Proteome analysis of maize pollen for allergy-relevant components

Over the last few decades, the cultivation of maize (Zea mays) has strongly increased in Central Europe. We therefore decided to study the allergen composition and the allergenic potency of its pollen in comparison with pollen from timothy grass (Phleum pratense), a typical representative of the native grasses. We found that 65% of the sera reactive to timothy pollen also bound to maize pollen proteins. By using 2-DE immunoblotting, followed by incubation with mAbs directed against known allergens or protein sequencing, those IgE-reactive components were further classified. Although novel, maize-specific pollen allergens could not be found, the presence of crossreacting allergens belonging to groups 1 and 13 (Zea m 1 and 13), both having high IgE prevalence, as well as the presence of the less important group 3 and 12 allergens was found. The structural variability of Zea m 1 and Zea m 13 was determined by sequencing clones isolated from a maize pollen cDNA library. This revealed sequence identities of 72 and 70%, respectively, to the corresponding Phl p 1 and Phl p 13 allergens of timothy grass pollen. IgE-crossreactivity was further studied using immunoblot inhibition tests. Here, timothy pollen extract completely blocked IgE binding to maize, whereas maize pollen extract blocked IgE reactivity to only some timothy pollen allergens.     

Allergenicity of Acroptilon repens and Juglans regia pollen in rats

Pollen proteins that are located in the cytoplasm or on the surface of the exine can function as allergens and evoke immune system responses in sensitive patients, leading to allergic rhinitis and asthma. In this research, the pollen allergenicity and ability to induce IgE response of the pollen of two plant species were studied in rats. Acroptilon repens is an herbaceous, invasive plant with entomophilous pollen, while Juglans regia which is a tree crop produces anemophilous pollen. Immunoblot analysis using sera of sensitised rats revealed IgE reactivity to three protein bands including the 70, 41 and 25.12 kDa bands present in the A. repens pollen extract, while only one single immunogenic band of 11 kDa was detected in J. regia pollen extract. Both pollen extracts increased the eosinophil content and caused some clinical signs of allergy in treated rats. The results showed that both entomophilous and anemophilous pollen can be allergenic.     

Twenty years of aerobiological monitoring in Trentino (Italy): assessment and evaluation of airborne pollen variability

The aim of the study is to report a reliable airborne pollen spectrum composition and seasonal timings for the monitored area as a basis for allergy management and to ascertain possible modifications through the detection of trends during the 20-year time series (1989–2008). Pollen was collected at San Michele all’Adige (Trento, Italy) by means of a Hirst-type spore trap. Sampling and counting of airborne pollen grains were carried out according to a national standard. Pollen concentration data for the period were processed in order to characterize the main pollen seasons for a subset of taxa, selected on the basis of their allergenicity and local representativeness. Variations in the pollen data over the years surveyed were analyzed using non-parametric tests. The results showed the presence of 63 pollen taxa, 40 of which belonged to tree and shrub species and 23 to herbaceous species. The local pollen spectrum was characterized by the presence of highly allergenic taxa, such as Urticaceae, Graminaceae, Ostrya sp., and Cupressaceae, in terms of percentage contribution as well as mean daily pollen count or peak values over the years surveyed. A significant upward trend was observed for daily mean pollen amount, mainly due to pollen from woody species and probably ascribable to a temperature-driven increase in pollen production. Evaluation of the results presented will form the basis of further research focussed on the climate change-related causes of modifications to vegetational dynamics as well as on the phenology of flowering and on pollen production.     

Efficient and sensitive identification and quantification of airborne pollen using next‐generation DNA sequencing

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen‐allergic patients. Current pollen monitoring methods are microscope‐based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost‐effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next‐generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.     

Mast cell alpha-chymase reduces IgE recognition of birch pollen profilin by cleaving antibody-binding epitopes.

In sensitized individuals birch pollen induces an allergic response characterized by IgE-dependent mast cell degranulation of mediators, such as alpha-chymase and other serine proteases. In birch and other plant pollens, a major allergen is profilin. In mammals, profilin homologues are found in an intracellular form bound to cytoskeletal or cytosolic proteins or in a secreted form that may initiate signal transduction. IgE specific to birch profilin also binds human profilin I. This cross-reactivity between airborne and endogenous proteins may help to sustain allergy symptoms. The current work demonstrates that cultured mast cells constitutively secrete profilin I, which is susceptible to degranulation-dependent proteolysis. Coincubation of chymase-rich BR mastocytoma cells with Ala-Ala-Pro-Phe-chloromethylketone (a chymase inhibitor) blocks profilin cleavage, which does not occur in degranulated HMC-1 mast cells, which are rich in tryptase, but chymase deficient. These data implicate chymase as the serine protease cleaving secreted mast cell profilin. Sequencing of chymase-cleaved profilins reveals hydrolysis at Tyr(6)-Val(7) and Trp(35)-Ala(36) in birch profilin and at Trp(32)-Ala(33) in human profilin, with all sites lying within IgE-reactive epitopes. IgE immunoblotting studies with sera from birch pollen-allergic individuals demonstrate that cleavage by chymase attenuates binding of birch profilin to IgE. Thus, destruction of IgE-binding epitopes by exocytosed chymase may limit further mast cell activation by this class of common plant allergens, thereby limiting the allergic responses in sensitized individuals.     

The Effects of Air Pollution on Structures, Proteins and Allergenicity of Pollen Grains

The prevalence of allergic disease has increased world wide during the last decades. Pollen allergy is the most typical form of allergic disease. The increase in its frequency during recent years is the most evident. Environmental factors play an important role in the problem of pollen allergy in large cities. The aim of this research is determination of allergenicity of Canna pollen in polluted and non-polluted conditions, detection of their allergenic proteins and also elucidation of some microscopic effects of air pollutants on pollen structure and proteins. Mature and immature pollen grains of Canna indica were collected from polluted and non-polluted areas. Pollen grains were studied by scanning electron microscopy. Mice were sensitized by injection of pollen extract and an adjuvant for five times. Allergy potency of different pollen extracts were compared by means of skin test, blood eosinophills number and IgE levels in sensitized and treated animals. Pollen proteins were studied by SDS-PGE and allergenic proteins were detected by immunoblotting techniques. Scanning electron microscope study of the pollen grains showed that in polluted areas, air born particles accumulated on the surface of pollen and changed both pollen's shape and pollen's tectum. Also many vesicles were released out of polluted pollen and the pollen material agglomerated on the surface of pollen. SDS-PAGE showed that different proteins exist in mature and immature pollen. In pollen collected from polluted area, some of protein bands between 22 and 45 kDa were disappeared . Also in all polluted pollen grains, protein content of pollen decreased in response to air pollution causing the release of pollen proteins. According to our experiments and regarding induction of allergic symptoms, the polluted pollen is more effective than non-polluted one, and mature pollen has more allergy potency than immature one.     

Proteomic profiling of birch (Betula verrucosa) pollen extracts from different origins

Pollen of the European white birch is a major source of spring pollinosis in Europe. Pollen-allergy diagnosis and treatment by specific immunotherapy commonly rely on extracts of natural origin. To gain insight into the protein content and its variability, we evaluated the profile of allergenic and non-allergenic proteins in extracts of pollen from different origins by MS-based proteomics. Aqueous extracts prepared from commercially available Swedish birch pollen, pollen collected from Austrian trees and a commercial skin prick extract were analyzed by 1-DE, 2-DE, immunoblotting and mass spectrometry, resulting in a complete inventory of extractable, disease-relevant pollen proteins. A main focus of this study was on the isoform distribution of Bet v 1, the major allergen of birch pollen. Using a combination of intact mass determination and peptide sequencing, five isoforms (a, b, d, f and j) were unequivocally identified in Swedish and Austrian birch pollen extracts, while the skin prick extract contained only isoforms a, b and d. Using the same methods as for Bet v 1, divergencies in the sequence of birch profilin (Bet v 2), a plant panallergen, were solved. The molecular characterization of pollen extracts is relevant for standardization and development of new reagents for specific immunotherapy.     

Sub-proteome analysis of novel IgE-binding proteins from Bermuda grass pollen

Bermuda grass (Cynodon dactylon) pollen (BGP) is one of the most common causes of airway allergic disease, and has been shown to contain over 12 allergenic proteins on 1-D immunoglobulin E (IgE) immunoblots. However, only a few allergens have been identified and characterized. Cyn d 1 is a major allergen and the most abundant protein in BGP, representing 15% of the whole-pollen extract. To investigate variability in the IgE-reactive patterns of BGP-sensitized patients and to identify other prevalent allergens, a BGP extract was passed through an affinity column to remove Cyn d 1, and the non-bound material was collected and analyzed by 2-DE. IgE-reactive proteins were subsequently characterized by immunoblotting using serum samples from ten BGP-allergic patients. The prevalent IgE-reactive proteins were identified by MALDI-TOF MS, N-terminal sequence similarity, and LC-MS/MS. Here, we present a sub-proteome approach for allergen investigation and its use for determining BGP 2-DE profiles and identifying six novel allergens.     

Analysis of the pollen allergen content of twelve olive cultivars grown in Portugal

Olive pollen presents intercultivar variability as regards to its antigenic and allergenic composition. In this study, we report the presence of differences among the SDS-PAGE pollen protein profiles of twelve Portuguese olive cultivars. Though most soluble proteins from these extracts seemed similar, three bands of about 18, 20 and 22 kDa presented sharp differences in intensity among the cultivars analyzed. The dissimilarity of patient’s sera reactivity to these protein extracts and the presence of several allergens already characterized (Ole e 1, Ole e 2, Ole e 5 and Ole e 9) in the extracts were also investigated. Epidemiological data indicated that 53.3 % out of the 428 patients analyzed with reactivity to pollen extracts, presented specific IgE levels to Olea europaea. A representative number of these sera were assayed in immunoblotting experiments. The cultivars ‘Galega’ and ‘Conserva de Elvas’ displayed low reactivity to the sera of atopic patients, whereas the extracts corresponding to the cultivars ‘Cobrançosa’, ‘Ascolana’ and ‘Verdeal de Serpa’ led to higher IgE reactivity. The use of antibodies to the allergens Ole e 1, Ole e 2, Ole e 5 and Ole e 9 in immunoblotting experiments also allowed cultivar discrimination. The cultivar ‘Verdeal de Serpa’ presented the highest Ole e 1, Ole e 5 and Ole e 9 allergen loads but the lowest Ole e 2. ‘Carrasquenha’ was the second cultivar in terms of the higher allergen content. Oppositely, the lowest allergen loads were those of the cultivars ‘Galega’ and ‘Conserva de Elvas’ coincidentally with their low IgE reactivity. These data may help interpret physiological differences in pollen performance for successful olive fertilization and, moreover, to better define future strategies for allergy diagnosis and treatment by specific immunotherapy.     

Biochemical and immunochemical characterization of hop-hornbeam (Ostrya Carpinifolia Scop.) pollen

In the last few years Ostrya carpinifolia pollen is consideredas an important cause of respiratoryallergy in Mediterranean areas. The concentration ofthe pollen was measured over a period of fifteen yearsfrom 1981 to 1996 in an area around Genoa; the resultsof this study have clearly indicated an increasingtrend that correlate with persons sensitization.In this study we sought to define the immunochemical andbiochemical properties of hop-hornbean pollen. Soluble proteins extracted from Ostryacarpinifolia pollen and from the taxonomicallyrelated species Corylus Avellana, were analyzedby polyacrylamide gel electrophoresis (SDS-PAGE), byhorizontal isoelectrofocusing (IEF) and by twodimensions electrophoresis (2D-PAGE). Allergenicproteins were identified with sera of sensibilizedpatients and cross-reactivity was evaluated byimmunoblotting techniques. The electrophoreticanalysis showed a partial identity between theproteins from Ostrya and Corylus extracts. The immunoblotting assay, developed withhuman IgE from subjects allergic to hop-hornbeampollen, displayed the major IgE reactivity for acomponent with a molecular weight of 17 kDa expressedin both Ostrya and Corylus extracts. This reactivity is consistent with the presence ofBet v 1 that is described as the major pollen allergenin the Betulaceae and Corylaceae families. Sera fromsubjects allergic to Ostrya were then preadsorbed with recombinant Bet v 1 immobilized in the Pharmacia CAP System; a significant reduction ofthe IgE binding activity was observed after thetreatment. We therefore suggest that Bet v 1 couldbe one of the allergenic proteins present in theOstrya pollen possibly being responsible forcross-reactivity with other members of taxonomicallyrelated families.     

Reproducibility between counts of airborne allergenic pollen from two cities in the East Midlands,UK

Historically in the East Midlands, UK, airborne pollen has been monitored in two cities, Derby and Leicester, situated 41 km (25 miles) apart. The aim of the present study was to compare aerobiological data from both sites to determine if a forecast based on data from one site would be sufficient for both, and to address the wider issue of reproducibility between geographically separated sites. Pollen types recorded could be split into two groups according to annual abundance, maximum daily concentration and the number of high count days. Six taxa made up the abundant group; ash, birch, grass, oak, nettle-type and yew-type, representing 90 and 88% of the total air spora for Derby and Leicester, respectively. Three consecutive years of grass and nettle pollen data are presented, supported by one year of abundant tree pollen data. There were highly significant positive correlations between the counts obtained. Line charts showing the average number of pollen grains m⁻³ air day⁻¹ show similar trends, and Bland–Altman plots show little discrepancy between the amounts of pollen counted on any given day. Each day was classified according to the UK accepted threshold levels for grass. Weighted kappa statistics showed substantial or almost perfect agreement between the forecast classifications. With the caveat that this would not apply in a region with restrictions to air flow such as a mountain range or with extreme fluctuations such as a coastline site, this study suggests that data from a single site is suitable for forecasting a distance of up to 41 km.     

On the causes of variability in amounts of airborne grass pollen in Melbourne,Australia

In Melbourne, Australia, airborne grass pollen is the predominant cause of hay fever (seasonal rhinitis) during late spring and early summer, with levels of airborne grass pollen also influencing hospital admissions for asthma. In order to improve predictions of conditions that are potentially hazardous to susceptible individuals, we have sought to better understand the causes of diurnal, intra-seasonal and inter-seasonal variability of atmospheric grass pollen concentrations (APC) by analysing grass pollen count data for Melbourne for 16 grass pollen seasons from 1991 to 2008 (except 1994 and 1995). Some of notable features identified in this analysis were that on days when either extreme (>100 pollen grains m⁻³) or high (50–100 pollen grains m⁻³) levels of grass pollen were recorded the winds were of continental origin. In contrast, on days with a low (<20 pollen grains m^-3) concentration of grass pollen, winds were of maritime origin. On extreme and high grass pollen days, a peak in APC occurred on average around 1730 hours, probably due to a reduction in surface boundary layer turbulence. The sum of daily APC for each grass pollen season was highly correlated (r = 0.79) with spring rainfall in Melbourne for that year, with about 60% of a declining linear trend across the study period being attributable to a reduction of meat cattle and sheep (and hence grazing land) in rural areas around Melbourne. Finally, all of the ten extreme pollen events (3 days or more with APC > 100 pollen grains m⁻³) during the study period were characterised by an average downward vertical wind anomaly in the surface boundary layer over Melbourne. Together these findings form a basis for a fine resolution atmospheric general circulation model for grass pollen in Melbourne’s air that can be used to predict daily (and hourly) APC. This information will be useful to those sectors of Melbourne’s population that suffer from allergic problems.     

Aerobiological, clinical and immunobiochemical studies on Lantana camara pollen and cross-reactivity with other Verbenaceae pollen species

Airborne pollen is an important and potent source of aeroallergens. The aim of the study was to conduct a 2-year aerobiological survey in Calcutta, India, for knowing the concentration and seasonal periodicity of Lantana camara (LC) pollen. The sensitization due to this pollen among seasonal respiratory allergic patients and its chemical composition was studied. An aerobiological survey was conducted with a volumetric Burkard sampler from 2004 to 2006. Protein components of LC pollen were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and IgE immunoblotting. Allergenic activities were determined by in vivo (skin prick test) and in vitro (enzyme-linked immunosorbent assay. In vitro inhibition tests were performed to evaluate cross-reactivity. LC pollen was present from March to May and from September to December contributing up to 10.5% to the total aeropollen load during peak month. Horizontal profile showed highest concentration for nearest (0.5 m) rotorod and it was decreased by half in a distance within 4.5–6.5 m from plot edge. LC pollen contained 7.5% carbohydrate, 19.3% lipid with proline and valine as dominant amino acid. Among 1,500 adult respiratory allergic patients tested, 7.93% showed higher level of positive reaction. IgE binding proteins of 22, 42, 45 and 95 kD were revealed. LC pollen showed remarkable cross-reactivity with other local Verbenaceae pollen taxa (Clerodendron viscosum, Tectona grandis and Vitex negundo). This is the first study on LC pollen regarding its aerobiological, clinical and immuno-biochemical aspects; it should be helpful for the diagnosis and therapy of patients susceptible to LC pollen.     

Pollen grains of queen sago (Cycas circinalis L.), a source of aeroallergen from West Bengal, India: an immunochemical approach

Cycas circinalis L. or queen sago is a common ornamental gymnosperm in tropics and subtropics. The objectives of the study were (a) to observe the seasonal variation of queen sago pollen in the atmosphere of a rural and an industrial area of West Bengal, India, (b) to visualize its allergenic potential on local population, and (c) to identify and isolate the important IgE-binding protein component present in the pollen extract. A two-year aerobiological survey was performed with Burkard personal volumetric sampler, and Cycas pollen was found to be present in air during April–July. Among 172 respiratory allergic patients of study area, 25.58% showed skin reaction to Cycas pollen extract. The allergenicity of the pollen extract was confirmed by in vivo (skin reaction test) and in vitro (IgE-ELISA and dot blotting) analyses and immunoblotting. Two components of 39.6 and 20.7 kDa were found to be the important IgE-binding proteins in pollen extract. The 20.7 kDa component was purified by two-step gel electrophoresis and it was found to retain its IgE reactivity. This component can be used for further work in diagnostic and therapeutic purpose in susceptible individuals. The overall study demonstrated that the pollen grains of Cycas circinalis is one of the important aeroallergen source of West Bengal, India,     

Air pollen dispersal in a tropical area

Volumetric data on airborne pollen have been gathered for two consecutive years at a neotropical location (Caracas). Among the 65 taxa which were identified, pollen from aCupressus species (introduced) and from aCecropia species (indigenous) were dominant. Less numerous but also abundant (daily averages ≥5 grains/m³ air) were pollen from Gramineae, Urticaceae,Alcalypha, Pinus, Piperaceae andMimosa. Pollen grains were recorded daily throughout the year. They increased in numbers during April–May and again during November–December. The first peak was contributed mainly by indigenous species, the second peak mainly by introduced species.     

Size distribution of allergenic Cry j 2 released from airborne <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> pollen grains during the pollen scattering seasons

Japanese cedar (Cryptomeria japonica) pollinosis is one of seasonal allergic rhinitis that mainly occurs in Japan. The pollinosis is caused by two main kinds of allergenic proteins called Cry j 1 and Cry j 2 which exist in Cryptomeria japonica pollen. In our previous study, we reported that the size-segregated of airborne fine allergenic Cry j 1 and morphological change of Cry j 1 due to the contact with rainfall. However, the study on airborne allergenic Cry j 2 in different particle sizes has not been identified until now. Therefore, the main aim of this study is to investigate the size distribution and scattering behavior of allergenic Cry j 2. The Cry j 2 particles were collected and determined in different particle sizes at the urban sampling points during the most severe pollination season of 2012 in Saitama, Japan. After the size-segregated Cry j 2 allergenic particles were collected using an Andersen high-volume (AHV) atmospheric sample, the airborne Cry j 2 concentrations were determined with a surface plasmon resonance (SPR) method. At the same time, the airborne Cryptomeria japonica pollens were also counted by the Durham pollen sampler. The higher concentrations of the allergenic Cry j 2 were detected even in particle sizes equal to or less than 1.1 μm (PM_1.1) than other particle sizes. The airborne particles ranges from 0.06 to 11 μm were also collected by a low-pressure impactor (LPI) atmospheric sampler. After that, the concentrations of Cry j 2 allergenic particles in fine particle sizes were measured by the SPR method either. With the help of this study, we have confirmed the existence of fine daughter allergenic particles, which clearly differ from the parent pollen grains in size, especially after the rainy days. It is possible that the daughter allergenic species will be released from the fractions of cell wall and burst pollen grains. We concluded that rainwater was one of the important factors that affects the release of pollen allergenic proteins of both Cry j 1 and Cry j 2 from the parent pollen grains.     

A 30-day-ahead forecast model for grass pollen in north London, United Kingdom

A 30-day-ahead forecast method has been developed for grass pollen in north London. The total period of the grass pollen season is covered by eight multiple regression models, each covering a 10-day period running consecutively from 21 May to 8 August. This means that three models were used for each 30-day forecast. The forecast models were produced using grass pollen and environmental data from 1961 to 1999 and tested on data from 2000 and 2002. Model accuracy was judged in two ways: the number of times the forecast model was able to successfully predict the severity (relative to the 1961–1999 dataset as a whole) of grass pollen counts in each of the eight forecast periods on a scale of 1 to 4; the number of times the forecast model was able to predict whether grass pollen counts were higher or lower than the mean. The models achieved 62.5% accuracy in both assessment years when predicting the relative severity of grass pollen counts on a scale of 1 to 4, which equates to six of the eight 10-day periods being forecast correctly. The models attained 87.5% and 100% accuracy in 2000 and 2002, respectively, when predicting whether grass pollen counts would be higher or lower than the mean. Attempting to predict pollen counts during distinct 10-day periods throughout the grass pollen season is a novel approach. The models also employed original methodology in the use of winter averages of the North Atlantic Oscillation to forecast 10-day means of allergenic pollen counts.     

Identification of specific IgE binding proteins in Castor bean (Ricinus communis) pollen obtained from different source materials

Allergenic components of Ricinus communis pollen obtained from different stages of inflorescence, different time intervals, different years and places were studied by immunoblot analysis. Proteins separated by SDS-PAGE and transferred to NC were identified using pooled sera from 15 skin and RAST positive patients. The IgE binding components in M.W. range of 14 to 70 kD were identified. The protein fractions of 70, 66, 64, 60, 50, 45, 36, 22 and 14 kD are the most prominent allergenic bands. Six samples collected during same pollination season from the same place showed similar allergenic profile. Of the samples collected from different stages of inflorescence, pollen of immature buds showed only three bands as compared to 18 from mature buds and flowers. Variability was seen in the IgE binding components of pollen stored for different years and obtained from different geographic regions of India. The IgE binding pattern of fifteen sera were heterogenous. The number of bands identified by different sera varied from 3 to 18. Two protein components of 66 and 36 kD were recognised by 14 (93.3%) of the 15 sera studied. The result suggests that there exists variations in the specific IgE binding pattern in pollen samples of Castor Bean, obtained from difference source materials.     

Profilin revealed in pollen nuclei: immuno-electron microscopy of high-pressure frozen Ledebouria socialis Roth (Hyacinthaceae)

 Cryoimmobilization by high-pressure freezing, combined with cryosubstitution and resin embedding, allowed accurate retention in situ of the small (12–15 kDa) water-soluble protein, profilin, in anthers of Ledebouria socialis Roth (Hyacinthaceae). The subcellular distribution of profilin was investigated by using post-embedding immunogold labelling with rabbit antisera raised against recombinant birch profilin (RP2) or birch COOH-terminal profilin peptide (RP3). The patterns observed in mature pollen grains are novel to eukaryotic organisms: profilin was consistently demonstrated within both the vegetative and generative nuclei, an addition to its well-known presence in the cytoplasm. Methodological and immunological aspects, as well as possible biological implications, of this finding are considered. Received: 17 March 1997 / Revision accepted: 10 July 1997     

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.