Identification and Purification of Novel Low-Molecular-Weight Lupine Allergens as Components for Personalized Diagnostics.

Lupine flour is a valuable food due to its favorable nutritional properties. In spite of its allergenic potential, its use is increasing. Three lupine species, Lupinus angustifolius, L. luteus, and L. albus are relevant for human nutrition. The aim of this study is to clarify whether the species differ with regard to their allergen composition and whether anaphylaxis marker allergens could be identified in lupine. Patients with the following characteristics were included: lupine allergy, suspected lupine allergy, lupine sensitization only, and peanut allergy. Lupine sensitization was detected via CAP-FEIA (ImmunoCAP) and skin prick test. Protein, DNA and expressed sequence tag (EST) databases were queried for lupine proteins homologous to already known legume allergens. Different extraction methods applied on seeds from all species were examined by SDS-PAGE and screened by immunoblotting for IgE-binding proteins. The extracts underwent different and successive chromatography methods. Low-molecular-weight components were purified and investigated for IgE-reactivity. Proteomics revealed a molecular diversity of the three species, which was confirmed when investigated for IgE-reactivity. Three new allergens, L. albus profilin, L. angustifolius and L. luteus lipid transfer protein (LTP), were identified. LTP as a potential marker allergen for severity is a valuable additional candidate for molecular allergy diagnostic tests.

Peanut oleosins associated with severe peanut allergy-importance of lipophilic allergens for comprehensive allergy diagnostics.

BACKGROUND: Peanut allergy is one of the most common and most severe food allergies in Western countries and its accurate diagnosis to prevent potential life-threatening allergic reactions is crucial. However, aqueous extracts used for routine diagnostic measurements are devoid of lipophilic allergens such as oleosins. We have recently succeeded in the isolation and purification of these unique proteins, and the present study evaluates their allergenic potential and clinical relevance. OBJECTIVE: We sought to assess allergenicity and sensitization prevalence of oleosins obtained from both raw and in-shell roasted peanuts. In addition, we tested the utilization of natural and recombinant oleosins for allergy diagnostic purposes. METHODS: Oleosin sensitization, prevalence, and impact of thermal processing were analyzed by immunoblot with sera from 52 peanut-allergic individuals displaying different clinical phenotypes. The application of natural and recombinant oleosins for allergy diagnostics was investigated by basophil activation test (BAT). IgE-binding epitopes were identified by oligopeptide microarray. RESULTS: Sensitization to oleosins was observed exclusively in peanut-allergic subjects suffering from severe systemic reactions. IgE-binding capacity of oleosins derived from in-shell roasted peanuts was increased as shown by immunoblot analysis and BAT. Both natural and recombinant molecules can be used to identify oleosin-sensitized patients by BAT. A linear epitope of Ara h 15 was determined that displays high similarity to other seed-derived oleosins. CONCLUSIONS: Oleosins are clinically relevant peanut allergens and most likely associated with severe allergic symptoms. In-shell roasting increases their allergenicity, which is consistent with the observation that most allergic reactions are in connection with roasted peanuts.

The BASALIT multicenter trial: Gly m 4 quantification for consistency control of challenge meal batches and toward Gly m 4 threshold data.

SCOPE: The BASALIT clinical trial (EudraCT 2009-011737-27) investigated efficacy of birch allergen immunotherapy on lowest observed adverse effect levels after soy food challenge in patients with birch-associated and Gly m 4 allergen mediated soy allergy. Thus, consistently stable Gly m 4 levels were required in standardized challenge meals. METHODS AND RESULTS: Soy meal included soy protein isolate (SPI, 88% total protein). A Gly m 4 specific ELISA was developed and validated. Six SPIs and 24 meal batches were analyzed for Gly m 4. (Repeated-measures) analyses of variance were done to identify potential changes between batches and time intervals. Gly m 4 was below the ELISA detection limit (2 ng/mL) in placebo batches. With <20% mean coefficient of variation, Gly m 4 levels were consistent in 24 soy meal batches and within individual 12-wk shelf-life. CONCLUSION: The novel Gly m 4 specific ELISA proved consistency of challenge meal batches over a 56-month study period. With an average of 178 µg/g Gly m 4 in SPI, Gly m 4 lowest observed adverse effect level can be calculated once clinical lowest observed adverse effect level data based on SPI are available. Hence, sensitivity of patients can be correlated to the relevant allergen content instead of total protein of the allergenic source.

Prospective investigation on the transfer of Ara h 2, the most potent peanut allergen, in human breast milk.

BACKGROUND: Peanut allergy is one of the most severe food allergies. Whether breastfeeding induces tolerance to peanuts or on the contrary, pre-disposes at risk-babies to occult allergic sensitization to peanuts is still a matter of discussion. We sought to investigate the transfer of the most potent peanut allergen Ara h 2 into human breast milk in a German breast milk study and to shed light on the time kinetics of Ara h 2 appearance. METHODS: We recruited 32 lactating, non-peanut-allergic women and collected breast milk samples at different time points after consumption of 100 g dry roasted peanuts. Breast milk samples were investigated for Ara h 2 with different immunological methods: by 2D immunoblotting with a patient's serum, by affinity enrichment using a monoclonal antibody against Ara h 2 followed by LC-MS/MS-based detection and by a competitive inhibition ELISA for the detection of Ara h 2 and its digestion-resistant peptides (DRP-Ara h 2). RESULTS: In a qualitative analysis, Ara h 2 could be identified in a breast milk sample by 2D immunoblot by means of a patient's serum and furthermore by immunoaffinity enrichment followed by LC-MS/MS analysis. In a semi-quantitative analysis, Ara h 2 and its digestion-resistant peptides were detected in the breast milk of 9 of 32 subjects. Evidence suggests that Ara h 2 is excreted individually either rapidly (after 1, 2, 3 or 4 h) or delayed (after 8 or 12 h) and in different concentrations. CONCLUSIONS: Time and concentration of secreted Ara h 2 in breast milk appears to be individually regulated. The identification of Ara h 2 in breast milk is the prerequisite for the investigation of its sensitizing or tolerogenic properties.

Peanut defensins: Novel allergens isolated from lipophilic peanut extract.

BACKGROUND: Peanut is one of the most hazardous sources of food allergens. Unknown allergens are still hidden in the complex lipophilic matrix. These allergens need to be discovered to allow estimation of the allergenic risk for patients with peanut allergy and to further improve diagnostic measures. OBJECTIVE: We performed detection, isolation, and characterization of novel peanut allergens from lipophilic peanut extract. METHODS: Extraction of roasted peanuts were performed under defined extraction conditions and examined by means of 2-dimensional PAGE. Subsequently, chromatographic methods were adapted to isolate low-molecular-weight components. Proteins were studied by using SDS-PAGE and immunoblotting with sera from patients with peanut allergy. For allergen identification protein sequencing, homology search and mass spectrometry were applied. Functional characterization for allergenicity was performed by using the basophil activation assay and for antimicrobial activity by using inhibition assays of different bacteria and fungi. RESULTS: IgE-reactive proteins of 12, 11, and 10 kDa were first detected after chloroform/methanol extraction in the flow through of hydrophobic interaction chromatography. The proteins were able to activate basophils of patients with peanut allergy. N-terminal sequencing and homology search in the expressed sequence tag database identified the allergens as peanut defensins, which was confirmed by using mass spectrometry. On microbial cell cultures, the peanut defensins showed inhibitory effects on the mold strains of the genera Cladosporium and Alternaria but none on bacteria. CONCLUSIONS: We identified defensins as novel peanut allergens (Ara h 12 and Ara h 13) that react in particular with IgE of patients with severe peanut allergy. Their antimicrobial activity is solely antifungal.

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

BACKGROUND: Peanut 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 RESULTS: A 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. CONCLUSION: Our 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.

Peanut allergens.

The earliest known evidence of peanut farming dates back 7,600 years. With a prevalence of roughly 1%, peanut allergy is a diagnostic and treatment challenge, but is also a very good model for studying all aspects of food allergy, including its molecular basis and pathomechanisms. Therefore, the very starting point for elucidating all these aspects is the identification of peanut allergens with subsequent clearing of their structure and their preparation as pure recombinant and/or natural allergens. This is the basis for in vitro diagnostic tests as well as the development of immunotherapeutic drugs. With regard to class I food allergy, peanut allergy affects by far the largest group of patients. In peanuts, 12 allergens have been identified and their molecular characteristics are described herein. Ara h 1, Ara h 3.01 and Ara h 3.02 (the former Ara h 4) belong to the cupin superfamily. The conglutins Ara h 2, Ara h 6 and Ara h 7, and the non-specific lipid transfer protein Ara h 9 belong to the prolamin superfamily. Ara h 5 (profilin) and Ara h 8 (Bet v 1-homologous protein) cause class II food allergies and are associated with inhalation allergy to pollen via the sequential and/or conformational similarity of molecules. Two peanut oleosins are listed as Ara h 10 and Ara h 11 and two defensins as Ara h 12 and Ara h 13 by the WHO/IUIS Allergen Nomenclature Subcommittee. The effect of the above-specified allergens has to be considered in the context of their matrix, which is influenced by processing factors and the individual's immune system.

Roasting and lipid binding provide allergenic and proteolytic stability to the peanut allergen Ara h 8.

Abstract Ara h 8 is the peanut allergen homologous to the birch pollen allergen Bet v 1. Because Bet v 1 has been shown to bind lipophilic ligands, the aim of this investigation was to determine the impact of lipid binding and roasting on the Ara h 8 structure and their influences on allergenicity. For the characterization of natural Ara h 8 (nAra h 8) from roasted and unroasted peanuts, circular dichroism spectroscopy, hydrophobic binding assay, immunohistochemistry, and immunoblot with sera of peanut allergic patients were performed and compared with results from recombinant Ara h 8 (rAra h 8) and Bet v 1. rAra h 8 displayed stronger hydrophobicity than rBet v 1. Patients' sera showed IgE reactivity with rAra h 8 and nAra h 8 from roasted peanuts, whereas fewer sera recognized nAra h 8 from unroasted peanuts. Simulated gastric digestion experiments demonstrated low proteolytic stability of rAra h 8, whereas the stability of nAra h 8 was increasingly higher in unroasted and roasted peanuts. The results demonstrate that IgE reactivity and thermal and proteolytic stability are reinforced in nAra h 8 after roasting, most likely due to Maillard reactions, lipid oxidations, and lipophilic associations. These aspects must be considered when estimating the allergenicity of Bet v 1-homologous proteins.

Development and in-house validation of allergen-specific ELISA tests for the quantification of Dau c 1.01, Dau c 1.02 and Dau c 4 in carrot extracts (Daucus carota).

Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6 ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosis.

Detection and structural characterization of natural Ara h 7, the third peanut allergen of the 2S albumin family.

In recent years, several novel relevant peanut allergens have been identified. Among those, a new member of the conglutin family was cloned by a phage display approach and initially annotated as Ara h 7.0101. Later, however, recloning of Ara h 7 revealed an alternate isoform, termed Ara h 7.0201. Because the natural Ara h 7 counterpart had not been found at the protein level in peanut extracts, the aim of the present study was to search for authentic natural Ara h 7 protein(s). To this end, enriched low molecular mass proteins (<20 kDa) from peanut extracts were separated by 2D electrophoresis and subjected to mass spectrometric analyses. Fifty of 65 analyzed spots were identified. Interestingly, Ara h 7.0101 was not identified, but Ara h 7.0201 and Ara h 7.0202, a different Ara h 7 isoallergen containing an additional pro-peptide cleavage site, were. In accordance with the conserved cysteine pattern of conglutins, Ara h 7.0201 possesses eight cysteine residues, in contrast to the six cysteines present in the previously cloned Ara h 7.0101. Moreover, a putative cleavage site in the Ara h 7.0202 isoform points to the characteristic biological function of conglutins as amylase/trypsin inhibitors.

Peanut varieties with reduced Ara h 1 content indicating no reduced allergenicity.

Peanut allergy is a major cause of food-induced severe anaphylactic reactions. To date, no medical care is available to prevent and treat peanut allergy and therefore hypoallergenic peanut varieties are of considerable health political and economic interest. Major allergens that induce IgE-responses in peanut-sensitive patients are Ara h 1, Ara h 2 and Ara h 3/4. In order to identify hypoallergenic peanuts, commercially locally available peanut varieties were screened for their allergen content. Ara h 1-deficient peanuts from Southeast Asia were identified by SDS-PAGE, immunoblotting, inhibition assays and ELISA. 2-D PAGE analyses demonstrated the different compositions of the tested extracts and revealed a number of variations of the allergen patterns of peanuts from different varieties. Mediator release experiments of these peanut extracts demonstrated similar allergenicities as compared with standard peanut extract. These results indicate that the allergenicity of peanuts with reduced Ara h 1 content might be compensated by the other allergens, and thus do not necessarily cause a reduction of allergenicity.

Lipid transfer protein (Ara h 9) as a new peanut allergen relevant for a Mediterranean allergic population.

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.

2-D DIGE analysis of the proteome of extracts from peanut variants reveals striking differences in major allergen contents.

Over the last decade, an increasing prevalence of peanut allergies was observed worldwide. Peanuts are meanwhile categorized among the most dangerous food allergens. This is particularly relevant since peanut-derived ingredients are widely used in industrial food production. To minimize the problem of hidden food allergens causing severe anaphylactic reactions, pre-packaged food containing peanut components needs to be classified according to European ruling since 2005. Food companies search for strategies to reduce the allergenicity of peanut-derived food additives either by genetically altering the allergen content or by identifying peanut varieties with low levels of major allergens. In our study, we focused on peanut extracts from Indonesia that apparently contain lower levels of the major Arachis hypogaea allergen 1 (Ara h 1). Basic extracts of Virginia-type and Indonesian peanuts were compared by 1- and 2-DE. We identified more than hundred individual components in these extracts by MS and provide a high-resolution allergen map that also includes so far unknown fragments of major peanut allergens. The reduced level of Ara h 1 associated with a significantly lower abundance of the most potent peanut allergen Ara h 2 in various Indonesian peanuts was also confirmed by Western blotting with monoclonal antibodies and sera of allergic patients.

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Human epithelial cells of the respiratory tract and the skin differentially internalize grass pollen allergens.

Epithelial cells of both the respiratory tract and the skin form a tight barrier against environmental harm. They represent the site of first contact for airborne allergen carriers. Consequently, in this study, we analyzed the uptake of grass pollen allergens by epithelial cells: Phl p 1 was selected as a glycosylated allergen containing disulfide bridges whereas Phl p 6 lacks post-translational modifications. Allergen uptake by the respiratory epithelial cell line A549 reached a plateau at 2 hours, and both allergens were localized intracellularly in non-acidic vesicles. In addition, in A549 cells allergens were exocytosed, suggesting a transcytosis mechanism in the passage of allergens over the respiratory epithelial barrier. In contrast, allergens were predominately localized in lysosomes in keratinocytes, and allergen uptake did not reach a plateau up to 24 hours. Notably, keratinocytes from atopic patients showed a significantly increased uptake of Phl p 1 as compared with healthy donors. Preincubation of epithelial cells with IL-4 and/or IFN-gamma to simulate inflammatory status led to an increased allergen uptake only in keratinocytes. This higher engulfment of allergens by inflammatory-type keratinocytes suggests a higher susceptibility of inflamed skin for the uptake of allergens and consequently a potentially higher risk for sensitization under natural exposure conditions, such as chronic atopic eczema.

The European Union CREATE project: a model for international standardization of allergy diagnostics and vaccines.

Allergen measurements are used extensively in the formulation of allergy diagnostics and vaccines, yet no purified international allergen standards are available for calibration purposes. The aims of the European Union CREATE project were to develop international standards with verifiable allergen content. Purified natural and recombinant allergens were analyzed by means of SDS-PAGE, mass spectrometry, circular dichroism spectra, and small-angle x-ray scattering. IgE reactivity was assessed by means of direct RAST, RAST inhibition, immunoblotting, and basophil histamine release with sera from 961 allergic patients. Three recombinant allergens, rBet v 1, rPhl p 5a, and rDer p 2, were structurally indistinguishable from their natural counterparts and showed excellent IgE reactivity suitable for use as certified reference materials. A second tier of allergens (rPhl p 5b, rOle e1, rDer p 1, rDer f 1, and rDer f 2) was identified that could provide suitable candidates for certified reference materials with minor improvements to the recombinant proteins. Only rPhl p 1 was considered unsuitable as a reference material. Quantitative ELISAs were identified that accurately measured each allergen, except for rPhl p 1. The CREATE project has provided a major step forward in allergen standardization and provides a model for the development of a comprehensive panel of international reference preparations that will harmonize allergen measurements worldwide.

Solution structure of Phl p 3, a major allergen from timothy grass pollen.

The major 97-aa timothy grass (Phleum pratense) allergen Phl p 3 was recently isolated from an extract of timothy grass pollen. Sequence comparison classifies this protein as a group 3 allergen. The solution structure of Phl p 3 as determined by nuclear magnetic resonance spectroscopy reveals that the protein consists of a core of hydrophobic amino-acid side chains from two beta-sheets of five and four anti-parallel beta-strands, respectively. This conformation is very similar to the crystal structure published for Phl p 2 and strongly resembles the known conformation of the carboxy-terminal domain of Phl p 1, the major difference being the loop orientations. Phl p 2 and Phl p 3 show virtually identical immunoreactivity, and comparison of the charged surface amino acids of the two proteins gives initial clues as to the IgE recognition epitopes of these proteins.

Quantification of the thaumatin-like kiwi allergen by a monoclonal antibody-based ELISA.

Thaumatin-like proteins (TLPs) have been established as a new family of fruit and pollen allergens. The aim of this study was to develop a two-site ELISA for the quantification of the thaumatin-like kiwi allergen (Act d 2) in kiwifruit extracts and kiwifruit-containing food products. Genomic DNA (gDNA) of Act d 2 was amplified and the deduced amino acid sequence was determined to obtain a primary structure. Act d 2 purified from kiwifruit extract by HPLC was identified by Edman degradation and MS. Balb/c mice were immunized with Act d 2 for the production of mAbs by hybridoma technology. The optimized ELISA measured Act d 2 concentrations ranging from 0.2 to 9.0 ng/mL, with intra- and interassay coefficients of variation of 3.65 and 10.44%, respectively. The developed ELISA is a useful method for the quantification of the thaumatin-like kiwi allergen in kiwifruit extracts as well as the allergen level in kiwifruit-containing food products. It may be a helpful analytical tool for the evaluation of the stability (integrity) of fruit allergen extracts for in vitro diagnosis.

Purification and characterization of natural Ara h 8, the Bet v 1 homologous allergen from peanut, provides a novel isoform.

The peanut allergen Ara h 8 is an important allergen for birch pollen allergic patients because of the cross-reactivity to the homologous Bet v 1. As the existence of Ara h 8 has been shown at the cDNA level so far (AY328088) and the allergen has indirectly been detected as natural protein, it was the aim of our study to identify natural Ara h 8 in peanut extract and to develop a purification strategy. This was achieved using a unique combination of purification steps, including optimized extraction conditions, size exclusion and ion exchange chromatography and treatment of the interfering contaminants with iodoacetic acid. A characterization of the protein by microsequencing showed discrepancies to the deduced amino acid sequence of AY328088. For this reason, we cloned and expressed a new Ara h 8 isoform from cDNA (EU046325). This IgE-reactive protein corresponds to the results of microsequencing, ESI-FTICR-MS and trypsin fingerprinting analysis of the authentic and purified nAra h 8. Apart from the ultimate use of recombinant allergens for diagnostic procedures, there is also a scientific need for the natural counterpart, as it represents an excellent reference point by which to compare protein characteristics and to standardize diagnostic and therapeutic allergens.