, indicating that the ELISA employed here determines total group 5 allergen concentrations. On average, 0.05 ng of group 5 allergens were detectable per grass pollen grain. Atmospheric group 5 allergen concentrations in particles > 7.2 microm were significantly correlated with grass pollen counts (rs = 0.842, P < 0. 001). On dry days, 37% of the total group 5 allergen load, whereas upon rainfall, 57% of the total load was detected in respirable particles. After rainfall, the number of starch granule equivalents increased up to 10-fold; starch granule equivalent is defined as a hypothetical potential number of airborne starch granules based on known pollen count data. This indicates that rainfall tended to wash out large particles and contributed to an increase in respirable particles containing group 5 allergens by bursting of pollen grains. Four day running means of group 5 allergens in respirable particles and of asthma attendances (delayed by 2 days) were shown to be significantly correlated (P < 0.001). CONCLUSION: Here we present, for the first time, an estimation of the total group 5 allergen content in respirable and nonrespirable particles in the atmosphere of Melbourne. These results highlight the different environmental risk factors for hay fever and allergic asthma in patients, as on days of rainfall following high grass pollen count, the risk for asthma sufferers is far greater than on days of high pollen count with no associated rainfall. Moreover, rainfall may also contribute to the release of allergens from fungal spores and, along with the release of free allergen molecules from pollen grains, may be able to interact with other particles such as pollutants (i.e. diesel exhaust carbon particles) to trigger allergic asthma.     

Identification of canary grass (Phalaris aquatica) pollen allergens by immunoblotting: IgE and IgG antibody-binding studies

The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen in the external environment of southern Australia. Seventeen allergenic fractions of canary grass pollen, ranging in mol. mass from 14 to 100 kDa. have been identified by immunoblotting, using IgE antibodies from sera of 24/30 grass-pollen-allergic subjects. The highest frequency of IgE binding (77%) was to a major 34-kDa fraction (tentatively designated Pha a I). This protein bas been partially purified and identified as a group I allergen by immunodepletion experiments, with partially purified Lol p I (from rye-grass pollen), atopic serum, and Lol p I-specific MAb. In addition, microsequencing of the N -terminus of Pha a I showed an amino acid sequence identical to Lol p I. In a separate study. IgE binding to Western blots of Pha a I, Lol p I. and Cyn d I was investigated in 24 sera and found to occur in 19/24. 18/24, and 9/24. respectively. IgE binding to ail three major allergens, and to both Pha a I and Lol p I, occurred in 8/24 sera. Our findings suggest that while tbe N -terminal sequence of Pha a I is identical to Lol p I, there may be specific allergenic epitopes exclusive to this allergen that are important for allergenicity in southern Australia.     

Spatial clustering of the IgE epitopes on the major timothy grass pollen allergen Phl p 1: importance for allergenic activity

BACKGROUND: The major timothy grass pollen allergen Phl p 1 is one of the most potent and frequently recognized environmental allergens. OBJECTIVE: We sought to study at a molecular and structural level the IgE recognition of Phl p 1 and its relation to allergenic activity. METHODS: Monoclonal human IgE antibody fragments specific for Phl p 1 and group 1 allergens from various grasses were isolated from a combinatorial library made of lymphocytes from patients with grass pollen allergy. Recombinant Phl p 1 fragments and the 3-dimensional structure of Phl p 1 were used to localize the major binding site for the IgE antibodies. A rPhl p 1 fragment containing this binding site was expressed in Escherichia coli, purified, and tested for IgE reactivity and allergenic activity with sera and basophils from patients with grass pollen allergy. RESULTS: Monoclonal antibodies, as well as polyclonal serum IgE, from patients with grass pollen allergy defined a C-terminal fragment of Phl p 1 that represents a sterically oriented portion on the Phl p 1 structure. This Phl p 1 portion bound most of the allergen-specific IgE antibodies and contained the majority of the allergenic activity of Phl p 1. CONCLUSION: IgE recognition of spatially clustered epitopes on allergens might be a general factor determining their allergenic activity. CLINICAL IMPLICATIONS: Geographic distribution of IgE epitopes on an allergen might influence its allergenic activity and hence explain discrepancies between diagnostic test results based on IgE serology and provocation testing. It might also form a basis for the development of low allergenic vaccines.