Component-resolved Allergy Diagnosis Research Articles

Food allergies and acute allergic reactions: diagnostic options

Introduction. Anaphylaxis is a systemic potentially life-threatening hypersensitivity reaction featuring the rapid onset, manifestations of the respiratory tract and/or hemodynamics and usually, but not always, accompanied by changes in the skin and mucous membranes [1]. The cause of developing an acute generalized reaction in food anaphylaxis may be the minimum culprit product amount that has entered the patient's body through the mouth or through the skin/mucous membranes. Patients like these are, as a rule, first seen by a paediatrician, a related specialist, or a paramedic. Since the doctors often consider the symptoms of foodborne anaphylaxis separately, the latter does not cause proper suspicion: angioedema and/or acute urticaria accompanying rhinorrhea, acute bronchospasm or laryngitis developed extremely quickly or immediately after contact with an allergen.The aim of the work was to acquaint paediatricians and doctors of related specialties with the food anaphylaxis symptoms, to justify the need for an allergologist-immunologist to consult these patients in order to resolve the issue of required component-resolved diagnosis of allergy and development of an individual elimination diet, taking into account the patient's sensitization profile.Materials and methods. Clinical observation of a child (boy, age — 12 months) with symptoms of atopic dermatitis and episodes of food anaphylaxis in the anamnesis; general clinical examination and allergological examination of the patient by component allergodiagnostics were performed.Results. Sensitization to ovalbumin (Gald 2 -16.60 kU/l) and ovomucoid (Gald 1-9.01 kU/l) was established. The detected sensitization is a predictor of severe systemic allergic reactions to eggs.Discussion. Component diagnostics performed as prescribed by an allergologist not only confirms the causative allergen, but also evaluates the risks of developing acute reactions if the allergen is accidentally introduced to the child and the timing of the patient's tolerance formation. This sensitization will cause sudden systemic allergic reactions for years to come.Conclusion. It is important for a specialist to timely suspect food anaphylaxis, differentiate it from other emergency and urgent conditions, and give the patient the correct recommendations on the need for consultation with an allergologist-immunologist. Only a thoughtful, comprehensive medical approach to each acute condition in a child will significantly reduce the risk of repeated episodes of food anaphylaxis.

HYMENOPTERA TOXINS: BIOLOGICAL ACTIVITY, PHARMACEUTICAL AND THERAPEUTIC USES

The present review article explains the salient features of hornet venom toxins, their physiological, biological and pharmacological effect on animals and man. Hornets sting very fast and inflict venom, which is more dangerous than those of bees. Hornet venom contains both proteinaceous and non-proteinaceous peptides i.e. scapin, adolapin, mellitin, mastoparans and enzymes, mainly phospholipase and hyaluronidase, which show multiple biological effects i.e. cytolytic, hemotoxic, neuro-inhibitor, anticancer, anti-parasitic, immune hypersensitive, inflammatory, antimicrobial and anti-insect activities. Hornet stings are more painful to humans than typical wasp stings because hornet venom contains a large amount (5%) of acetylcholine. Hornet toxin components interact with receptors, ion channels and gated channels and affect the permeability functions of cells. Heavy envenomation shows quick pathophysiological lethal effects in man and pet. This article emphasizes the use of various hornet venom components for the production of disease-modifying anti-rheumatic and analgesic, anticancer drugs and insecticides. Hornet venom allergens could be used to prepare the rational design of component-resolved diagnosis of allergy and venom immunotherapy of inflicting patients.

Оральный аллергический синдром и ангиоотеки в ротовой полости у пациентов с сенсибилизацией к березе: есть ли повышенные риски?

Aim: to study the sensitization profile in patients with sensitivity to Bet v1 allergen who have clinically significant respiratory symptoms of pollinosis during the birch flowering season, oral allergy syndrome (OAS), and angioedema to nuts and stone fruits. Patients and Methods: a prospective open-label study included 40 children (average age 7.52±0.64), 26 (65%) boys and 14 (35%) girls. All children had sensitization to the birch major allergen (Bet v1), symptoms of respiratory allergy during the birch flowering season, manifestations of OAS with angioedema in combination with hives or without it after eating fruits of Rosaceae family/nuts/peanuts. Children underwent general clinical and allergy researches, determination of total IgE and eosinophil cationic protein. By the method of component-resolved diagnosis of allergy (ISAC-112, ImmunoCAP), a comprehensive allergology examination for 112 allergen molecules was performed for each child. Results: the children of the study group were found to have increased values of total IgE (256.66±40.45 IU/mL) and eosinophil cationic protein (67.22±8.67 ng/mL). It was found that 57.5% of children were sensitized to tree nut storage proteins (rJug r1, rJug r2, NCoR a9, rAna o2, rBer e1), and some patients (27.5%) had increased specific IgE to non-specific lipid-transfer proteins (npLTPs) (rPru p3, NCoR a8, rAra h9, rJug r3). Antibodies to peanut storage proteins (rAra h1, rAra h2, rAra h3, rAra h6) were in 22.5% of children in the study group. Conclusion: the component-resolved diagnosis of allergy allows to determine the sensitization profile of patients. Sensitization detection to storage proteins and/or nsLTPs of nuts, peanuts and fruits is a predictor of possible severe allergic reactions. The exclusion of these products, both raw and heat-treated, reduces the number of severe acute allergic reactions in patients and prevents possible fatal outcomes. KEYWORDS: oral allergy syndrome, angioedema, children, birch, sensitization to Bet v1 allergen, storage proteins, nsLTPs. FOR CITATION: Lepeshkova T.S., Beltyukov E.K., Tsarkova S.A., Naumova V.V. Oral allergy syndrome and angioedema in patients with birch allergen sensitization: are there any high risks? Russian Medical Inquiry. 2021;5(1):21–24. DOI: 10.32364/2587-6821-2021-5-1-21-24.

CDNA Cloning and Characterization of the House Dust Mite Allergen Sui p 2

Suidasia pontifica (Sp) is a House Dust Mite (HDM) species found in tropical urban residential areas which causes sensitization of up to 75% of allergic patients. Cloning of allergens from Sp is a significant initial step in understanding the clinical significance of this HDM species. The study describes the cloning and in silico characterization of the group 2 allergen gene from Sp (Sui p 2) with significant homology to Myeloid Differentiation 2 (MD-2) lipid-binding (ML domain) protein allergens. Sui p 2 gene was amplified by Reverse Transcription – PCR using gene-specific primers designed from known HDM group 2 allergens. The amplicons were characterized using NCBI BLAST, ExPASy, MEGA7 and Chimera. Sui p 2 cDNA gene exhibits percent identity to Tyrophagus putrescentiae (65.84%), Aleuroglyphus ovatus (65.69%) and Blomia tropicalis (62.98%). Sui p 2 gene contains 405 bp open reading frame with untranslated regions on its 5’ and 3’ end. Furthermore, structure-based percent identity of Sui p 2 to the group 2 allergen of D. farinae and D. pteronyssinus is 47.62% and 46.83%, respectively. Predicted protein structure reveals a putative immunoglobulin fold consisting of preserved cysteine residues, hydrophobic core and two antiparallel β-sheets containing several TLR4, LPS and IgE binding sites. The results suggest that Sui p 2 may play an important role in HDM induced allergy and may be involved in the induction of the innate immune system. Characterization of Sui p 2 is important to evaluate properly its use for allergy component resolved diagnosis and allergen-specific immunotherapy.

Mold Sensitization in Asthmatic and Non-asthmatic Subjects Diagnosed with Extract-Based Versus Component-Based Allergens.

Asthmatic patients are suspected of having a higher risk of mold sensitization. Thus, precise diagnosis of fungal sensitization is important. Mold allergen extracts are difficult to standardize, but component-resolved allergy diagnosis may be an alternative to replace extract-based tests. In this research, asthmatic and non-asthmatic subjects were studied for their sensitization to Aspergillus fumigatus (Asp f), Cladosporium herbarum (Cla h), Penicillium chrysogenum (Pen ch), Alternaria alternata (Alt a), and Aspergillus versicolor (Asp v). Extract-based tests were applied using the skin prick test (SPT) and allergen-specific immunoglobulin E (sIgE). Subjects with extract-based sensitization to Asp f or Alt a were further investigated for sIgE response to recombinant (r) single mold allergens. At least one mold sensitization was found in about 50% of asthmatic and non-asthmatics with the most frequent sensitization to Alt a, followed by Pen ch, Asp f, Cla h, and Asp v. Interestingly, sensitization rate to individual mold species was always higher in asthmatics and was only significant for Pen ch. The component-resolved diagnosis with the sum of rAsp f 1 - rAsp f 4 plus rAsp f 6 matched the extract-based results (SPT and/or sIgE) in 50% of asthmatics and 46% of non-asthmatics, whereas, rAlt a 1 covered 59% of asthmatics and 50% non-asthmatics of extract-based Alt a sensitization. In conclusion, individual fungal sensitization rate was higher in asthmatics compared to non-asthmatics. Extract-based tests, especially SPTs, were most sensitive, but component-based tests covered 80% of extract-based serological sensitization to Alternaria and Aspergillus.

Employment of proteomic and immunological based methods for the identification of catalase as novel allergen from banana

Diagnostic reagents based on food allergen extracts often lack sufficient sensitivity. The introduction of well characterized food allergens in molecular allergy diagnosis has been recognized as valid approach to circumvent unstandardized allergen extracts. Banana fruit (Musa acuminata) is a well-established allergen source which besides six characterized allergens, contains unidentified IgE reactive proteins whose clinical relevance remains undefined. By employment of a combinatorial peptide ligand library (CPLL) methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis, a novel allergen from banana fruit was detected in banana as catalase. A recombinant homologue of natural catalase was produced, isolated and biochemically characterized. The recombinant protein showed IgE reactivity in 7 out of 13 tested patients with suspected allergy to banana in immunoblot. Novel banana fruit allergens should be added as components to allergen-microarrays for the diagnosis and the monitoring of banana allergy. SignificanceBy employment of CPLL methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis catalase from banana fruit is identified as a novel allergen, with proposed designation as Mus a 7. IgE reactive recombinant Mus a 7 was produced and should be included in a component-resolved allergy diagnosis.

Mass Barcode Signal Amplification for Multiplex Allergy Diagnosis by MALDI-MS.

A highly sensitive method based on mass-barcoded gold nanoparticles (AuNPs) and immunomagnetic separation has been developed for multiplex allergy diagnosis by MALDI mass spectrometry in a component-resolved manner. Different analytical probes were prepared by coating AuNPs with individual allergenic proteins and mass barcode, represented by polyethylene glycol molecules of various chain lengths. Magnetic beads (MBs) functionalized with antihuman IgE antibodies (Abs) were used as immunomagnetic capture probes. IgE Abs were extracted from a patient's blood serum by the formation of a sandwich structure between the AuNPs and MBs. Multiple specific IgE Abs were simultaneously identified by mass spectrometry detection of the mass barcodes, providing an efficient component-resolved allergy diagnosis. Because of the signal amplification provided by the mass barcodes, the developed diagnosis method is very sensitive, with a limit of detection down to picograms per milliliter level for specific IgE Abs. The method can be potentially useful when the sample amount is highly limited and a multiplex diagnostic procedure is required.

Development of a novel strategy to isolate lipophilic allergens (oleosins) from peanuts.

BackgroundPeanut allergy is one of the most severe class I food allergies with increasing prevalence. Especially lipophilic allergens, such as oleosins, were found to be associated with severe symptoms, but are usually underrepresented in diagnostic extracts. Therefore, this study focused on isolation, molecular characterization and assessment of the allergenicity of peanut oleosins.Methods and ResultsA comprehensive method adapted for the isolation of peanut oil bodies of high purity was developed comprising a stepwise removal of seed storage proteins from oil bodies. Further separation of the oil body constituents, including the allergens Ara h 10, Ara h 11, the presumed allergen oleosin 3 and additional oleosin variants was achieved by a single run on a preparative electrophoresis cell. Protein identification realized by N-terminal sequencing, peptide mass fingerprinting and homology search revealed the presence of oleosins, steroleosins and a caleosin. Immunoblot analysis with sera of peanut-allergic individuals illustrated the IgE-binding capacity of peanut-derived oleosins.ConclusionOur method is a novel way to isolate all known immunologically distinct peanut oleosins simultaneously. Moreover, we were able to provide evidence for the allergenicity of oleosins and thus identified peanut oleosins as probable candidates for component-resolved allergy diagnosis.

Generation of Recombinant FcεRIα of Dog, Cat and Horse for Component-Resolved Allergy Diagnosis in Veterinary Patients

Generation of Recombinant FcεRIα of Dog, Cat and Horse for Component-Resolved Allergy Diagnosis in Veterinary Patients

Identification of Sola l 4 as Bet v 1 homologous pathogenesis related-10 allergen in tomato fruits

The aim was to investigate the potential contribution of a major birch pollen Betv1-homologous allergen to birch pollen-associated tomato fruit allergy. Two isoforms of a Betv1-homologous protein (designated Solal4) from tomato fruit were identified by cDNA-cloning and produced as recombinant proteins. Allergen-specific IgE levels to tomato, birch pollen, Betv1, and Sola l 4 were determined in birch pollen allergic patients with allergy or tolerance to tomato. Solal4 was recognized in 76% of birch/tomato allergic patients, while tomato- and Betv1-specific IgE was detectable in 64% and 81% of sera. Almost all patients sensitized to Betv1 reacted with Solal4. Both Solal4 isoforms displayed allergenic potency and IgE-cross-reactivity with Betv1 as investigated by competitive ELISA and in vitro mediator release assay. Nevertheless, the reactivity pattern of patients' sera was diverse. Sola l 4, a novel pathogenesis related-10 protein, qualifies as major allergen in tomato fruits. Data suggest Sola l 4 as classII allergen. IgE-testing using Sola l 4 showed low clinical specificity, but high sensitivity in tomato allergic patients and will further improve component-resolved allergy diagnosis.

Molecular Characterization of Recombinant Mus a 5 Allergen from Banana Fruit

Allergy to banana fruit appears to have become an important cause of fruit allergy in Europe. Among five allergens that have been found, beta-1,3-glucanase denoted as Mus a 5 was identified as a candidate allergen for the component-resolved allergy diagnosis of banana allergy. Because of the variations in protein levels in banana fruit, in this study Mus a 5 was produced as a fusion protein with glutathione-S-transferase in Escherichia coli. The recombinant Mus a 5 was purified under native conditions by a combination of affinity, ion-exchange, and reversed phase chromatography. N-terminal sequence was confirmed by Edman degradation and 55% of the primary structure was identified by mass fingerprint, while the secondary structure was assessed by circular dichroism spectroscopy. IgG reactivity of recombinant protein was shown in 2-D immunoblot with anti-Mus a 5 antibodies, while IgG and IgE binding to natural Mus a 5 was inhibited with the recombinant Mus a 5 in immunoblot inhibition test. IgE reactivity of recombinant Mus a 5 was shown in ELISA within a group of ten persons sensitized to banana fruit. Recombinant Mus a 5 is a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy.

The crux with a reliable in vitro and in vivo diagnosis of allergy

This might be due to different reasons. Allergen-specific IgE in serum is a marker for sensitization and a prerequisite for the development of an allergy but is not sufficient for the induction of symptoms. We should be aware that all in vivo and in vitro tests are subject to inherent variation and potential interferences. In patients with symptoms suggestive of allergic rhinitis but negative results in skin test and serum soluble IgE, detection of local soluble IgE in nasal secretions during natural exposure to aeroallergens and a positive nasal allergen provocation test might explain the symptomatics 14. As discussed by Aalberse et al. 5 and reported elsewhere, there are good indications that recombinant allergens could be more efficient for the in vitro and in vivo diagnosis of allergy than extracts 15. Recombinant allergens can be produced as GMP pharmaceuticals satisfying the quality requirements for medical use 15, and technologically, it is straightforward to generate microarrays containing hundreds of different allergens 9. The concept of a component resolved diagnosis of allergy is not new but the problem is, as pointed out by Aalberse et al. 5, how to validate its clinical relevance. Even if the production of a microarray containing an optimal panel of perfectly standardized recombinant allergens is technically possible to date, which can contribute to improve the in vitro diagnosis of atopy in terms of sensitivity and specificity, it will still generate false-positive and false-negative results concerning the diagnosis of allergy. Thus, the allergologist should be prudent in judging the outcome of any in vivo or in vitro test that is inconsistent with a carefully collected clinical history, especially if patients are considered for inclusion in allergen-specific immunotherapeutic treatments. At the current state of the art, it is worthwhile to maintain a healthy scepticism about allergy diagnostic test results and to let the clinical history drive the diagnosis. The laboratory of the author is supported by the Swiss National Science Foundation grants No. 3200_146247 and 32NM30_136032/1 (EuroNanoMed) and by the European Community's Seventh Framework Program [FP7-2007-2013] under grant agreement no. HEALTH-F2-2010-260338 ‘ALLFUN’. The author has no conflict of interest relevant to this article.

Isolation and characterization of a 28 kDa major allergen from blackgram (Phaseolus mungo)

Legumes are the major elicitors of IgE-mediated food allergy in many countries of the world. Purified major allergens are prerequisite for component resolved diagnosis of allergy. The present study was aimed to isolate and characterize a major allergenic protein from blackgram (Phaseolus mungo). Respiratory allergy patients with history of blackgram allergy were skin prick tested (SPT) and sera were collected from SPT positive patients. The blackgram extract was fractionated using a combination of anion exchange and hydrophobic interaction chromatography. The purified protein was characterized by indirect ELISA, immunoblot, ELISA inhibition, SPTs, stripped basophil histamine release, lymphoproliferation assay and digestibility assay. The purified protein separated at 28kDa on 12% gel and showed IgE binding with 81% of blackgram hypersensitive patients’ sera on immunoblot indicating it to be a major allergen. Periodic Acid Schiff's and meta-periodate treatment staining detected it to be a glycoprotein. The 28kDa protein recognized 7/9 (77.8%) of blackgram positive patients by SPT, where as all 9 patients showed significant histamine release on stimulation with protein as compared to controls. The 28kDa protein remained stable up to 15min on incubation with SGF. Bands of 14–16kDa appeared after 15min of pepsin digestion that remained stable up to 60min of incubation. However, purified protein degraded within 5min after incubation with SIF. The N-terminus-12 residues sequence of 28kDa protein was GRREDDYDNLQL. A stretch of residues ‘DDYDNLQL’ showed homology with Rho-specific inhibitor of transcription termination (E=0.42, Identity=87%) and NBS-LRR type disease resistant protein from peanut (Arachis hypogaea) (E=2, Identity=77%). In conclusion, the purified 28kDa protein is a potent major allergen that may have implication in diagnosis of blackgram allergy.

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.

Molecular and immunological characterization of Mus a 5 allergen from banana fruit

Banana fruit has become an important cause of fruit allergy in the recent years. Among the five registered IUIS allergens, Mus a 1 and Mus a 2 have been characterized in detail. In this study, molecular characterization and evaluation of the allergenic properties of β-1,3-glucanase from banana (Musa acuminata), denoted as Mus a 5, were performed. The gene of Mus a 5 was cloned and sequenced. The obtained cDNA revealed a novel Mus a 5 isoform with an open reading frame encoding a protein of 340 amino acids comprising a putative signal peptide of 28 amino acid residues. By MALDI-TOF analysis Mus a 5 isolated from banana fruit revealed a molecular mass of 33451±67 Da. Two Mus a 5 isoforms (pI 7.7 and 8.0) were detected by 2D immunoblot with an identical N-terminal sequence. By mass fingerprint, 76 and 83% of the primary structure was confirmed for the two mature Mus a 5 isoforms, respectively. IgE reactivity to Mus a 5 was found in 74% of patients sensitized to banana fruit. Upregulation of basophil activation markers CD63 and CD203c was achieved with Mus a 5 in a concentration-dependent manner. Mus a 5 is a functional allergen and a candidate for the component-resolved allergy diagnosis of banana allergy.

Les allergènes moléculaires : évolution ou révolution dans le diagnostic de l’allergie

The recent developments in molecular biology made it possible to produce recombinant allergens. These allergens together with natural purified allergens are now available for specific IgE measurement either under an individual or a microarray format. Compared to natural extracts these standardized reagents represent an important breakthrough for the in vitro allergy diagnosis and they allow the precise characterisation of the sensitization profile of a given patient. Thus, we are now entering the molecular allergy era which will permit to conduct epidemiological studies, to improve the specificity of IgE determination (decrease of cross-reactivity and better understanding of polysensitization), to develop the component resolved diagnosis of allergy, to identify severity, persistence or recovery markers of allergy. These new technologies will be rapidly extended to skin tests, which will use molecular allergens and to a tailored immunotherapy defined according to the individual sensitization profile of the allergic patient.

Diagnostic value of Alt a 1, fungal enolase and manganese-dependent superoxide dismutase in the component-resolved diagnosis of allergy to pleosporaceae

Over the last decades, genomics and proteomics have contributed to the current knowledge of individualized allergenic components and their potential use in the diagnosis of IgE-mediated allergies. Recent investigations have demonstrated that Alt a 1 should be considered as a relevant allergen of the Pleosporaceae group and that enolase is the main allergen involved in the cross-reactivity to fungi. However, the real utility of these allergens as tools for the diagnosis of allergy to Alternaria is still unclear. To demonstrate the current value of the available fungal allergen panel and the need to build an accurate mould allergen array for the diagnosis of allergy to Pleosporaceae. Specific IgEs to individual mould allergens and allergenic mould extracts were evaluated using the ImmunoCAP™ system in 30 patients allergic to Alternaria and in 100 blood donors. Cross-reactivity studies were performed by Fluoro Enzyme ImmunoAssay (FEIA) and FEIA inhibition using individual allergens and allergenic extracts. Two-dimensional electrophoresis associated with a MALDI-TOF analysis was carried out to identify new allergen molecules. All allergic patients had positive specific IgE responses to several moulds from different taxonomical families. Classic and molecular diagnosis demonstrated that 23% of patients had multi-sensitization. The current commercially available fungal allergen array was not sufficient to establish an accurate diagnosis. Unexpected correlations between Alternaria or Alt a 1 and Curvularia or Cladosporium stimulated the investigation of a more accurate allergen panel. A manganese-dependent superoxide dismutase (MnSOD) homologous to Asp f 6 was identified as a new IgE-binding molecule from Alternaria alternata. Alt a 1 is the marker for allergy to Pleosporaceae, not including Curvularia. MnSOD can explain 6.6% of allergy to Alternaria without Alt a 1 sensitization and should be included together with Alt a 1 and fungal enolase in the molecular array for the diagnosis of allergy to Pleosporaceae.

Component resolved allergy diagnosis using microarray:past, present and future

Component resolved allergy diagnosis using microarray:past, present and future

Characterization of recombinant Mal d 4 and its application for component‐resolved diagnosis of apple allergy

Profilins are ubiquitous panallergens that have been extensively characterized; yet, their clinical relevance is still unclear. The aim of the present study was to produce recombinant apple profilin (rMal d 4) and to evaluate its allergenic activity and its potency for component-resolved allergy diagnosis. Complementary DNA-derived Mal d 4 was cloned, expressed in Escherichia coli and subsequently purified via poly (l-proline) sepharose. A total of 28 sera from apple-allergic patients were used for IgE-ELISA, immunoblot, RAST and basophil histamine release (BHR) test. In addition, skin prick tests (SPTs) were performed in five patients. Four different complementary DNA coding for apple profilin, Mal d 4, each with an open reading frame of 393 nucleotides, were identified. One isoform Mal d 4.0101 was expressed in Escherichia coli and subsequently purified. Mass spectroscopy revealed the expected mass of 13.826 for rMal d 4.0101, and circular dichroism analysis data were typical for a folded protein and small-angle X-ray scattering measurement identified the protein as a monomer. All the serum samples displayed IgE binding to rMal d 4.0101 in IgE ELISA, immunoblot and RAST. In immunoblotting, IgE binding to natural Mal d 4 was partially/completely inhibited by preincubation with rMal d 4.0101, and RAST values to apple extract were significantly reduced upon serum pretreatment with rMal d 4.0101. SPTs and BHR assays using purified rMal d 4.0101 were positive. Purified rMal d 4.0101 was destroyed within seconds when subjected to pepsin digestion. Apple profilin complementary DNAs were identified. The physicochemical and allergenic properties of purified recombinant Mal d 4.0101 were evaluated showing that the recombinant protein was equal to the natural protein as shown by inhibition assays. Thus, Mal d 4 represents another example suitable for component-resolved diagnosis of food allergy.

Rapid method for arrayed investigation of IgE-reactivity profiles using natural and recombinant allergens.

The availability of increasing numbers of purified natural and recombinant allergens offer the possibility for component-resolved characterization of IgE binding. To make use of this potential, fast and simple methods with high capacity have to be developed. A laboratory multiscreen device was used in an innovative two-dimensional approach. In the first step, natural and recombinant allergens were immobilized onto the membrane using the sample chambers as application mask and, after blocking and rotating the membrane through 90 degrees, the same device was used to apply and incubate sera of allergic patients. Enzyme-linked immunoassay (ELISA) quantification of specific IgE was performed for purposes of comparison. Proteins were most efficiently bound onto nitrocellulose in 20 mM sodium hydroxide (NaOH). Up to 45 proteins or extracts could be investigated with a maximum of 45 sera in a single application, resulting in a resolution of 2,025 spots on one membrane with a size comparable to a standard Western blot. A high correlation for IgE-binding between natural and recombinant allergens was observed. Development of the membrane resulted in very evenly distributed square patterns. The results corresponded with the conventional ELISA measurements of specific IgE. The innovative usage of a standard incubation device for both application of proteins as well as screening of sera provides a simple high throughput method for the characterization of IgE binding to allergens. The results are important for component resolved diagnosis of allergy by means of fast monitoring of IgE- and IgG-reactivity spectra. Recombinant allergens may be used as targets for these purposes.