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Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70–90% coverage of the allergenic epitopes from mugwort pollen–allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free (15)N-labeled antigen whose (1)H-(15)N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3–specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

Oral allergy syndrome (OAS) is one of the most common IgE-mediated allergic reactions. It is characterized by a number of symptoms induced by the exposure of the oral and pharyngeal mucosa to allergenic proteins belonging to class 1 or to class 2 food allergens. OAS occurring when patients sensitized to pollens are exposed to some fresh plant foods has been called pollen food allergy syndrome (PFAS). In the wake of PFAS, several different associations of allergenic sources have been progressively proposed and called syndromes. Molecular allergology has shown that these associations are based on IgE co-recognition taking place between homologous allergens present in different allergenic sources. In addition, the molecular approach reveals that some allergens involved in OAS are also responsible for systemic reactions, as in the case of some food Bet v 1-related proteins, lipid transfer proteins and gibberellin regulated proteins. Therefore, in the presence of a convincing history of OAS, it becomes crucial to perform a patient’s tailored molecule-based diagnosis in order to identify the individual IgE sensitization profile. This information allows the prediction of possible cross-reactions with homologous molecules contained in other sources. In addition, it allows the assessment of the risk of developing more severe symptoms on the basis of the features of the allergenic proteins to which the patient is sensitized. In this context, we aimed to provide an overview of the features of relevant plant allergenic molecules and their involvement in the clinical onset of OAS. The value of a personalized molecule-based approach to OAS diagnosis is also analyzed and discussed.

Background: Laboratory Animal Allergy (LAA) has been considered a risk for the workers since 1989 by the NIOSH. About one third of the Laboratory Animal Workers (LAWs) can manifest symptoms to LAA as asthma, rhinitis, conjunctivitis and cutaneous reactions. The prevalence of LAA-induced clinical symptoms has been estimated with a great variability (4–44%) also due to the different methodologies applied. Objective: Evaluate the prevalence of IgE positivity to mouse and rat allergens in LAWs and assess which factors are predisposing to sensitization among subjects exposed to laboratory animals in the workplace. Methods: One hundred LAWs were invited to fill out a questionnaire regarding current allergic symptoms, atopic history, home environment, previous and current occupational history. IgE reactivity versus specific allergens was evaluated with ImmunoCAP ISAC. Results: Out of one hundred LAWs, 18% had a serum susceptibility to mouse and/or rat allergens and 42% reported to have occupational allergy symptoms. Combining the results acquired by ImmunoCAP ISAC and questionnaire, 17% of LAWs have been defined as LAWs-LAA positive since they present a positive IgE response and allergy symptoms, 1% LAWs-LAA sensitized, 25% LAWs-LAA symptomatic and 57% LAWs-LAA negative. Presence of previous allergy symptoms in work and life environment were significantly related to LAWs-LAA positive/sensitized. Conclusions: The study aimed to define the immunological profile of LAWs using the proteomic array as an innovative approach in the study of environmental and occupational exposure to allergens. We suggested a definition of LAWs-LAA considering serum IgE response and presence of allergy symptoms. The proposed approach has the advantage to provide a standard methodology for evaluating the specific IgE responsiveness to animal allergens in specific workplace also considering the immunological profile of workers referred to exposure in life and occupational environment.

Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensin–proline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.

Allergy to pomegranate is often associated with severe symptoms. Two allergens have previously been described: 9k-LTP Pun g 1 and pommaclein Pun g 7. This study describes the isolation of a chitinase III, identified by direct protein sequencing and mass spectrometry. It is a 29-kDa protein showing 69% sequence identity with the latex hevamine and IgE binding in dot blotting, immunoblotting and FABER(center dot) test. Chitinase-specific IgE were detected in 69 of 357 patients sensitized to one or more pomegranate allergenic preparations present on the FABER(center dot) test. Using this test, 19.2% of the patients sensitized to kiwifruit chitinase IV were also sensitized to pomegranate chitinase III, rather than to latex chitinase I (7.2%) with which it shares the N-terminal hevein-like domain. In conclusion, a new allergen has been identified, contributing to improving food allergy diagnosis. This study reveals the important role of chitinases III and IV as allergy sensitizers and prompts further investigations.

Background Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides. Objective We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense ). Methods The total pollen transcriptome, proteome, and allergome of JGP were documented. Serum IgE reactivities with pollen and purified allergens were assessed in 64 patients with grass pollen allergy from a subtropical region. Results Purified Sor h 1 and Sor h 13 were identified as clinically important allergen components of JGP with serum IgE reactivity in 49 (76%) and 28 (43.8%), respectively, of patients with grass pollen allergy. Within whole JGP, multiple cDNA transcripts and peptide spectra belonging to grass pollen allergen families 1, 2, 4, 7, 11, 12, 13, and 25 were identified. Pollen allergens restricted to subtropical grasses (groups 22-24) were also present within the JGP transcriptome and proteome. Mass spectrometry confirmed the IgE-reactive components of JGP included isoforms of Sor h 1, Sor h 2, Sor h 13, and Sor h 23. Conclusion Our integrated molecular approach revealed qualitative differences between the allergenic components of JGP and temperate grass pollens. Knowledge of these newly identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with grass pollen allergy in subtropical regions and reduce the burden of allergic respiratory disease globally.

Summary Background Bullous pemphigoid (BP) is an autoimmune skin disease in which patient autoantibodies react with BP180 and BP230 proteins. In addition to IgG, IgE has been shown to play a role in the disease. Objectives To evaluate the feasibility of detecting IgE and IgG against the immunodominant BP180 NC16A domain (BP180) using a microarray system. Methods BP180 was immobilized on an experimental version of the ISAC® microarray (Exp96). The BP study group and the controls were all tested on the commercial ISAC 103 version and on the Exp96. IgG and IgE were measured in a single run. BP180 IgG and IgE results were compared with those using an enzyme-linked immunosorbent assay (ELISA). Results All results obtained using the IgG ELISA on the 31 patients with BP were replicated with the ISAC IgG. Five of eight BP sera tested by ELISA showed similar results with ISAC IgE. Twenty-nine (94%) and 19 (61%) of the 31 patients with BP were IgG and IgE positive to BP180, respectively, whereas four (3%) and six (4%) of 138 normal donors were IgG and IgE positive, respectively. Interestingly, the levels of IgG against BP180 detected using the ISAC system were related to the disease severity. Patients with BP showed a peculiar profile of IgE recognition toward some groups of allergens, which was absent in a group of allergic individuals. A significant, higher prevalence of hen’s egg recognition was observed in patients with BP who had specific IgE to BP180. Conclusions The present preliminary study indicates that the ISAC microarray system is suitable for detecting IgG and IgE autoantibodies in patients with BP. Notably, this system allows the assessment of IgE and IgG autoantibodies at the same time, could be employed for the detection of autoantibodies to other autoantigens, and allows profiling for specific IgE to allergens.

IgE-mediated allergic diseases are among the most prevalent diseases worldwide. The use of extracts in the skin test and the additional use of IgE testing still represent the current basis for the diagnostic work-up. During the past 30 years, knowledge of the molecular structure of allergens has increased dramatically, and the characterization and production of allergenic molecules, as natural purified compounds or recombinant products, is allowing us to approach the allergy diagnostic work-up differently. Much of this is based on the adoption of microtechnology since the first release of a biochip for IgE detection. Its use has prompted the development of new concepts linked to the diagnosis of allergic diseases. This review describes the background of allergy diagnosis and the tools currently used for specific IgE detection. It gives insight into the most recent advancement in the field of biotechnology leading to allergenic molecule availability, microtechnology leading to the routine use of protein biochips for IgE detection, and how they should be combined with information technology.

Summary Background The availability of allergenic molecules and high-throughput microtechnologies allow the collection of a large number of IgE results at the same time in a single test. This can be carried out applying the test in the routine diagnostic work-up. Objective The aim of this study was to make a cross-sectional evaluation of the raw prevalence of IgE reactivity to allergenic molecules in serum samples from a cohort of Italian patients using an innovative tool. Methods The ISAC, a microarray system, has been used for specific IgE detection using 75 different allergenic molecules. Sera were collected from 23 077 unselected consecutive individuals complaining about any allergic disease. Results Sixteen thousand four hundred and eight of 23 077 patients had IgE to at least one of 75 allergenic molecules. The top-ranked molecules in this cohort were Cup a 1 (42.7%), Der f 2 (38.7%), and Phl p 1 (37.9%), whereas all the other allergens tested scored in a range between 36.8% and 0.04%, including the first food allergen, Pru p 3, ranked 15th (9.79%). Prevalence varied quite markedly depending on the age range considered, and showing a different behaviour in the lifetime sensitization process. Unsupervised two-way hierarchical clustering analysis generated distinctive patterns of reactivity as the result of IgE recognition of either homologous allergens belonging to different biological sources or non-homologous belonging to the same biological source. Conclusions Allergen-based microarray is a tool for the detection of IgE-related sensitization to panels of allergens and gives a more precise and comprehensive evaluation for an IgE-based epidemiology. This insight brings data for better understanding of the sensitization process.

To characterise occupational wheat allergic phenotypes (rhino-conjunctivitis, asthma and dermatitis) and immunoglobulin (IgE) sensitisation to particular wheat allergens in bakers.We conducted clinical and immunological evaluations of 81 consecutive bakers reporting occupational symptoms using commercial tests (skin prick test (SPT), specific IgE, ISAC microarray) and six additional dot-blotted wheat allergens (Tri a 39, Tri a Trx, Tri a GST, Tri a 32, Tri a 12, Tri a DH).Wheat SPT resulted positive in 29 bakers and was associated with work-related asthma (p0.01). Wheat IgE was detected in 51 workers and was associated with work-related asthma (p0.01) and rhino-conjunctivitis (p0.05). ISAC Tri a 30 was positive in three workers and was associated with work-related dermatitis (p0.05). Wheat dot-blotted allergens were positive in 22 bakers. Tri a 32 and Tri a GST were positive in 13 and three bakers, respectively, and both were associated with work-related dermatitis (p0.05). This association increased (p0.01) when Tri a 32, Tri a GST and Tri a 30 were analysed together (p0.01). Wheat IgE levels were associated with work-related dermatitis (p0.01).Wheat IgE levels and wheat microarrayed allergens may be associated with some occupational allergic phenotypes. The extension of the panel of wheat allergens may be promising for discriminating the clinical manifestations of baker's allergy.

Background From patients’ reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow’s milk (CM) allergic patients. Objective and Methods To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), β-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. Results The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly αS1-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. Conclusions 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected.

Summary Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a rare syndrome due to a mutation in the forkhead box protein 3 gene (FOXP3) leading to an impaired regulatory T cell (Treg) activity associated both with skewed T helper type 2 (Th2) response and autoreactive phenomena. The purpose of this study was to describe a combined proteomics and genomics approach to comprehensively evaluate clinical and immunological phenotypes of patients affected by IPEX. T cell receptor (TCR)-Vβ repertoire and peripheral blood lymphocytes phenotype from three brothers affected by IPEX were studied by flow cytometry. Specific immunoglobulin (Ig)E were evaluated by means of an allergenic molecules microarray [immuno solid-phase allergen chip (ISAC)]. Total RNA was extracted and hybridized to Affymetrix oligonucleotide arrays to obtain quantitative gene-expression levels. No FOXP3 protein was detectable within CD127-CD25highCD4+ T cells from peripheral blood. A T cell-naive phenotype (CD62L+CD45R0-) associated with a reduction of both CD26 and CD7 expression and a TCR-Vβ 8 and 22 family expansions were found. B lymphocytes were mainly CD5+ (B1) cells expressing a naive phenotype (tcl1+CD27-). The three IPEX patients had severe food allergy and specific IgE reactivity to cow's milk allergens, a hen's egg allergen and a wheat allergen. Gene expression profile analysis revealed a dysregulation associated mainly with Th1/Th2 pathways. The multiplexing evaluation reported in this study represents a comprehensive approach in the assessment of genetic conditions affecting the immune system such as the IPEX syndrome, paving the way for the development of diagnostic tools to improve the standard clinical and immunological profiling of the disease.

Methods Pulp and seeds of green kiwifruit were manually separated and homogenized. Act d 10 was purified from seed extracts by ion exchange chromatography and RPHPLC, and sequenced by an automatic amino acid sequencer. Act d 10 was evaluated in vivo by skin prick test (SPT) and in vitro by detecting IgE using streptavidin-conjugated CAP (Phadia, Sweden). Act d 10 was spotted on a customized ISAC microarray (PMD, Austria) for evaluating IgE prevalence in a larger cohort of allergic patients.

BackgroundFood allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.ObjectiveUsing the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.MethodsTwo new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.ResultsAlignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients' diet.ConclusionThe biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention.

BACKGROUND: Hen's egg allergy ranks among the most frequent primary food allergies in children. We aimed to investigate sensitization profiles of egg allergic patients and compare in vitro IgE reactivities of eggs from ancient chicken breeds (Araucana and Maran) with those from conventional laying hen hybrids. METHODOLOGY: Egg allergic children (n = 25) were subjected to skin prick test, double blind placebo controlled food challenge, and sensitization profiles to Gal d 1-5 were determined by allergen microarray. IgE binding and biological activity of eggs from different chicken breeds were investigated by immunoblot, ELISA, and mediator release assays. PRINCIPAL FINDINGS: We found that Gal d 1 and Gal d 2 are generally major egg allergens, whereas Gal d 3-5 displayed high sensitization prevalence only in patients reacting to both, egg white and yolk. It seems that the onset of egg allergy is mediated by egg white allergens expanding to yolk sensitization in later stages of disease. Of note, egg white/yolk weight ratios were reduced in eggs from Auraucana and Maran chicken. As determined in IgE immunoblots and mass analysis, eggs from ancient chicken breeds did not differ in their protein composition. Similar IgE-binding was observed for all egg white preparations, while an elevated allergenicity was detected in egg yolk from Araucana chicken. CONCLUSION/SIGNIFICANCE: Our results on allergenicity and biological activity do not confirm the common assumption that aboriginal food might be less allergenic. Comprehensive diagnosis of egg allergy should distinguish between reactivity to hen's egg white and yolk fractions to avoid unnecessary dietary restrictions to improve life quality of the allergic child and its family.

Background Nonspecific lipid transfer proteins (LTPs) represent potent pollen and food allergens. However, the allergenic properties of peanut LTP have not been studied. Objective To identify LTP in peanut extract using sera from subjects with peanut allergy and Pru p 3–sensitized subjects from Southern Europe, clone and express this protein, and obtain information on the importance as allergen for these selected patients. Methods Peanut LTP (Ara h 9) was cloned and sequenced by using a combination of bioinformatic and molecular biology tools (PCR, immunoblotting, Basic Local Alignment Search Tool [BLAST] searches). The immunologic properties of Ara h 9, Ara h 1, Ara h 2, and Ara h 3 were studied by using sera from subjects with peanut and peach allergy from Italy by immunoblotting and allergen microarray technology. Results Two Ara h 9 isoforms—Ara h 9.01 and Ara h 9.02—were cloned and expressed. Ara h 9 represented a minor allergen for subjects with peanut allergy. However, including Ara h 9 as single component for serologic detection of sensitization to peanut by component-resolved diagnosis seems crucial, because the frequency of sensitization to the classic major peanut allergens Ara h 1, Ara h 2, and Ara h 3 was low in these patients from Southern Europe. Conclusion Ara h 9 is a new member of the LTP allergen family that seems to play an important role in peanut allergy for patients from the Mediterranean area.

To cite this article: Ciardiello MA, Palazzo P, Bernardi ML, Carratore V, Giangrieco I, Longo V, Melis M, Tamburrini M, Zennaro D, Mari A, Colombo P. Biochemical, immunological and clinical characterization of a cross-reactive nonspecific lipid transfer protein 1 from mulberry. Allergy 2010; 65: 597–605. Abstract Background: Mulberry (Morus spp.) is a genus comprising several species of deciduous trees whose fruits are commonly eaten in southern Europe. Subjects with severe systemic reaction have been described. The aim of this study was to isolate the allergens of this species. Methods: A nonspecific lipid transfer protein 1 (ns-LTP1) was purified from black mulberry by ion exchange and reverse phase high-performance liquid chromatography, and the primary structure was elucidated by direct protein sequencing. Its allergenic activity was evaluated in vivo by skin prick test and in vitro by Western Blot, CD203c basophil activation assay and high throughput multiplex inhibition method on immunosolid-phase allergen chip (ISAC). Results: Mulberry ns-LTP (Mor n 3) comprises 91 amino acids producing a molecular mass of 9246 Da. This protein shows high sequence identity with several allergenic ns-LTP1. Immunoblot analysis and CD203c activation assay demonstrated its allergenic activity in symptomatic subjects and in ns-LTP allergic patients who are not mulberry consumers. Immunological co-recognition was studied in vivo on a selected group of well-characterized ns-LTP allergic patients showing a high percentage of nMor n 3+ subjects (88.46%) even in patients who have never eaten mulberry before. IgE inhibition on ISAC micro-array demonstrated an almost complete cross-reactivity to nArt v 3, rCor a 8 and a very high percentage of inhibition to nPru p 3. Conclusions: Mor n 3 is the first allergen isolated in black mulberry and immunologically characterized. It displayed allergenic activity among symptomatic and nonconsumer patients and a pattern of cross-reactivity to other plant-derived LTPs.

Background IgE recognition of panallergens having highly conserved sequence regions, structure, and function and shared by inhalant and food allergen sources is often observed. Methods We evaluated the IgE recognition profile of profilins (Bet v 2, Cyn d 12, Hel a 2, Hev b 8, Mer a 1, Ole e 2, Par j 3, Phl p 12, Pho d 2), PR-10 proteins (Aln g 1, Api g 1, Bet v 1.0101, Bet v 1.0401, Cor a 1, Dau c 1 and Mal d 1.0108) and tropomyosins (Ani s 3, Der p 10, Hel as 1, Pen i 1, Pen m 1, Per a 7) using the Immuno-Solid phase Allergen Chip (ISAC) microarray system. The three panallergen groups were well represented among the allergenic molecules immobilized on the ISAC. Moreover, they are distributed in several taxonomical allergenic sources, either close or distant, and have a route of exposure being either inhalation or ingestion. Results 3,113 individuals (49.9% female) were selected on the basis of their reactivity to profilins, PR-10 or tropomyosins. 1,521 (48.8%) patients were reactive to profilins (77.6% Mer a 1 IgE+), 1,420 (45.6%) to PR-10 (92.5% Bet v 1 IgE+) and 632 (20.3%) to tropomyosins (68% Der p 10 IgE+). A significant direct relationship between different representative molecules within each group of panallergens was found. 2,688 patients (86.4%) recognized only one out of the three distinct groups of molecules as confirmed also by hierarchical clustering analysis. Conclusions Unless exposed to most of the allergens in the same or related allergenic sources, a preferential IgE response to distinct panallergens has been recorded. Allergen microarray IgE testing increases our knowledge of the IgE immune response and related epidemiological features within and between homologous molecules better describing the patients' immunological phenotypes.

Identification, characterization, and purification of allergens are essential for the structural and immunologic studies needed to understand how these molecules induce specific IgE antibody production by the human immune system. Advances in molecular biology techniques have led to the production of recombinant allergens having constant properties, allowing detection of specific IgE directed against different molecular components of an allergenic source. Presence of homologous allergens in different sources is the reason for cross-reaction. Molecule-based diagnostic tools can lead to better interpretation of poly-sensitizations, observed by ST and in vitro tests using allergenic extracts as they were made before. Some examples IgE sensitization to major genuine allergens and panallergens will be presented.