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Background The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5-specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. Methods A Phl p 5-specific ELISA system was assessed with respect to accuracy, precision, inter-assay (within laboratory) and inter-laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. Results The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter-assay variation (max. GCV 24%) and especially inter-laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. Conclusions Based on the collaborative study results, the assessed Phl p 5-specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method.

Background Although general practitioners (GPs) are frequently the first healthcare professionals whom asthma patients refer to for their symptoms, few studies have explored the extent of adherence to guidelines for asthma management based on data provided directly by GPs. Aims of the present study were to assess drug prescriptions for asthma by GPs and to evaluate prescriptive adherence to GINA guidelines (GL) and its relationship with disease control in real life. Methods 995 asthmatic patients (45% males, mean age 43.3 ± 17.7 yrs) were enrolled by 107 Italian GPs distributed throughout the country. Data on diagnosis, disease severity, prescribed anti-asthmatic drugs and control were collected through questionnaires filled out by GPs taking into consideration the 2009 GINA Guidelines. Data on drug use and chronic sinusitis, nasal polyposis, chronic bronchitis, emphysema were reported by patients through a self-administered questionnaire. Results The large majority of patients were classified by GPs as having intermittent (48.4%) or mild persistent asthma (25.3%); 61% had co-morbid allergic rhinitis (AR). The prevalent therapeutic regimen used by patients was a combination of inhaled corticosteroids (ICS) plus long-acting β2-agonists (LABA) (54.1%), even in the intermittent/mild persistent group. ICS as mono-therapy or in combination with other drugs but LABA, was the second most frequently adopted treatment (14.4%). In general, the GPs adherence to GL treatment indications was 28.8%, with a significant association with a good asthma control (OR 1.85, 95% CI 1.18–2.92). On the other hand, comorbidity (OR 0.52, 95% CI 0.32–0.84), moderate (0.44, 0.28–0.69) and severe (0.06, 0.02–0.20) persistent asthma showed significant negative effects on asthma control. Conclusions Our results show that over-treatment of intermittent/mild persistent asthma is frequent in the GPs setting while therapeutic regimens are more appropriately applied for moderate/severe asthma. In general, we found low adherence to GINA GL treatment recommendations even if its relevance in asthma control was confirmed.

In 2014, the Italian severe/uncontrolled asthma (SUA) registry was released to improve asthma care, awareness, knowledge on natural history, phenotypes. Aim: to characterize Italian SUA patients. Methods: The web-based registry, containing demographic, clinical, functional, inflammatory data, included 492 SUA patients (by GINA guidelines) from 3 Italian centres (Pisa, Perugia, Ancona). Results: SUA patients were more frequently females (60.9%), obese/overweight (64.7%), with a mean age of 53.8 yrs. 36.9% had asthma familiarity, 83.1% allergic asthma, 77.2% perennial symptoms. A late-onset of asthma symptoms (30yr) and a late first diagnosis (34yr) were observed. Other than the combination bronchodilator/corticosteroid, 51.6% of patients received antileukotrienes, 19.5% anticholinergics, 15.8% oral corticosteroids, 69.4% anti-IgE. In the last 12 months, SUA patients had 1.5±2.4 mean number of exacerbations, 9.7% had an emergency department visit and 7.3% hospitalization, 57.5% took oral steroids for exacerbations. The median blood eosinophil count was 300/mm 3 . The median FENO was 22 ppb. 65 patients performed induced sputum: 64.6% had an eosinophilic asthma phenotype (sputum Eos ≥ 3%), 13.8% neutrophilic (sputum Neu ≥ 76%), 16.9% paucigranulocytic. Comorbidities included allergic rhinitis (62.4%), gastro-oesophageal reflux (42.1%), nasal polyposis (30.2%), allergic conjunctivitis (30.2%), aspirin hyper-sensitivity (22%). Conclusion: Italian SUA patients are characterized by late symptoms onset and diagnosis, indices of eosinophilic inflammation, high burden in terms of health care costs and comorbidity. Supported by: the Italian Medicines Agency; an unrestricted grant of Roche.

Although asthma is usually considered to originate in childhood, adult-onset is being increasingly reported. Aim: to assess factors associated to early- (

To cite this article: Brunetto B, Tinghino R, Braschi MC, Antonicelli L, Pini C, Iacovacci P. Characterization and comparison of commercially available mite extracts for in vivo diagnosis. Allergy 2010; 65: 184–190. Abstract Background: Assessment of sensitization by allergen-specific IgE testing and skin prick testing (SPT) are primary tools in routine clinical diagnosis of allergies. To perform a correct diagnosis, it is critical that the allergen reagent used contains an adequate amount of all relevant components. This study aimed at evaluating commercially available mite extracts for in vivo diagnosis from eight manufacturers. Methods: Eight extracts from Dermatophagoides pteronyssinus and eight from Dermatophagoides farinae were analysed for total protein content by Bradford and for major allergen content by ELISA. SDS-PAGE, immunoblotting and SPT were also carried out. Results: The protein amount ranged from 27.7 μg/ml extract to 361.1 μg/ml (D. pteronyssinus) and from 20.3 to 353.0 μg/ml (D. farinae). In regards major allergen concentration, Der p 1 ranged from 9.6 to 36.2 μg/ml, Der f 1 26.5–196.1 μg/ml, mite group 2 0.7–31.7 μg/ml in D. pteronyssinus and 1.3–10.4 μg/ml in D. farinae. SDS-PAGE experiments showed that some components are poorly represented or absent in extracts from most manufacturers. Similar results were obtained by IgE-immunoblotting and SPT with 10 mite allergic patients confirmed a broad spectrum of reactivity of the extracts in the same subject. Conclusions: Immunochemical analysis showed a heterogeneous amount of component/s among mite extracts from different manufacturers. These data were confirmed by in vivo testing, suggesting that, for some of the patient tested, the absence of relevant allergens could strongly affect the diagnosis.

The aim of the study was to assess the symptoms prevalence of allergic diseases in a population of 11-15 yr old schoolchildren, to evaluate the associations between asthma and other symptoms and identify risk factors for asthma, rhinitis and eczema syndromes. A sample of 481 students was studied using an International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. Prevalence of different kind of self-reported symptoms was calculated. Using a logistic regression approach, we tried to identify risk factors for three syndromes - rhinitis, eczema and asthma. The highest and the lowest prevalence rates of self-reported symptoms were recorded for rhinitis (43.6%) and for eczema (8.1%), respectively. The prevalence of asthma was 15.7%. Univariate analysis showed a mutual association between wheeze and rhinitis symptoms. Multivariate logistic regression model for eczema syndrome revealed female gender as a significant risk factor. The polytomic logistic multivariate regression revealed female gender and family history of allergy as significant risk factors for rhinitis syndrome only, and maternal smoking and familial allergy for rhinitis and asthma together. In particular, familial allergy yields a 400% higher chance of developing asthma and rhinitis together. The synergistic effect of familial allergy on rhinitis and asthma syndromes suggests the implementation of preventive measures in children with family history of these diseases.

Summary Background With the development of genetically modified crop plants there has been a growing interest in the approaches available to assess the potential allergenicity of novel gene products. For additional assessment of the potential allergenicity of expressed proteins, informative data can be generated using animal models. Soybean is one of the major source of protein in human and animal nutrition, and has also been well characterized as a major allergenic source. Advances in biotechnology have resulted in an increasing number of genetically engineered foods, and among these soybean is one of the most widespread. Objective To develop and characterize a murine model of IgE-mediated soybean sensitization induced by intragastric immunization, in the presence of Cholera Toxin, with wild-type soybean extract (wt-SE) or with genetically modified soybean extract (gm-SE). Methods Balb/c mice born in our animal facilities, from females fed on soy-free food, were fed with the same soy-free food and used in all the experiments. Mice were sensitized by gavages with soybean extracts, and allergen-specific IgE and IgG responses were studied by direct ELISA and ELISA inhibition. Antigen-specific cell proliferation and cytokine production were evaluated in spleen cell cultures. Results Sensitization with both soybean extracts induced high levels of antigen-specific IgE and IgG1 and low levels of specific IgG2a. Both wt-SE and gm-SE were able to inhibit the binding of specific IgE from mice immunized with gm-SE to the same antigen used for the ELISA coating. A comparable proliferative response was obtained with the homologous as well as with the heterologous extracts. Conclusion In sensitized mice, we observed a predominantly T-helper type 2 (Th2)-type immune response, with increased soybean-specific IgE and IgG1 antibodies and a concomitant increase of IL-4 and IL-5 production. Results obtained by specific IgE ELISA inhibition and by antigen-specific T cell proliferation demonstrated that wt-SE and gm-SE shared B and T epitopes. The present murine model of soybean sensitization established by the oral route should provide valuable information about risk assessment for food allergy from new proteins of genetically modified foods.

Background: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. Methods: cDNA of Ole e 5 was amplified by nested 3′-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients’ IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). Results: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. Conclusions: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

Summary 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.

Background: Airborne insect antigens represent important aeroallergens which have been widely investigated. Although it has been demonstrated that house dust contains significant silverfish (Lepisma saccharina) levels, none of the extracts obtained so far has been extensively characterized. Thus, we have prepared and characterized a silverfish extract and investigated its IgE-reactive components by testing the reactivity of sera from patients allergic to inhalant insect allergens. Methods: The extract from silverfish insect bodies was prepared by homogenizing frozen silverfish in Tris-HCl buffer. The soluble material (Sup) was filtered and the insoluble material (Ppt) was resuspended in 100 mM Tris pH 10.6. The two fractions were characterized by biochemical and immunochemical methods. IgE reactivity was investigated on both fractions before and after periodate treatment. Results: Protein content and total carbohydrates was 2 and 3% w/w for Sup and 1 and 0.3% w/w for Ppt. The SDS-PAGE profile of the two fractions showed a different pattern in the MW range of 5–175 kD. Sup and Ppt, probed with allergic sera, showed a complex pattern of IgE reactivity. When periodate-treated fractions were tested, IgE reactivity was either completely abrogated, reduced or not affected, depending on the allergic serum employed. Conclusions: The results obtained indicate that the classic aqueous-extraction procedures that have been used up to now for other insects might not be completely satisfactory, since several allergenic components are not soluble at the normally used pH. We developed a dedicated extraction procedure allowing the detection of a certain degree of reactivity in sera negative to allergens extracted following classic procedures.

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.

Background: Cow's milk allergy is a common disease of infancy and early childhood. If the baby is not breast-fed, a substitute for cow's milk formula is necessary. Objective: The aim of this study was to investigate, in vitro and in vivo, the allergenicity of mare's milk in a population of selected children with severe IgE-mediated cow's milk allergy. Methods: Twenty-five children (17 male and 8 female) aged 19 to 72 months (median age 34 months) with IgE-mediated cow's milk allergy were selected for this study. All the children underwent skin prick tests with cow's milk and mare's milk and double-blind placebo-controlled oral food challenge (DBPCOFC) with fresh cow's milk, fresh mare's milk, and, as placebo, a soy formula (Isomil, Abbott, Campoverde, Italy). We performed immunoblotting of cow's and mare's milk developed with IgE from allergic children. Results: All the children showed strong positive skin test responses to cow's milk (4+); 2 children had positive skin test responses to mare's milk (2+). All children had positive DBPCOFCs to cow's milk; one child had a positive DBPCOFC to mare's milk. No children reacted to the placebo (Isomil). In the cow's milk, some proteins are able to strongly react with human IgE; when the sera are tested with mare's milk, the bands corresponding to the same proteins are recognized by a lower percentage of sera. Conclusion: These data suggest that mare's milk can be regarded as a good substitute of cow's milk in most children with severe IgE-mediated cow's milk allergy. It would be prudent, however, to confirm its tolerability by a supervised titrated oral challenge test. (J Allergy Clin Immunol 2000;105:1031-4.)

Background: Cupressaceae allergy is a worldwide pollinosis caused by several species. Some species in limited geographic areas pollinate in fall and winter. Juniperus oxycedrus matches these features. Objective: We sought to define the immunochemical, allergologic, and environmental aspects of J. oxycedrus pollen. Methods: Pollen extract from J. oxycedrus was prepared and characterized by biochemical analysis and human specific IgE binding by means of ELISA and immunoblotting. A 3-year phenological study was conducted to define the pollinating period of J. oxycedrus . Forty consecutive patients allergic to cypress were recruited in two areas and divided into two groups according to their exposure to J. oxycedrus pollen. Clinical evaluation, skin prick tests, and specific IgE determination with J. oxycedrus , J. ashei , and Cupressus arizonica extracts were carried out on both groups. Results: J. oxycedrus pollen extract was obtained, and it showed specific IgE binding and wide cross-reactivity with other Cupressaceae species. The extract caused a positive skin test response in all the patients tested, with about 80% of them having detectable specific IgE. Symptoms related to J. oxycedrus pollen exposure were recorded in 72% of the directly exposed patients and occasionally in 9% of the nonexposed patients. In the Mediterranean coastal area considered, J. oxycedrus was the first Cupressaceae species that started to pollinate at the beginning of November and ended in the first part of December. Conclusions: J. oxycedrus represents a newly characterized pollen species of the Cupressaceae family that cross-reacts with other members of the same family. Subjects with cypress allergy have in vivo and in vitro positive test responses for J. oxycedrus and can show symptoms when exposed to its pollen. Finally, the most important feature of J. oxycedrus is its early pollinating period in southern Europe (Italy), causing a further extension of the cypress pollen season in areas where other Cupressaceae species are present. (J Allergy Clin Immunol 1998;101:755-61.)

Exposure to indoor allergens can occur both at home and in public places such as schools and workplaces. To investigate and compare the presence of indoor allergens in different kind of environments (schools, offices and homes), dust samples were collected from furniture, desks, mattresses and floors with a standardized procedure. Samples were analyzed for Der p 1, Der f 1, Mite group 2 (mites) and Fel d 1(cat) by monoclonal antibody ELISA assay. Mite allergens were detected with low frequencies in schools and workplaces and with high frequency in homes. Fel d 1 was found with high frequency in every examined environment. Homes rather than public places can represent the environment where people can easier incur in mite allergy. All environments could be at risk for cat allergen exposure.

Summary Background The presence of specific IgE antibodies to invertebrates is common among patients with rhinitis and asthma. Tropomyosin has been described as an invertebrate cross-reactive allergen. We have recently characterized an allergenic extract from silverfish (Lepisma saccharina). Since this insect could be a new source of tropomyosin in the indoor environment, we have thought important to clone and characterize the tropomyosin from it. Methods Recombinant tropomyosin was cloned and characterized by means of immunoblotting with tropomyosin-specific monoclonal antibodies, rabbit polyclonal antibodies and IgE from allergic patients. Its allergenic activity was investigated in histamine release assays. Immunoblotting and ELISA inhibition were carried out to identify the natural tropomyosin in the silverfish extract and to study the cross-reactivity among other arthropod tropomyosins. Results Tropomyosin-specific antibodies recognized in immunoblotting the natural tropomyosin in the insoluble fraction of silverfish extract. The silverfish tropomyosin (Lep s 1) was cloned and fully expressed. It shared high homology with other arthropod tropomyosins. rLep s 1 was recognized by tropomyosin-specific monoclonal and polyclonal antibodies and by IgE of allergic patients. It was able to inhibit the IgE binding to the insoluble fraction of silverfish extract, and to induce histamine release by an arthropod-allergic serum. Inhibition experiments revealed IgE cross-reactivity between rLep s 1 and other arthropod tropomyosins. Conclusion rLep s 1 is the first allergen cloned and characterized from silverfish extract. It enabled us to identify the natural counterpart in the insoluble fraction of silverfish extract, suggesting that the tropomyosin is not readily extractable with a classic aqueous extraction procedure. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.

Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity.cDNA of Ole e 5 was amplified by nested 3'-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients' IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition).The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein.Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.

Background: Calcium-binding plant allergens can be grouped in different families according to the number of calciumbinding domains (EF hands). Objective: We sought to identify pollens containing crossreactive calcium-binding allergens and to investigate structural and immunologic similarities of members belonging to different families of calcium-binding allergens. Methods: By means of multiple sequence alignment and molecular modeling, we searched for structural similarities among pollen allergens with 2 (Phl p 7, timothy grass; Aln g 4, alder), 3 (Bet v 3, birch) and 4 EF hands (Jun o 4, prickly juniper). Purified recombinant Aln g 4 and Jun o 4 were used to determine the prevalence of IgE recognition in 210 patients sensitized to different pollens and to search, by means of ELISA competition, for the presence of cross-reactive epitopes in pollens from 16 unrelated plant species. IgE cross-reactivity among the allergen families was studied with purified rPhl p 7, rAln g 4, rBet v 3, and rJun o 4 and 2 synthetic peptides comprising the N-terminal and C-terminal EF hands of Phl p 7 by means of ELISA competition. Results: Structural similarities were found by using molecular modeling among the allergens with 2, 3, and 4 EF hands. Pollens from 16 unrelated plants contained Aln g 4- and Jun o 4-related epitopes. Twenty-two percent of the patients with multiple pollen sensitization reacted to at least one of the calcium-binding allergens. A hierarchy of IgE cross-reactivity (rPhl p7 > rAln g 4 > rJun o 4 > rBet v 3) could be established that identified rPhl p 7 as the EF-hand allergen containing most IgE epitopes in the population studied. Conclusion: The demonstration that members of different families of calcium-binding plant allergens share similarities suggests that it may be possible to use representative molecules for the diagnosis and therapy of allergies to EF-hand allergens. (J Allergy Clin Immunol 2002;109:314-20.)

Background: A rapid method for the purification of the major 43-kDa all_ergen 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 all_ergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and all_ergenic 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 all_ergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF3 (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF3/GnGXF3 (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF3/Gn(GF)XF3 (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. Conclusions: The rapid purification process of Cup a 1 all_owed 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.

Background: Cross-reacting carbohydrate determinants (CCDs) are antigenic structures shared by allergenic components from taxonomically distant sources. The case history of a patient with a great discrepancy between skin test and specific IgE results led us to investigate the role of these determinants in his specific case and in an allergic population. Objective: We sought to determine the role of CCDs in causing false-positive and clinically irrelevant results in in vitro tests. Methods: The involvement of CCDs was studied by specific IgE inhibition by using glycoproteins with a known carbohydrate structure. Direct and inhibition assays were performed by commercially available systems, in-house ELISA, and the immunoblotting technique. The binding to the periodate-oxidated carbohydrate structure of glycoproteins and allergenic extracts was also evaluated. A comparative study between skin test and specific IgE responses to the antigens studied was carried out in 428 consecutive allergic subjects. Results: All the tests performed suggested that cross-reacting carbohydrate epitopes were the cause of false-positive specific IgE results in one of the commercial systems and the high reactivity in all the solid-phase in vitro tests. None of the cross-reacting carbohydrate allergens yielded a positive skin test response. Periodate treatment caused variable degrees of reduction of IgE binding to the different antigens studied, indicating that CCDs played a different role in each of them. About 41% of patients allergic to pollen had specific IgE for a glycoprotein, without a positive skin test response to the same molecule. Conclusions: CCDs must be taken into account when evaluating the clinical relevance of positive results in in vitro specific IgE assays, at least in the diagnosis of patients with pollen allergy. Commercial systems should be carefully assessed for the ability to detect specific IgE for carbohydrate determinants to avoid false-positive or clinically irrelevant results. (J Allergy Clin Immunol 1999;103:1005-11.)

Species of the Cupressaceae family are a worldwide cause of respiratory allergies. We used monoclonal antibodies (mAbs) to investigate the presence and the nature of cross-reacting epitopes shared by various components within Cupressus arizonica pollen extract (CaE) or by CaE and pollen extract from C. sempervirens (CsE). mAbs were produced in mice immunized with whole CaE (4A6 and 5E6) or with the major allergen components (2D5). Their reactivity was investigated by ELISA and immunoblotting before and after CaE periodate treatment. Cross-reactivity was evaluated by ELISA inhibition and immunoblotting. mAbs 2D5 and 4A6 recognized periodate-resistant epitopes, whereas the mAb 5E6 reacted with a periodate-sensitive determinant. The former mAbs recognized epitopes present on CaE major allergen and also shared by other components. mAb 5E6 showed a spread reactivity on CaE, with exclusion of the major allergen. When the three mAbs were tested with CsE, a restricted pattern of reactivity to mAbs 2D5 and 4A6 was obtained, whereas mAb 5E6 maintained a spread reactivity. The CaE major allergen is represented by two components recognized by human IgE and sharing common epitopes, as proven by mAbs reactivity. The use of these mAbs demonstrates that cross-reactivity within CaE components and between CaE and CsE is due to the presence of periodate-sensitive as well as -resistant epitopes.

Background: Species belonging to the Cupressaceae family are a relevant source of allergens that are present in a wide number of countries. Objective: We sought to identify, purify, and characterize recombinant allergens from Juniperus oxycedrus , a species belonging to the Cupressaceae family. Methods: Double-stranded cDNA was synthesized from mRNA and cloned into the lambda-ZAP expression vector. IgE screening of the library was performed with a pool of sera from subjects allergic to Cupressaceae. A recombinant 6×His-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni 2+ affinity chromatography. It was studied further by immunoblotting inhibition with pollen extracts from other Cupressaceae, Oleaceae, Urticaceae, and Graminaceae. The role of protein-bound calcium on the allergen's IgE-binding capacity was tested in a plaque assay in the presence or absence of EGTA. Results: A cDNA coding for a newly identified Juniperus oxycedrus pollen allergen, rJun o 2, was isolated. The deduced amino acid sequence contained four typical Ca 2+ binding sites and showed a significant sequence similarity to calmodulins. Depletion of Ca 2+ in the plaque assay led to a loss of IgE-binding capacity of rJun o 2. Immunoblotting inhibition revealed that J. oxycedrus , J. ashei , Cupressus arizonica , C. sempervirens , Parietaria judaica , Olea europaea , and Lolium perenne pollen extracts were able to inhibit IgE binding to blotted rJun o 2 at different concentrations. Conclusion: rJun o 2 contains IgE-binding epitopes shared by taxonomically unrelated species, and therefore it can be regarded as a new panallergen. These findings could contribute to an explanation for the phenomenon of multiple positive test results in polysensitized patients and the potential symptom-eliciting role of allergenic sources previously not encountered. (J Allergy Clin Immunol 1998;101:772-7)

Exposure to indoor allergens is an important risk factor for sensitisation and respiratory allergy. The aim of this paper was to evaluate the levels of mite, cat and latex allergens in dust collected from an indoor workplace and to assess whether the exposure to these allergens was associated with the allergy symptoms reported by employees. Sixty dust samples were collected. Allergen concentrations were measured with antibody based ELISAs. All 144 participants compiled a questionnaire exploring possible symptoms of allergy. No association between latex allergen exposure and symptoms was found in spite of the high frequency of latex allergens. Mite allergens were detected in a minority of rooms. Cat allergen was the most important indoor allergen in the sampled workplace and exposure to this allergen could represent a risk for employees.