The allergen profile of beech and oak pollen

Background Beech and oak pollen are potential allergen sources with a world‐wide distribution. Objective We aimed to characterize the allergen profile of beech and oak pollen and to study cross‐reactivities with birch and grass pollen allergens. Methods Sera from tree pollen‐allergic patients with evidence for beech and oak pollen sensitization from Basel, Switzerland, (n=23) and sera from birch pollen‐allergic patients from Vienna, Austria, (n=26) were compared in immunoblot experiments for IgE reactivity to birch (Betula pendula syn. verrucosa), beech (Fagus sylvatica) and oak (Quercus alba) pollen allergens. Subsequently, beech and oak pollen allergens were characterized by IgE inhibition experiments with purified recombinant and natural allergens and with allergen‐specific antibody probes. Birch‐, beech‐ and oak pollen‐specific IgE levels were determined by ELISA. Results Beech and oak pollen contain allergens that cross‐react with the birch pollen allergens Bet v 1, Bet v 2 and Bet v 4 and with the berberine bridge enzyme‐like allergen Phl p 4 from timothy grass pollen. Sera from Swiss and Austrian patients exhibited similar IgE reactivity profiles to birch, beech and oak pollen extracts. IgE levels to beech and oak pollen allergens were lower than those to birch pollen allergens. Conclusion IgE reactivity to beech pollen is mainly due to cross‐reactivity with birch pollen allergens, and a Phl p 4‐like molecule represented another predominant IgE‐reactive structure in oak pollen. The characterization of beech and oak pollen allergens and their cross‐reactivity is important for the diagnosis and treatment of beech and oak pollen allergy.     

Identification of italian cypress (Cupressus sempervirens) pollen allergen Cup s 3 using homology and cross-reactivity.

BACKGROUND: The prevalence of seasonal allergic diseases of the upper airways is increasing in industrialized countries. The Cupressaceae are important causes of pollinosis, particularly in Europe. OBJECTIVE: To determine whether the pollen from Cupressus sempervirens (Italian cypress) contains a pathogenesis-related group 5 (PR-5) protein, similar to that found in other allergenic Cupressaceae pollens. METHODS: Messenger RNA was purified from Italian cypress pollen, and complementary DNA (cDNA) was synthesized. cDNAs for PR-5 proteins were amplified by polymerase chain reaction and extended by rapid amplification of cDNA ends methods. Recombinant Cup s 3 was expressed in Escherichia coli as a fusion protein. Inhibition enzyme-linked immunosorbent assays were used to test the allergenicity of Cup s 3. RESULTS: Three cDNAs were cloned. These clones had approximately 95% identity to Jun a 3 and Cup a 3. Recombinant Cup s 3.0102 maltose-binding protein inhibited the IgE from most patients from binding to an extract of Italian cypress. The extent of inhibition suggested that antibodies to Cup s 3 were a prominent component of the IgE response to Italian cypress pollen. CONCLUSION: Cup s 3, an allergen of Italian cypress pollen, was identified based on cross-reactivity and homology with other pollen PR-5 proteins, despite an apparently low level of protein expression. Variations in the content of Cup s 3 in the pollen from different regions or trees should be considered in the choice of extracts for diagnosis and specific immunotherapy for Italian cypress pollen hypersensitivity.     

Allergenic relevance of Cupressus arizonica pollen extract and biological characterization of the allergoid

BACKGROUND: Cupressaceae (cypress) pollens can cause pollinosis in winter. However, the lack of specific commercial extracts combined with the early pollination period of cypress trees make a precise diagnosis difficult. The need for a reliable and effective cypress extract for diagnostic and therapeutic purposes is increasingly felt. METHODS: Mixed or single Cupressus arizonica, lusitanica and sempervirens pollen extracts precipitated with ammonium sulfate (PPT) were compared by direct RAST, RAST inhibition and SDS-PAGE techniques. The major allergen of C. arizonica (Cup a 1), purified by anion exchange chromatography, was checked by immunoblotting experiments before chemical modification, in parallel with a C. arizonica extract, with potassium cyanate (KCNO) to obtain a monomeric allergoid. The allergoid extract was characterized for its biological, chemico-physical and immunological features by RAST inhibition, SDS-PAGE and ELISA assays. RESULTS: Direct RAST, RAST inhibition, and SDS-PAGE data indicated that the PPT C. arizonica pollen extract showed the most allergenic potential, and it can be considered representative of the Cupressus spp. Immunoblotting data confirmed Cup a 1 as a major allergen. RAST inhibition and ELISA showed that modified PPT C. arizonica extract had less IgE reactivity than the native, non-modified extract, while preserving the immunogenic capacity typical for an allergoid. Finally, the SDS-PAGE profile of Cup a 1 allergoid was similar to native Cup a 1 allergen, suggesting the modified C. arizonica extract shows the characteristics of a monomeric allergoid. CONCLUSIONS: The PPT C. arizonica pollen extract shows good in vitro diagnostic potential and its chemically modified form offers the features of a monomeric allergoid. It might therefore lend itself to the development of a product to be administered by the sublingual or oromucosal route for immunotherapy of individuals with cypress pollinosis.     

Multiple grass mixes as opposed to single grasses for allergen immunotherapy in allergic rhinitis

Grass pollen allergy affects approximately 40% of allergic patients. Subcutaneous allergen immunotherapy (SCIT) is the only allergen‐specific and disease‐modifying treatment available. Currently available therapeutic vaccines for the treatment of grass pollen allergy are based on natural grass pollen extracts which are either made from pollen of one cross‐reactive grass species or from several related grass species. Clinical studies have shown that SCIT performed with timothy grass pollen extract is effective for the treatment of grass pollen allergy. Moreover, it has been demonstrated that recombinant timothy grass pollen allergens contain the majority of relevant epitopes and can be used for SCIT in clinical trials. However, recent in vitro studies have suggested that mixes consisting of allergen extracts from several related grass species may have advantages for SCIT over single allergen extracts. Here, we review current knowledge regarding the disease‐relevant allergens in grass pollen allergy, available clinical studies comparing SCIT with allergen extracts from timothy grass or from mixes of several related grass species of the Pooideae subfamily, in vitro cross‐reactivity studies performed with natural allergen extracts and recombinant allergens and SCIT studies performed with recombinant timothy grass pollen allergens. In vitro and clinical studies performed with natural allergen extracts reveal no relevant advantages of using multiple grass mixes as opposed to single grass pollen extracts. Several studies analysing the molecular composition of natural allergen extracts and the molecular profile of patients' immune responses after SCIT with allergen extracts indicate that the major limitation for the production of a high quality grass pollen vaccine resides in intrinsic features of natural allergen extracts which can only be overcome with recombinant allergen‐based technologies.     

Impact of cross‐reactive carbohydrate determinants on wood dust sensitization

Background Occupational wood dust exposure can induce allergy and may be one cause of respiratory health problems among woodworkers. Objective The objective was to determine the prevalence and quantitative level of specific immunoglobulin E (sIgE) to beech and pine wood in exposed workers. Wood sensitization was specified with regard to cross‐reactivity and was correlated to the reported symptoms. Methods Danish workers (n=701) were investigated for sIgE to beech and pine. Wood samples from workplaces were analysed and coupled to ImmunoCAPs. Workers sensitized to wood were tested for cross‐reactive carbohydrate determinants (CCDs) and environmental allergens. IgE binding was specified for glycogenic vs. proteinogenic epitopes by inhibition tests. Results The prevalence of wood sensitization among all workers was 3.7%. There was no association between sensitization prevalence or sIgE concentrations and self‐reported allergic symptoms. Beech‐ and pine‐sensitized workers showed a high prevalence of CCD sensitization (73%). However, workers with a single sensitization to wood had no sIgE to CCDs. Specifying IgE epitopes demonstrated that sera of workers reporting allergic symptoms recognized proteinogenic IgE‐epitopes on wood allergens, whereas workers without allergic symptoms had primarily sIgE‐epitopes to glycogenic structures. Although 96% of the wood‐sensitized workers were atopic, no significant correlation was found between wood sensitization and sIgE to beech and birch pollen, but an association was found between sIgE against CCDs and pine pollen. Conclusion Sensitization prevalence to beech and pine wood measured by tailored ImmunoCAPs was not correlated to allergic symptoms. We recommend the application of CCD tools to assess the relevance of individual wood sensitization. Cite this as: S. Kespohl, V. Schlünssen, G. Jacobsen, I. Schaumburg, S. Maryska, U. Meurer, T. Brüning, T. Sigsgaard and M. Raulf‐Heimsoth, Clinical & Experimental Allergy, 2010 (40) 1099–1106.     

Characterization of IgE-reactive autoantigens in atopic dermatitis. 2. A pilot study on IgE versus IgG subclass response and seasonal variation of IgE autoreactivity.

Previously we reported that patients with severe forms of atopy (e.g. atopic dermatitis, AD) frequently display IgE reactivity against autoantigens. Here we investigated the effects of periodate treatment and reducing versus nonreducing conditions on IgE recognition of nitrocellulose-blotted human cell extracts. IgE and IgG subclass reactivities of AD patients to blotted human cellular extracts as well as to ELISA plate-bound purified endogenous (myosin, histones) antigens and an environmental allergen (timothy grass pollen allergen, Phl p 5) were compared. Serum samples were collected over a period of 12 months from 3 autoreactive AD patients with pollen allergy and tested for IgE reactivity to nitrocellulose-blotted human epithelial and endothelial cell extracts as well as to birch and timothy grass pollen allergens. Results obtained indicate that (1) IgE autoantibodies may be directed primarily against protein and not carbohydrate epitopes, (2) reducing conditions seem to expose or better extract epitopes recognized by IgE autoantibodies, (3) IgE and IgG1-4 autoantibody responses were poorly associated and (4) IgE responses to autoallergens may reflect skin manifestations and may be boosted by seasonal exposure to pollen allergens. Our results thus indicate that IgE autoreactivity may represent a true form of autoimmunity directed against partly denatured peptide epitopes which may be boosted by exogenous allergen contact.     

Comparison between the native glycosylated and the recombinant Cup a1 allergen: role of carbohydrates in the histamine release from basophils

BACKGROUND: Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract. OBJECTIVE: To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one. METHODS: Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils. RESULTS: Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity-after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation. CONCLUSION: A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy.     

Allergenic relationship between taxonomically diverse pollens

Background Skin tests and tests for IgE antibodies show that subjects are usually sensitive to a number of different pollens, frequently from taxonomically diverse species which are assumed to be allergenically non-crossreactive. This suggests that the presence of IgE antibody-reactivity to an individual pollen may not necessarily have resulted from contact with that pollen or even with a taxonomically closely related species. Objective Since this has important consequences for allergen avoidance and desensitization of patients, we attempted to define allergenic relationships between diverse pollen species. Methods Sera from subjects were examined in direct IgE antibody binding experiments and by quantitative inhibition, protein blotting and adsorption and edition studies. Results Sera from subjects diagnosed as allergic to white cypress pine. Italian cypress. ryegrass or birch pollen were shown to have IgE antibodies that reacted with pollens from these four species and from cocksfoot, couch grass, lamb's quarter, wall pellitory. olive, plantain and ragweed. These reactions were confirmed in protein blotting and adsorption and elution studies where numerous IgE-binding bands were detected in all 11 different pollen extracts with sera from each of the different allergic categories, further evidence of allergenic (i.e. IgE-binding crossreactivity between the different pollens was provided by inhibition studies in which clear-cut inhibitions of IgE binding to the different pollen allergen discs were obtained with comparable amounts of the different pollen extracts. Conclusion We conclude that the presence of pollen reactive IgE antibodies may not necessarily be a true reflection of sensitizing pollen species.     

Allergenic cross-reactivity of olive pollen

BACKGROUND: Sera of patients allergic to olive (Olea europaea) pollen were used to analyze the IgE cross-reactivity between olive-pollen extract and other pollens obtained from phylogenetically unrelated species. METHODS: We used IgE immunostaining of pollen extracts blotted to nitrocellulose membranes after SDS-PAGE and inhibition analysis of this binding. RESULTS: A high inhibition of the IgE binding on olive-pollen extract was exhibited by birch, mugwort, pine, and cypress pollens, suggesting that these extracts contain proteins which share common epitopes and thus can be recognized by olive-allergic sera. IgE binding to Gramineae pollen extracts was not inhibited by olive-pollen extract, indicating a primary sensitization of the patients to these species. From the inhibition assays, the presence of an allergen of 45 kDa in the olive pollen, which has no homologous counterparts in other allergenic species, has been inferred. CONCLUSIONS: Olive pollen contains allergens which cross-react with pollens from unrelated species, a fact that could simplify the diagnosis and treatment of pollinosis.     

Similar localization of conformational IgE epitopes on the house dust mite allergens Der p 5 and Der p 21 despite limited IgE cross‐reactivity

Background

Due to high IgE recognition frequency and high allergenic activity, Der p 5 and Der p 21 are clinically important house dust mite (HDM) allergens. The objective of this study was to characterize the immunodominant IgE epitopes of Der p 5 and Der p 21 responsible for their high allergenic activity.

Methods

A panel of 12 overlapping peptides spanning the Der p 5 and Der p 21 sequence were synthesized to search for sequential IgE epitopes by direct testing for allergic patients' IgE reactivity. Peptide‐specific antibodies raised in rabbits were used in inhibition studies for localizing conformational IgE epitopes which were visualized on the surfaces of the allergen structures by molecular modelling. IgE cross‐reactivity between the allergens was investigated by IgE inhibition studies.

Results

Immunodominant IgE epitopes defined by allergic patients' IgE on Der p 5 and Der p 21 were primarily of the conformational, discontinuous type including N‐ and C‐terminal portions of the protein. They could be located on each allergen on one area with similar localization, but despite similar structure of the allergens, no relevant IgE cross‐reactivity could be detected.

Conclusion

Our study shows that Der p 5 and Der p 21 contain a major conformational IgE epitope‐containing area located on similar portions of their structure, but they lack relevant IgE cross‐reactivity. These data are important for the development of modern allergy vaccines based on defined molecules for allergen‐specific immunotherapy of HDM allergy.     

Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen

The knowledge of IgE-binding epitopes on allergen molecules is important for better understanding allergen-antibody interactions and, thus, for developing new strategies for immunotherapy. Our purpose was to more precisely define the number and structure of IgE-binding epitopes of a paradigmatic major grass pollen allergen. We performed an IgE-binding epitope mapping of rHol l 5, a group V pollen allergen of velvet grass (Holcus lanatus), with overlapping fragments (length between 15 and 186 amino acids), which were expressed in E. coli as MBP fusion proteins. Using sera of 65 grass pollen allergic patients, the fragments were analysed by immunoblotting for IgE reactivity. Specificity of antibody binding was confirmed by competitive blot inhibition assays. At least four different continuous IgE-binding epitopes were identified on small fragments (about 30 amino acids), and at least five different discontinuous IgE-binding epitopes on larger fragments, which were destroyed by further fragmentation. The fragments were differentially recognized by individual patients' sera. By investigating IgE-binding to one of the small fragments in more detail, we found further epitope regions on this fragment. It was noteworthy that IgE reactivity to small fragments was weak compared to large fragments or to the complete molecule. Competitive blot inhibition experiments showed that binding of IgE antibodies to the small fragments was specific but with lower avidity than to the complete rHol l 5. rHol l 5 harbours multiple discontinuous as well as continuous IgE-binding epitopes spread over the whole molecule, which were individually recognized by IgE antibodies from different patients. Low avidity of IgE antibodies to small fragments suggests that the continuous epitope regions do not represent the complete epitope and are most probably parts of discontinuous epitopes.     

Characterization of peptidic and carbohydrate cross-reactive determinants in pollen polysensitization

Background Cross‐reactivity may be due to protein sequence or domain homologies and/or the existence of cross‐reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross‐reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross‐reactive determinants. Material and methods Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS‐PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass‐sensitive patient, followed by anti‐human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. Results The results showed two different patterns of cross‐reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA ‐binding pattern. The IgE binding was abolished by pre‐incubation with sugar residues only in the case of the second pattern. Discussion This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross‐sensitization to a wide range of glycoproteins. Two‐D blots allow to characterize a cross‐sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.     

Heterogeneity of atopy. I. Clinical and immunologic characteristics of patients allergic to cypress pollen

The heterogeneity of pollen-allergic persons is well known but poorly characterized. Cypress is one of the major pollen-producing plants of the Mediterranean area. A study was undertaken to characterize the symptoms presented by patients allergic to cypress pollen and the heterogeneity of the IgE immune response between patients allergic only to cypress pollen and those who are polysensitized. Eighty-nine patients allergic to cypress pollen were studied, 26 being allergic only to cypress pollen. The IgE response was assessed by skin prick tests and the titration of serum total IgE and cypress-specific IgE by RAST. Clinical reactivity was assessed by symptom scores during the cypress pollen season and skin tests. Pollen counts were obtained. The clinical reactivity was similar in both patient groups. Rhinitis was present in all patients, conjunctivitis in 73.7–88.5%, and asthma in only 7.4–19.2%. The age of onset of symptoms caused by cypress pollen allergy was significantly greater in patients allergic to cypress pollen only. Total serum IgE was within the normal range in the cypress pollen group and significantly lower than in the polysensitized groups. Cypress pollen RAST was higher in the polysensitized group. We concluded that conjunctivitis is particularly common in cypress pollen allergy. Patients allergic only to cypress pollen may be unique in their way of expressing serum total IgE levels.     

Antigenic cross‐reactivity between Schistosoma mansoni and peanut: a role for cross‐reactive carbohydrate determinants (CCDs) and implications for the hygiene hypothesis

The antigenic reactivity of constituents of Schistosoma mansoni and peanut (Arachis hypogaea) was investigated to determine whether identical antigenic epitopes possessed by both organisms provided a possible explanation for the negative correlation between chronic schistosome infection and atopy to allergens. Aqueous extracts of peanuts were probed in Western immunoblots with rabbit IgG antibodies raised against the egg, cercarial and adult worm stages of S. mansoni. Several molecules in the peanut extract were antigenically reactive with antibodies from the various rabbit anti‐schistosome sera. A pair of cross‐reactive peanut molecules at ~30 000–33 000 molecular weight was purified and both proteins were identified by mass spectrometric analysis as the peanut allergen Ara h 1. Anti‐S. mansoni soluble egg antigen antibodies that were eluted off the peanut molecules reacted with two S. mansoni egg antigens identified by mass spectrometry as IPSE/α‐1 and κ‐5. Alignments of the amino acid sequences of Ara h 1 and either IPSE/α‐1 or κ‐5 revealed a low level of peptide sequence identity. Incubation of nitrocellulose paper carrying electrophoresed peanut molecules, six constituents of other allergic plants and S. mansoni egg antigens in a mild solution of sodium metaperiodate before probing with antibodies, inhibited most of the cross‐reactivities. The results are consistent with the antigenic cross‐reactive epitopes of S. mansoni egg antigens, peanut and other allergic plants being cross‐reactive carbohydrate determinants (CCDs). These findings are novel and an explanation based on ‘blocking antibodies’ could provide an insight for the inverse relationship observed between schistosome infection and allergies.     

Can component‐based microarray replace fluorescent enzimoimmunoassay in the diagnosis of grass and cypress pollen allergy?

Background Few data on the diagnostic accuracy in pollinosis of the microarray ISAC of allergens are available. Objective We aim to comparatively analyse ISAC CRD103 with the whole‐extract ImmunoCAP in grass and cypress pollen allergy, evaluating the suitability of the manufacturer's recommended cut‐off points for both techniques. Methods We studied 120 atopic patients grouped into grass and cypress pollen‐allergic patients and controls based on clinical history and skin prick tests. Specific IgE against Phleum pratense and Cupressus arizonica by ImmunoCAP and ISAC CRD103 were performed on all subjects. Results In the grass pollen group (43 allergic/26 controls), both microarray and CAP showed high sensitivity (Se) and specificity (Sp) values (ISAC: Se 97.7, Sp 92.3; CAP: Se 95.3, Sp 96.1) for recommended cut‐off points. Comparing the optimal (ISAC: 0.4 ISU; CAP: 0.33 kU/L) with the recommended cut‐off points within the same technique, diagnostic agreement was observed in both techniques. Thus, CAP and ISAC showed similar diagnostic performance in grass pollen allergy when using recommended cut‐off points. In cypress pollen group (12 allergic/92 controls), the microarray (Se: 91.7, Sp 91.3) showed similar Se but significantly higher Sp (P=0.034) than CAP (Se: 91.7, Sp: 80.4) using recommended cut‐off points. However, although diagnostic performance of the microarray did not change when comparing the optimal (0.82 ISU) with the recommended cut‐off point, CAP improved diagnosis of cypress pollen allergy, when applying the optimal (0.66 kU/L)(CAP Se: 91.7, Sp: 89.1) instead of the manufacturer's recommended cut‐off point. Thus, when the most suitable cut‐off point for both techniques (ISAC: 0.3 ISU; CAP: 0.66 kU/L) is selected, microarray and CAP provide equivalent diagnoses. Conclusions and Clinical Relevance Component‐based microarray ISAC CRD103 and whole‐allergen CAP showed high Se and Sp diagnosing equally grass and cypress pollen allergy. The cut‐off point for each allergen should be properly applied for both techniques. Cite this as: P. Cabrera‐Freitag, M. J. Goikoetxea, C. Beorlegui, P. Gamboa, G. Gastaminza, M. Fernández‐Benítez, M. Ferrer, M. Blanca and M. L. Sanz, Clinical & Experimental Allergy, 2011 (41) 1440–1446.     

Fruit-pollen-latex cross-reactivity: implication of profilin (Bet v 2).

BACKGROUND: An association between allergy to fruits and latex, and between pollen and plant-derived food has been described. The cross-reactive structures responsible for these associations have not yet been completely elucidated. METHODS: IgE reactivity to the recombinant allergens Bet v 1 and Bet v 2, different pollens, natural latex, papain, and bromelain was investigated in 29 patients with allergy to fruits or vegetables who lived in an area without birch trees. RESULTS: Exactly 79.3% of patients were allergic to grass pollen, and two of them had clinical allergy to latex. Serum IgE reactivity (CAP) to birch pollen was found in 65% of patients, to Bet v 2 in 51.7%, to Bet v 1 in 3.4%, to latex in 58.6%, to bromelain in 51.7%, and to papain in 17.2% of patients. All subjects with positive IgE to Bet v 2 had also reactivity to latex, grass, olive tree, birch, and mugwort pollens. The six patients not allergic to pollen did not show IgE reactivity to latex, Bet v 1, or Bet v 2. A significant correlation was found between CAP to latex with Bet v 2 (r=0.86, P<0.001), with birch (r=0.86, P<0.001), and with ryegrass (r=0.81, P<0.001). Immunoblotting using nine sera with positive CAP to birch pollen showed IgE-binding to a 15-kDa band that was recognized by antiprofilin monoclonal antibody. Bet v 2 CAP could be inhibited up to 52% by ryegrass and up to 23% by mugwort. CAP to latex was almost completely inhibited by ryegrass pollen with sera from five subjects without symptoms due to latex, whereas no inhibition was observed with serum from one patient with allergy to latex. CONCLUSIONS: Patients with allergy to plant-derived food and associated pollinosis showed a high frequency of IgE reactivity to Bet v 2, which may cause positive serum IgE determinations to latex and birch pollen due to the presence of cross-reactive epitopes. IgE reactivity to Bet v 2 may serve as an indicator of broad sensitization.     

Cross-reactivity of IgE antibodies to allergens

The cross‐reactivity of IgE antibodies is of interest for various reasons, three of which are discussed. Firstly, from the clinical view, it is important to know the patterns of cross‐reactivity, because they often (but not always) reflect the pattern of clinical sensitivities. We discuss the cross‐reactivities associated with sensitization to pollen and vegetable foods: PR‐10 (Bet v 1‐related), profilin, the cross‐reactive carbohydrate determinant (CCD), the recently described isoflavone reductase, and the (still elusive) mugwort allergen that is associated with celery anaphylaxis; cross‐reactivities between allergens from invertebrates, particularly tropomyosin, paramyosin, and glutathione S‐transferase (GST); and latex‐associated cross‐reactivities. Clustering cross‐reactive allergens may simplify diagnostic procedures and therapeutic regimens. Secondly, IgE cross‐reactivity is of interest for its immunologic basis, particularly in relation to the regulation of allergic sensitization: are IgE antibodies to allergens more often cross‐reactive than IgG antibodies to “normal” antigens? If so, why? For this discussion, it is relevant to compare not only the structural relation between the two allergens in question, but also the relatedness to the human equivalent (if any) and how the latter influences the immune repertoire. Thirdly, prediction of IgE cross‐reactivity is of interest in relation to allergic reactivity to novel foods. Cross‐reactivity is a property defined by individual antibodies to individual allergens. Quantitative information (including relative affinity) is required on cross‐reactivity in the allergic population and with specific allergens (rather than with whole extracts). Such information is still scarce, but with the increasing availability of purified (usually recombinant) allergens, such quantitative information will soon start to accumulate. It is expected that similarity in short stretches of the linear amino‐acid sequence is unlikely to result in relevant cross‐reactivity between two proteins unless there is similarity in the protein fold.     

Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen

BACKGROUND: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. METHODS: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. RESULTS: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF(3)/Gn(GF)XF(3) (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. CONCLUSION: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.     

Quantification of the major allergen from cypress (Cupressus arizonica) pollen, Cup a 1, by monoclonal antibody-based ELISA

BACKGROUND: Cypress pollen allergy is an important cause of rhinoconjunctivitis and asthma in Mediterranean countries. Cypress allergenic extracts are difficult to produce since they have low protein and high carbohydrate content, thus accurate standardization of them is essential to guarantee their quality. The aim of this study is to develop a sandwich ELISA for the quantification of Cup a 1, the major allergen of cypress (Cupressus arizonica) pollen extract. METHODS: Monoclonal antibodies directed to purified Cup a 1 were produced. Two of them (9C7 as capture antibody and 3D2 as the tracer) were selected to develop a quantitative sandwich ELISA. This ELISA was subsequently evaluated and compared with other techniques. RESULTS: The described ELISA is very sensitive with a detection limit of 8.7 ng/ml and a practical working range of 62.5-1,000 ng/ml. The assay is also highly reproducible with intra-assay and interassay coefficients of variation of less than 10%. The purified Cup a 1, used as standard, presents pectate lyase enzymatic activity. The assay also detected Cup a 1-like proteins in pollen from other Cupressaceae. A good correlation was obtained between Cup a 1 content of 12 C. arizonica pollen extracts and their IgE-binding activity. CONCLUSIONS: The described Cup a 1 ELISA is sensitive, specific and reproducible and can be used for the quantification of Cup a 1 in C. arizonica and other related pollen extracts. It also provides a reliable indication of the allergenic activity of the whole cypress pollen extract.