Mediator Release Assay Research Articles

In Vitro Assessment of the Allergenicity of Novel H1N1 Influenza Vaccine Produced in Dog Kidney Cells in Subjects with Dog Allergy

A licensed inactivated seasonal influenza vaccine produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and did not trigger degranulation in rat basophilic leukemia (RBL) cells passively sensitized with human anti-dog IgE, supporting its safe use in dog-allergic individuals. The canine kidney cell-derived H1N1 pandemic vaccine, however, was adjuvanted with the emulsion adjuvant MF59®, and support for its similar safe use was sought. We evaluated allergenicity of the adjuvanted canine kidney cell-derived H1N1 vaccine in subjects with dog allergy, with utilization of an in vitro mediator release assay.

In vitro diagnosis of IgE-mediated allergy: breakthroughs in the last decade

Background This article briefly reviews the historical development, present status and perspectives of the diagnosis of IgE-mediated allergy. After more than a century of relying on skin testing and 40 years of relying on quantification of specific IgE antibodies for the diagnosis of IgE-mediated allergy, clinicians now have access to different novel in vitro diagnostics in the form of component-based assays and flow-assisted quantification of activated basophils. Although these techniques already constitute key advances in the diagnosis of allergy, it is anticipated that future developments, such as epitope mapping and identification of novel basophil activation markers, might further improve management of the allergic patient. In this context, the recent development of a flow cytometric technique to analyze histamine release at a single cell level might represent a significant step forward. IgE-mediated allergies constitute an important and increasing health problem with significant morbidity and potential mortality. Correct diagnosis with identification of all offending allergens is an absolute prerequisite for appropriate and potentially life-saving preventive measures or the initiation of allergen-specific immunotherapy. The diagnostic algorithm for IgE-mediated allergy involves a thorough clinical history that is documented by the demonstration of allergen-specific IgE (specific IgE) antibody by either in vivo skin testing (prick/intradermal) or a serologic assay. Although these tests can provide useful information, they do not demonstrate an absolute predictive value and the results are not always unequivocal. In these cases, where there exists a discrepancy between history and results from quantification of sIgE and/or skin tests, additional tests such as mediator release assays or (double-blind placebo-controlled) challenge tests have long been used to establish correct diagnosis. During the last decade two novel in vitro assays have entered the allergy diagnostics arsenal, that is, component-resolved allergy diagnosis (CRD) and the basophil activation test by flow cytometry.

A modified RBL-2H3 mediator release assay for the detection of polyclonal IgE antibody

RBL-2H3 mediator release assay, developed for specific IgE screening studies, was not as sensitive as passive cutaneous anaphylaxis (PCA) assay in the polyclonal antibody detection. In the present investigation, the detection sensitivity of RBL-2H3 assay was elevated by modifying the experiment protocols from choosing the proper releasing medium and optimizing the sensitization manner. The polyclonal antibody was generated from Brown Norway (BN) rats exposed to Ovalbumin (OVA). In contrast to Tyrode buffer A, RBL-2H3 cells cultured in DMEM had a lower spontaneous secretion and a higher response to antigen stimulation, both of which could help to increase the detection sensitivity. The rat sera used in the sensitization process should be diluted appropriately to avoid the proliferation-promoting effect on RBL-2H3 cells. The results of the kinetics of sensitization showed that prolonging the sensitization time and then reculturing the cells in IgE free medium for a further 24 h after the removal of rat sera could reach a marked increase in the degree of sensitization. The highest anti-OVA antibody titer detected by the modified RBL-2H3 assay was 4096, while PCA assay was 1024. These data provide evidence that the modified RBL-2H3 mediator release assay has a promising prospect in the determination of the biologic activity of polyclonal antibody.

Close-up of the Immunogenic α1,3-Galactose Epitope as Defined by a Monoclonal Chimeric Immunoglobulin E and Human Serum Using Saturation Transfer Difference (STD) NMR

Anaphylaxis mediated by carbohydrate structures is a controversially discussed phenomenon. Nevertheless, IgE with specificity for the xenotransplantation antigen α1,3-Gal (α-Gal) are associated with a delayed type of anaphylaxis, providing evidence for the clinical relevance of carbohydrate epitopes in allergy. The aim of this study was to dissect immunoreactivity, interaction, and fine epitope of α-Gal-specific antibodies to obtain insights into the recognition of carbohydrate epitopes by IgE antibodies and their consequences on a molecular and cellular level. The antigen binding moiety of an α-Gal-specific murine IgM antibody was employed to construct chimeric IgE and IgG antibodies. Reactivity and specificity of the resulting antibodies were assessed by means of ELISA and receptor binding studies. Using defined carbohydrates, interaction of the IgE and human serum was assessed by mediator release assays, surface plasmon resonance (SPR), and saturation transfer difference NMR analyses. The α-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference antibody, anti-Gal IgE did not induce mediator release, potentially reflecting the delayed type of anaphylaxis. The α1,3-Gal epitope fine structures of both the recombinant IgE and affinity-purified serum were defined by saturation transfer difference NMR, revealing similar contributions of carbohydrate residues and participation of both galactose residues in interaction. The antibodies generated here constitute the principle underlying α1,3-Gal-mediated anaphylaxis. The complementary data of affinity and fine specificity may help to elucidate the recognition of carbohydrates by the adaptive immune response and the molecular requirements of carbohydrate-based anaphylaxis.

Bet v 1‐like pollen allergens of multiple Fagales species can sensitize atopic individuals

In the temperate climate zone of the Northern hemisphere, Fagales pollen allergy represents the main cause of winter/spring pollinosis. Among Fagales trees, pollen allergies are strongly associated within the Betulaceae and the Fagaceae families. It is widely accepted that Fagales pollen allergies are initiated by sensitization against Bet v 1, the birch pollen major allergen, although evidence is accumulating that the allergenic activity of some Bet v 1-like molecules has been underestimated. To investigate the allergenic potential of the clinically most important Fagales pollen allergens from birch, alder, hazel, hornbeam, hop-hornbeam, oak, beech and chestnut. To obtain the full spectrum of allergens, the three previously unavailable members of the Bet v 1-family, hop-hornbeam Ost c 1, chestnut Cas s 1 and beech Fag s 1, were identified in the respective pollen extracts, cloned and produced as recombinant proteins in E. coli. Together with recombinant Bet v 1, Aln g 1, Car b 1, Cor a 1 and Que a 1, the molecules were characterized physicochemically, mediator release assays were performed and IgE cross-reactivity was evaluated by ELISA and Immuno Solid-phase Allergen Chip (ISAC) IgE inhibition assays. All allergens showed the typical Bet v 1-like secondary structure elements, and they were all able to bind serum IgE from Fagales allergic donors. Strong IgE binding was observed for Betuloideae and Coryloideae allergens, however, cross-reactivity between the two subfamilies was limited as explored by inhibition experiments. In contrast, IgE binding to members of the Fagaceae could be strongly inhibited by serum pre-incubation with allergens of the Betuloideae subfamily. The data suggest that Bet v 1-like allergens of the Betuloideae and Coryloideae subfamily might have the potential to induce IgE antibodies with different specificities, while allergic reactions towards Fagaceae allergens are the result of IgE cross-reactivity.

Is aboriginal food less allergenic? A study on the IgE-binding capacity of egg white and yolk from modern and ancient chicken breeds investigated in a cohort of hen’s egg allergic children

Results In our cohort, 48% of patients were sensitized to egg white and yolk, while 52% were reacting to egg white exclusively. In allergen microarray, Gal d 1 and 2 were identified as major allergens for all patients, whereas Gal d 3-5 displayed high sensitization prevalence only in patients reacting to both egg components. Mean egg white-specific IgE was significantly higher in patients displaying additional sensitization to yolk compared to yolk-negative individuals (6.31 μg/ml and 1.53 μg/ml, respectively). Eggs from ancient chicken breeds demonstrated reduced egg white/yolk ratios compared to those from conventional laying he nh ybrids but did not differ in their allergen composition as determined by mass spectrometry. Accordingly, we observed no significant differences in IgE-binding and basophil mediator release assays comparing egg white and yolk from different chicken breeds. Conclusions The onset of egg allergy seems to be mediated by egg white allergens expanding to yolk sensitization in later stages of the disease. Notably, our results on allergenicity and biological activity do not confirm the common assumption that aboriginal food might be less allergenic.

Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen

Allergen-specific immunotherapy for food allergies, including peach allergy, has not been established. Use of allergens with reduced allergenic potential and preserved immunogenicity could improve the safety and efficacy of allergen-specific immunotherapy. We sought to create a hypoallergenic derivative of the major peach allergen Pru p 3 and to characterize its biochemical and immunologic properties. A Pru p 3 folding variant generated by means of reduction and alkylation was investigated for structural integrity and stability to gastrointestinal enzymes. IgE reactivity and allergenic potency were determined by means of immunoblotting, ELISA, and in vitro mediator release assay with sera from patients with peach allergy. T-cell immunogenicity was investigated by using human allergen-specific T cells and CBA/J mice immunized with either native Pru p 3 (nPru p 3) or reduced and alkylated (R/A) Pru p 3. Pru p 3 processing by endolysosomal fractions of dendritic cells and antigenicity was examined in mice. Unfolding of Pru p 3 reduced its high resistance to gastrointestinal proteolysis and almost completely abrogated its IgE reactivity and allergenic potency. However, R/A Pru p 3 was capable of stimulating human and murine T cells. Endolysosomal degradation of R/A Pru p 3 was accelerated in comparison with nPru p 3, but similar peptides were generated. IgG and IgE antibodies raised against nPru p 3 showed almost no cross-reactivity with R/A Pru p 3. Moreover, the antigenicity of R/A Pru p 3 was strongly reduced. Unfolded Pru p 3 showed reduced allergenicity and antigenicity and preserved T-cell immunogenicity. The hypoallergenic variant of Pru p 3 could be a promising vaccine candidate for specific immunotherapy of peach allergy.

Evaluation of two new recombinant guinea‐pig lipocalins, Cav p 2 and Cav p 3, in the diagnosis of guinea‐pig allergy

Guinea-pigs, classical laboratory animals now often kept as pets, regularly elicit important allergic reactions. Two guinea-pig allergens have been described previously to some extent; however, biomolecular and immunological data are lacking. To identify major guinea-pig allergens, produce them as recombinant proteins and define their allergenic potential and clinical importance in allergic patients. Protein extracts were prepared from various guinea-pig tissues and allergens were purified by ion exchange chromatography. The N-termini of two immunoglobulin E (IgE)-reactive proteins were determined and degenerate primers were designed for cDNA amplification by RT-PCR. Allergenic properties of recombinant proteins were assayed by immunoblotting, ELISA and mediator release assays. Specific IgE were quantified in the sera of 26 guinea-pig-allergic patients. Major IgE-reactive proteins were detected in submaxillary and harderian gland extracts. Two proteins were identified and the cDNAs were cloned. The 17 kDa protein expressed in the harderian gland corresponds to the previously described Cav p 2. The 19 kDa protein, Cav p 3, is a new allergen expressed in the submaxillary gland. Recombinant Cav p 2 and Cav p 3 were recognized by IgE antibodies from 65% and 54% of guinea-pig-allergic patients, respectively. Both proteins demonstrated equivalent allergenic activity in the mediator release assays. Two major guinea-pig allergens, Cav p 2 and Cav p 3, have been characterized and produced as recombinant proteins. Combined to guinea-pig serum albumin, the new allergens proved to be valuable in the component-resolved diagnosis of guinea-pig allergy.

Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a

The scarcity of monoclonal human IgE antibodies with specificity for defined allergens is a bottleneck for the molecular characterisation of allergens and their epitopes. Insights into the characteristics of such antibodies may allow for analyses of the molecular basis underlying allergenicity and cross-reactivity, standardisation of allergens as well as improvement of allergy diagnostics and therapeutics. Here we report the generation and application of the first set of authentic human IgG, IgE and IgA antibodies. On the basis of a Phl p 5a specific antibody fragment, a lambda light chain and the IgG1, IgG4, IgE, IgA1, and IgA2 heavy chains, the corresponding human immunoglobulins were constructed and produced in mammalian cells. In parallel, a murine hybridoma line with specificity for Phl p 5a was established, recloned and produced as human chimeric IgE. After purification, immunoreactivity of the antibodies with the allergen was assessed. Applicability in allergy diagnostics was confirmed by establishment of artificial human sera. Functionality of both antibodies was further demonstrated in receptor binding studies and mediator release assays using humanised rat basophil leukaemia cells (RBL-SX38) suggesting the presence of spatially separate epitopes. By using Phl p 5 fusion proteins and recombinant IgE in immunoblotting and mediator release assays we assigned the epitope of the authentic IgE to a looped stretch exclusively present in Phl p 5a. In summary, the Phl p 5-specific antibodies are the first full set of allergy-related antibody isotypes of their kind and represent valuable tools for studies of fundamental mechanisms and structure/function relationships in allergy.

Molecular characterization of Api g 2, a novel allergenic member of the lipid‐transfer protein 1 family from celery stalks

Celery represents a relevant cross-reactive food allergen source in the adult population. As the currently known allergens are not typical elicitors of severe symptoms, we aimed to identify and characterize a non-specific lipid transfer protein (nsLTP). MS and cDNA cloning were applied to obtain the full-length sequence of a novel allergenic nsLTP from celery stalks. The purified natural molecule consisted of a single isoallergen designated as Api g 2.0101, which was recombinantly produced in Escherichia coli Rosetta-gami. The natural and recombinant molecules displayed equivalent physicochemical and immunological properties. Circular dichroism revealed a typical α-helical fold and high thermal stability. Moreover, Api g 2 was highly resistant to simulated gastrointestinal digestion. As assessed by ELISA, thermal denaturation did not affect the IgE binding of Api g 2. Natural and recombinant Api g 2 showed similar allergenic activity in mediator release assays. Api g 2-specific IgE antibodies cross-reacted with peach and mugwort pollen nsLTPs. Based on our results, it can be anticipated that inclusion of recombinant Api g 2 in the current panel of allergens for molecule-based diagnosis will facilitate the evaluation of the clinical relevance of nsLTP sensitization in celery allergy and help clinicians in the management of food allergic patients.

Diclofenac Hypersensitivity: Antibody Responses to the Parent Drug and Relevant Metabolites

BackgroundHypersensitivity reactions against nonsteroidal antiinflammatory drugs (NSAIDs) like diclofenac (DF) can manifest as Type I-like allergic reactions including systemic anaphylaxis. However, except for isolated case studies experimental evidence for an IgE-mediated pathomechanism of DF hypersensitivity is lacking. In this study we aimed to investigate the possible involvement of drug- and/or metabolite-specific antibodies in selective DF hypersensitivity.Methodology/Principal FindingsDF, an organochemically synthesized linkage variant, and five major Phase I metabolites were covalently coupled to carrier proteins. Drug conjugates were analyzed for coupling degree and capacity to crosslink receptor-bound IgE antibodies from drug-sensitized mice. With these conjugates, the presence of hapten-specific IgE antibodies was investigated in patients' samples by ELISA, mediator release assay, and basophil activation test. Production of sulfidoleukotrienes by drug conjugates was determined in PBMCs from DF-hypersensitive patients. All conjugates were shown to carry more than two haptens per carrier molecule. Immunization of mice with drug conjugates induced drug-specific IgE antibodies capable of triggering mediator release. Therefore, the conjugates are suitable tools for detection of drug-specific antibodies and for determination of their anaphylactic activity. Fifty-nine patients were enrolled and categorized as hypersensitive either selectively to DF or to multiple NSAIDs. In none of the patients' samples evidence for drug/metabolite-specific IgE in serum or bound to allergic effector cells was found. In contrast, a small group of patients (8/59, 14%) displayed drug/metabolite-specific IgG.Conclusions/SignificanceWe found no evidence for an IgE-mediated effector mechanism based on haptenation of protein carriers in DF-hypersensitive patients. Furthermore, a potential involvement of the most relevant metabolites in DF hypersensitivity reactions could be excluded.

In vitro assessment of the allergenicity of a novel influenza vaccine produced in dog kidney cells in individuals with dog allergy

An inactivated influenza vaccine produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used to immunize egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. To evaluate the allergenicity of the inactivated influenza vaccine produced in cell culture in a mediator release assay. Rat basophil leukemia (RBL) cells transfected with human IgE receptor-1 were sensitized with sera from dog-allergic adults with positive skin prick test reactions to dog extract and detectable dog dander IgE and were stimulated with serial dilutions of vaccine and dog dander extract. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation. Western blots were performed, and UniCAP was used to measure dog-specific IgE antibody levels. The median (interquartile range) level of dog dander IgE was 8.31 kU(A)/L (1.895-14.5 kU(A)/L) and of dog epithelium IgE was 3.19 kU(A)/L (0.835-6.27 kU(A)L). Median (range) maximum NHR (at the first 10-fold dilution) was 0% (0%-1.4%) to vaccine and 10.2% (0%-35.9%) to dog dander (P < .001). In an egg-allergic control subject, the maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to vaccine on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from an egg-allergic control subject strongly bound embryonated egg-derived vaccine. An influenza vaccine produced in continuous canine kidney cells did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE.

Rat Basophil Leukemia (RBL) cell-based Mediator Release Assay to Egg White in Children with Egg Allergy

Rat Basophil Leukemia (RBL) cell-based Mediator Release Assay to Egg White in Children with Egg Allergy

Targeting the cysteine-stabilized fold of Art v 1 for immunotherapy of Artemisia pollen allergy

Plants of the genus Artemisia domestic in Northern and Central Europe, USA and parts of Asia are a major cause of allergic symptoms from late summer to autumn. Art v 1, the major mugwort pollen allergen appears as two-domain glycoprotein, consisting of an N-terminal defensin-like and a proline/hydroxyproline-rich C-terminal part. Patients sensitized to Art v 1 commonly display IgE antibodies against the cysteine-stabilized defensin fold. Site-directed mutagenesis of eight cysteines was used to disrupt disulfide bonds to generate molecules with altered IgE-binding capacity. Engineered constructs were expressed in E. coli and recombinant proteins were tested for their allergenic and T cell reactivity as well as for their physicochemical characteristics. Three cysteine variants (C22S, C47S, and C49S) exhibited extremely low IgE-binding activity in immunoblot and ELISA using sera from Art v 1-allergic patients. Mediator release assays using rat basophil leukemia cells showed that these variants displayed a 1×105-fold reduced allergenic potency as compared to wild-type protein. All variants were able to activate allergen-specific T cells in PBMC, as well as Art v 1-specific T cell lines and clones. Variant C49S displayed an increased hydrophobic surface potential which correlated with an advanced activation of allergen-specific T cells. The low allergenicity and high immunogenic activity of Art v 1 variant C49S renders the molecule an attractive candidate for hypoallergen-based immunotherapy of Artemisia pollen allergy.

Relevance of IgE binding to short peptides for the allergenic activity of food allergens

Analysis of IgE antibody binding to epitopes provides information for food allergy diagnosis and management and construction of hypoallergenic candidate vaccines, but the contribution of sequential epitopes to functionally relevant IgE binding is not fully understood. We sought to study the impact of IgE-binding peptides described as major sequence epitopes in the literature on IgE-binding capacity of 2 selected food allergens. IgE-binding peptides of the food allergens Ara h 2 (peanut) and Pen a 1 (shrimp) were identified. Synthetic soluble peptides representing the identified sequences were assessed for their capacity to inhibit IgE binding to the parent allergens by means of ELISA and in mediator release assay. The IgE-binding capacity of unfolded recombinant (r) Ara h 2 was analyzed. A hybrid tropomyosin carrying the IgE-binding regions of Pen a 1 grafted into the structural context of the nonallergenic mouse tropomyosin was applied in ELISA inhibition experiments and ImmunoCAP analysis. Although IgE-binding peptides representing sections of the allergen sequences were detected, no relevant capacity to inhibit the IgE binding to the parent allergen in ELISA or basophil activation test was observed. Unfolded rAra h 2 showed reduced IgE-binding capacity compared with folded rAra h 2 and failed to elicit mediator release. Hybrid tropomyosin bound less IgE than rPen a 1 in ImmunoCAP analysis and revealed marginal inhibitory capacity. Peptides identified as major sequence epitopes on Pen a 1 and Ara h 2 show little contribution to the IgE binding of the allergens studied.

Mediator release assay for assessment of biological potency of German cockroach allergen extracts

Cockroach is an important allergen in inner-city asthma. The diagnosis and treatment of cockroach allergy has been impeded by the lack of standardized cockroach extracts. We investigated the utility of a mediator release assay based on rat basophil leukemia (RBL) cells for comparing the potency of German cockroach extracts. RBL cells (line 2H3) transfected with human FcepsilonRI were passively sensitized with sera from subjects with cockroach allergy and stimulated with serial dilutions of 3 commercial cockroach extracts (1:10 weight/volume). In addition, the in-house prepared extract was tested in separate experiments with pooled sera that produced optimal performance in the RBL assay. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation and was examined in relation to the intradermal skin test (ID(50)EAL) and serum cockroach-specific and total IgE levels. The median cockroach-specific IgE concentration in 60 subjects was 0.72 kU(A)/L (interquartile range, 0.35-2.97 kU(A)/L); 19 sera (responders) produced a minimum 10% NHR to more than 1 extract. Responders had higher median cockroach-specific IgE (7.4 vs 1.0 kU(A)/L) and total IgE (429 vs 300 kU/L) levels than nonresponders. Ranking of extract potency was consistent between the mediator release assay and the ID(50)EAL. For the in-house prepared cockroach extract, the dose-response curves were shifted according to the concentration of the extract. NHR was reproducible between different experiments by using pooled sera. The mediator release assay measures biologic potency and correlates with the ID(50)EAL. It should be further evaluated to determine whether it could be used to replace intradermal skin test titration for assessing the potency of cockroach extract.

Immunologic characterization of isoforms of Car b 1 and Que a 1, the major hornbeam and oak pollen allergens

Birch pollen allergy is one of the most common causes of spring pollinosis often associated with hypersensitivity reactions to pollen of other Fagales species. Yet, only the major disease eliciting allergens of alder and hazel have been fully characterized. Therefore, the aim of this study was to perform cloning, expression and immunologic characterization of the Bet v 1 homologues from oak (Que a 1) and hornbeam (Car b 1). The isoform pattern of Car b 1 and Que a 1 was analyzed by proteomics using 2D gel electrophoresis and LC ESI-QTOF MS. Isoallergens showing high IgE-binding were cloned and expressed in Escherichia coli. IgE-binding activity of the recombinant proteins was determined by enzyme-linked immunosorbent assay (ELISA) and basophil mediator release assays using serum samples from patients mainly exposed either to oak and hornbeam or to birch pollen. Cross-reactivity of the allergens was further investigated at the T-cell level. Dominant isoforms of Car b 1 and Que a 1, identified by mass spectrometry, showed different IgE-binding properties when testing Fagales pollen-allergic patients living in birch-free areas as compared to birch-sensitized individuals. Tree pollen-allergic patients who are primarily exposed to Fagales pollen other than birch reacted stronger with rCar b 1 and rQue a 1 than with rBet v 1, as determined by inhibition ELISA and basophil mediator release assays. Thus, rCar b 1 and rQue a 1 allergens should be considered for improving molecule-based diagnosis and therapy of tree pollen allergies manifesting in birch-free areas.

Immune recognition of novel isoforms and domains of the mugwort pollen major allergen Art v 1

Allergen isoforms can differ in their IgE and T cell recognition patterns, and thus might have an impact on the selection of candidates for molecule-based diagnostic and therapeutic approaches. The present study aimed at the identification and characterization of isoforms of Art v 1, the mugwort pollen major allergen. In addition, single Art v 1 domains were physicochemically and immunologically characterized. For this purpose, the Art v 1 cDNA was radiolabeled and used to screen a mugwort pollen cDNA library. Positive clones were sequenced and used for the production of recombinant proteins in Escherichia coli using the pHIS-Parallel2 vector. Protein purification was performed by affinity- and ion exchange chromatography. Antibody binding to the recombinant proteins was determined by immunoblot, ELISA, cross-inhibition experiments, and mediator release assays. We could identify 7 Art v 1 isoforms differing in 1–6 amino acid residues. Interestingly, all amino acid variations were restricted to the proline domain carrying the molecule's post-translational modifications. No significant difference in IgG or IgE reactivity could be observed between Art v 1 isoforms and the defensin domain produced in E. coli. When expressed in E. coli, the proline domain was not recognized by Art v 1-specific antibodies. Our results demonstrated that the relevant IgE epitopes of Art v 1 are located on the defensin domain and suggest the involvement of carbohydrates in the allergenicity of natural Art v 1. Plant-based expression systems could help to reveal possibly different glycosylation patterns and IgE binding properties of Art v 1 isoforms. These findings have direct implications on the development of novel tools for mugwort pollen allergy diagnosis and therapy.

In-vitro-Testverfahren bei Analgetikaintoleranz

Acute intolerance reactions to aspirin or nonsteroidal antiinflammatory drugs are referred to as aspirin-exacerbated respiratory disease (AERD), a nonallergic hypersensitivity reaction with potentially anaphylactoid character. AERD is essentially based on a continuous and progressive subclinical airway inflammation, due to an altered arachidonic acid metabolism. Intake of COX-1 inhibitors induces an excessive production of leukotrienes due to an insufficient and dysbalanced production of prostaglandin E 2 . Clinically, the resulting reactions comprise attacks of rhinitis and asthma. Todays gold standard for confirming the diagnosis of AERD is provocation testing with COX-1 inhibitors. However, due to the potentially severe complications resulting from provocation testing, it should only be performed in centers with suitable emergency facilities. Based on the known pathophysiological mechanisms of the disease during the last decade, many attempts have been made to establish a reliable in vitro testing approach to substitute provocation testing. Current assays can be classified according to the underlying methodology: mediator release assays, tests based on flow cytometry, or combinations of these methods. Accordingly, the present article reviews existing mediator release assays: the cellular antigen stimulation test (CAST), the Analgetikaintoleranztest (AIT), and the aspirin-sensitive patients identification test (ASPItest) as well as tests based on flow-cytometric quantification of CD63 expression (CAST-Flow, Basotest), and the hybrid test system CAST-Combi. Based on the most recent data concerning their sensitivity and specificity, the review provides an overview on the current status of this challenging field of in vitro diagnostic testing.

Molecular characterisation of Lac s 1, the major allergen from lettuce ( Lactuca sativa)

Background IgE sensitisation to non-specific lipid transfer proteins (nsLTP), e.g., Pru p 3 the major allergen from peach and most important allergenic LTP, is strongly associated with severe symptoms in food allergic patients. Lac s 1, a member of the nsLTP protein family, was recently identified as major allergen in lettuce ( Lactuca sativa), but has not yet been investigated on the molecular basis. Objective Molecular characterisation and immunological comparison of Lac s 1 to peach allergen Pru p 3. Methods Lac s 1 cDNA was cloned by RT-PCR and natural (n) Lac s 1 was purified by a two-step chromatography. Protein structure was verified by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy. Immunoblotting, ImmunoCAP™, and competitive IgE binding experiments were performed to study the IgE sensitisation pattern and cross-reactivity with Pru p 3. Allergenic potency was analysed by histamine release assay. Results Twenty-nine lettuce allergic patients, with or without concomitant peach allergy, and 19 peach allergic patients without lettuce allergy were included in this study. IgE reactivity to lettuce was due to mono-sensitisation to Lac s 1 or cross-reactive glycan structures. Two Lac s 1 isoforms were identified which showed amino acid identitiy (aa-id) of 62% to each other, up to 66% to Pru p 3, and 72% to the N-terminal peptide of plane pollen LTP Pla a 3. The prevalence of IgE binding to nLac s 1 was 90% using lettuce extract in immunoblotting experiments. Enhanced sensitivity was observed in ImmunoCAP™ using purified nLac s 1 in comparison to extracts (93% versus 76%). Although IgE sensitisation to Lac s 1 and Pru p 3 was strongly associated, the two LTPs showed different IgE binding properties. Sensitisation to LTPs does not necessarily reflect the clinical disease, but Lac s 1 was capable of triggering histamine release as shown by positive skin test results in Lac s 1 mono-sensitised patients and by in vitro mediator release assays. Conclusion Purified nLac s 1 will enhance the sensitivity in component resolved diagnosis of lettuce allergy. Similar to other cross-reactive food allergies, exclusive testing of IgE reactivities to LTP cannot be used as biomarker for clinical relevance. Our data provide indirect evidence that Pru p 3 might act as the primary sensitising agent in patients allergic to both lettuce and peach.