Meat allergy associated with galactosyl-α-(1,3)-galactose (α-Gal)-Closing diagnostic gaps by anti-α-Gal IgE immune profiling.

BACKGROUND: Glycoproteins and glycolipids of some mammalian species contain the disaccharide galactosyl-α-(1,3)-galactose (α-Gal). It is known that α-Gal is immunogenic in humans and causes glycan-specific IgG and also IgE responses with clinical relevance. α-Gal is part of the IgE-reactive monoclonal therapeutic antibody cetuximab (CTX) and is associated with delayed anaphylaxis to red meat. In this study, different α-Gal-containing analytes are examined in singleplex and multiplex assays to resolve individual sensitization patterns with IgE against α-Gal. METHODS: Three serum groups, α-Gal-associated meat allergy (MA) patients, idiopathic anaphylaxis (IA) patients with suspected MA, and non-meat-allergic healthy control individuals (HC), were analyzed via singleplex allergy diagnostics and a newly established immunoblot diagnostic system. The new dot blot detection system resolved individual IgE sensitization profiles for α-Gal-containing analytes CTX, bovine thyroglobulin (Bos d TG), and human serum albumin (HSA)-conjugated α-Gal. RESULTS: Singleplex allergy diagnostics using the α-Gal analytes CTX and Bos d TG confirms the history of MA patients in 91% and 88% of the cases, respectively. A novel dot blot-based assay system for the detection of IgE against α-Gal reveals individual IgE sensitization profiles for α-Gal-containing analytes. An α-Gal-associated IgE cross-reactivity profile (IgE against CTX, Bos d TG, and HSA-α-Gal) was identified, which is associated with MA. CONCLUSIONS: Detection of individual sensitization patterns with different α-Gal-containing analytes provides the basis for an individual allergy diagnosis for α-Gal-sensitized patients. Higher amounts of α-Gal in pork and beef innards compared to muscle meat as indicated by a higher staining intensity are a plausible explanation for the difference in allergic symptom severity.

Peanut allergens: new consolidated findings on structure, characteristics, and allergome.

Immunoglobulin E-mediated food allergy is the result of a complex pathomechanism. Factors contributing to the dysfunction of the immune system are the allergenic sources and the variable matrix effects arising from the processes involved in interaction with the gastrointestinal tract, the allergens themselves through their structural features, and the specific behavior of the individual immune system. The starting point for elucidating the pathomechanism of food allergy is the identification of allergens and the description of their structure. They are the basis for in vitro diagnostics 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. 11 allergens have been identified in peanuts. Ara h 1, Ara h 3, and Ara h 4 belong to the cupin superfamily, Ara h 2, Ara h 6, and Ara h 7 to the prolamin superfamily; Ara h 5 (profilins) and Ara h 8 (superfamily of Bet v 1-homologous proteins) are associated with aeroallergens. Peanut lipid transfer proteins (LTP) and two peanut oleosins are listed as Ara h 9, Ara h 10, and Ara h 11 by the IUIS Allergen Nomenclature Subcommittee. Peanut agglutinin (PNA) and a third oleosin have been shown to possess allergenic properties. The effect of the above specified allergens has to be considered in the context of their matrix, which is influenced by processing factors.

Gram-positive bacteria on grass pollen exhibit adjuvant activity inducing inflammatory T cell responses.

BACKGROUND: Recently, it has been established that pollen grains contain Th2-enhancing activities besides allergens. OBJECTIVE: The aim of this study was to analyse whether pollen carry additional adjuvant factors like microbes and what immunological effects they may exert. METHODS: Timothy pollen grains were collected and disseminated on agar plates, and the growing microorganisms were cultivated and defined. Furthermore, the immunologic effects of microbial products on DC and T cell responses were analysed. RESULTS: A complex mixture of bacteria and moulds was detected on grass pollen. Besides Gram-negative bacteria that are known to favour Th1-directed immune responses, moulds were identified as being sources of allergens themselves. Herein, we focused on Gram-positive bacteria that were found in high numbers, e.g. Bacillus cereus and Bacillus subtilis. Contact of immature dendritic cells (DC) from grass pollen allergic donors with supernatants of homogenized Gram-positive bacteria induced maturation of DC as measured by up-regulation of CD80, CD83 and CD86 and by enhanced production of IL-6, IL-12p40 and TNF-α, which was less pronounced compared with effects induced by lipopolysaccharide (LPS). Consequently, stimulation of autologous CD4(+) T cells with supernatants of homogenized Gram-positive bacteria plus grass pollen allergen-pulsed DC led to an enhanced proliferation and production of IL-4, IL-13, IL-10, IL-17, IL-22 and IFN-γ production compared with T cells that were stimulated with allergen-pulsed immature DC alone, whereas production of the transcription factor for regulatory T cells FoxP3 was not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that grass pollen is colonized by several microorganisms that influence the immune response differently. Similar to LPS, supernatants of homogenized Gram-positive bacteria may serve as adjuvants by augmenting DC maturation and inflammatory Th1, Th2 and Th17 responses helping to initiate allergic immune responses.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

In contrast to specific B cells, human basophils are unaffected by the toxic activity of an allergen toxin due to lack of internalization of immunoglobulin E-bound allergen.

BACKGROUND: Specific immunotherapy is the only curative therapy for type I allergies and the alarming increase in allergy prevalence emphasizes the need for additional/alternative strategies for curative treatment. Allergen toxins (AT), fusion products of an allergen with an apoptosis inducing cytotoxin, are a new kind of immunotoxin. OBJECTIVE: AT should allow allergen-specific targeting and elimination of allergy-relevant cells, with B cells being the primary target. An important question is the fate of the effector cells, e.g. mast cells and basophils, which carry allergen-specific IgE: the immunotoxin might even prove to be harmful. METHODS: We established a reliable in vitro B cell model (using two mouse hybridoma cell lines) for testing specificity and toxicity of P5-ETA', a fusion protein of the major timothy grass pollen allergen Phl p 5b and truncated Pseudomonas Exotoxin A. In a second step, we investigated the impact of the AT on human basophils. RESULTS: P5-ETA' reliably eliminated Phl p 5-specific cells in the in vitro B cell model, leaving unspecific B cells unharmed. Human basophils of grass pollen allergic donors specifically bound P5-ETA', released IL-4 and up-regulated the activation marker CD203c, but were not subject to the toxic effect because of lack of internalization of IgE-bound allergen. CONCLUSION: According to our data, basophils are pure effector cells in the context of IgE-bound allergen and not involved in classical antigen presentation.

Birch pollen-related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1.

BACKGROUND: Recently allergic reactions to legumes mediated by Bet v 1-homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1-homologous allergen Vig r 1. METHODS: Ten patients were selected who had a history of allergic reactions to mungbean seedlings and a respiratory allergy to birch pollen. The Bet v 1 homologue in mungbean seedlings, Vig r 1, was cloned by a PCR strategy, expressed in Escherichia coli, and purified by preparative SDS-PAGE. In all sera, specific IgE against birch pollen, Bet v 1, Bet v 2, Vig r 1, and the Bet v 1 homologues in soy (Gly m 4) and cherry (Pru av 1) was determined by CAP-FEIA. Cross-reactivity of specific IgE with Vig r 1, Bet v 1, Gly m 4, and Pru av 1 was assessed by immunoblot inhibition. Expression of Vig r 1 during development of mungbean seedlings and under wounding stress was analysed by immunoblotting. The Vig r 1 double band was analysed by matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: All patients were sensitized to birch pollen and Bet v 1, 20% to Bet v 2, and 90% to Gly m 4. Seventy percent of the patients showed IgE binding to a double band at 15 kDa in mungbean extract that was inhibited after pre-incubation of sera with rBet v 1. PCR cloning revealed that the mungbean homologue of Bet v 1 had a molecular weight of 16.2 kDa, a calculated pI of 4.6% and 42.8% amino acid sequence identity with Bet v 1. MS analysis confirmed similarity of the double band with the deduced Vig r 1 sequence, but also indicated the existence of other Vig r 1 isoforms. ImmunoCAP analysis detected IgE against Vig r 1 in 80% of the sera. IgE binding to Vig r 1 was inhibited with Gly m 4 in six of six and with rPru av 1 in four of six patients. Vig r 1 expression occurred during development of seedlings and was increased by wounding stress. CONCLUSIONS: Food allergy to mungbean seedlings can be caused by primary sensitization to birch pollen and is mediated by Vig r 1 in the majority of the patients with birch pollen-related allergy to mungbean seedlings.

The early allergic response in small airways of human precision-cut lung slices.

To study the role of small airways in the early allergic response (EAR), the method of human precision-cut lung slices (PCLS) was developed and used to examine the bronchoconstriction elicited by passive sensitisation and allergen provocation. Viable human PCLS of 250-microm thickness containing airways <1.5 mm in outer diameter were prepared from lung lobes obtained from lung resection and taken into culture. According to the low release of lactate dehydrogenase and the constant ciliary beat frequency, human PCLS were viable for at least 3 days. Following overnight passive sensitisation with serum from allergic individuals, administration of grass-pollen extract or activating immunoglobulin E antibody resulted in immediate airway contraction that was quantified by videomicroscopy. The extent of the EAR increased with decreasing airway size (outer airway diameter), with the strongest response occurring in the terminal bronchioles. Histamine receptor antagonism was ineffective, and leukotriene or thromboxane receptor antagonism attenuated the early allergic response only in some cases. However, simultaneous blockade of leukotriene and thromboxane receptors almost completely prevented the early allergic response in the precision-cut lung slices from all individuals, suggesting such a dual treatment as a potential future asthma therapy.

Characterization of immunoglobulin E responses in Balb/c mice against the major allergens of timothy grass (Phleum pratense) pollen.

BACKGROUND: Grass pollen, such as that from timothy grass (Phleum pratense), represents a major cause of type I allergy. OBJECTIVE: To characterize the IgE immune response and to identify the major allergens eliciting an IgE response in a mouse model using pollen extract of P. pratense for sensitization, in order to assess analogies to human hyperreactivity and to gain information on the allergenic potential as determined by the IgE-reactivity kinetics of defined allergens. METHODS: Balb/c mice were sensitized with pollen extract or with purified natural allergens. Serum IgE levels, the induction of specific IgE antibodies and immediate hypersensitivity were monitored by ELISA, Western blot and a skin test, respectively. RESULTS: The sensitized mice mounted a strong IgE response and showed IgE-reactivity first against Phl p 5a and 5b, then Phl p 4 and 13 and lastly against Phl p 6. No IgE response was mounted against Phl p 1. However, all purified fractions examined (Phl p 5a, 5b, 6 and 1) induced specific IgE and showed similar kinetics of IgE induction as pollen extract (first Phl p 5a and 5b, then Phl p 6). Skin test experiments demonstrated positive reactivity only in sensitized mice. CONCLUSION: The IgE reactivity induced by the major allergens in Balb/c mice was very similar to that found in allergic patients, with the exception of Phl p 1. The kinetics of the specific IgE response was comparable using either pollen extract or the purified major allergens, indicating that the intrinsic properties of the allergens are of importance rather than their proportionate amounts in pollen extract. This model should prove to be suitable for investigations regarding the mechanisms of induction and manifestation of timothy grass pollen allergy and for the evaluation of therapeutic strategies.

Localization, release and bioavailability of pollen allergens: the influence of environmental factors.

Allergens are integral constituents of plants or animals and their normal functions and localization are being characterized. To trigger responses in humans, allergens must become bioavailable and the role of air pollutants--for example diesel-exhaust particles --in this process is causing concern. Finally, the fact that some pollen releases eicosanoid-like proinflammatory mediators may have wide implications.

Optimization of electrophoresis for the identification of low molecular mass allergens in hazelnuts.

Conventional electrophoresis techniques used to identify food allergens are insufficient to separate low molecular mass proteins and peptides. In this paper we performed three different methods which provided an extended resolving power for small proteins. Applying the improved techniques, we were able to separate hazelnut proteins into distinct bands below 10 kDa.

Immunological identification and characterization of individual food allergens.

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.

Patient-tailored cloning of allergens by phage display: peanut (Arachis hypogaea) profilin, a food allergen derived from a rare mRNA.

A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10(11) cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-(L-proline)-Sepharose and yielded 30 mg l(-1) culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.

Group 13 grass allergens: structural variability between different grass species and analysis of proteolytic stability.

BACKGROUND: Determination of the allergen composition of an extract is essential for the improvement of hyposensitization therapy. Surprisingly, although grass pollen extracts have been studied intensively for 20 years, a further major allergen, Phl p 13, was detected recently in timothy grass pollen. OBJECTIVES: We sought to determine the occurrence and importance of group 13 allergens in various grass species and to investigate their proteolytic stability. METHODS: The group 13 allergens were determined by means of 2-dimensional PAGE blotting with patient sera and group 13-specific mAbs. The allergens were isolated chromatographically from several pollen extracts and analyzed by means of microsequencing. Cross-reactivity among various grass species was studied by using Western blots and immunoblot inhibition tests. The stability of the allergens was tested under defined extraction conditions. RESULTS: Group 13 allergens are detectable in all common grasses and show IgE cross-reactivity among them. The allergenic components were identified in the neutral pH range with molecular masses of 50 to 60 kd, and in the case of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5. Protein sequencing clearly confirms structural identities between different grass species, although individual variations are found. If low-molecular-mass components were depleted by means of gel filtration, a rapid degradation of group 13 allergens was observed. This is in contrast to other pollen allergens described thus far. CONCLUSION: Group 13 allergens are widespread and are major allergens in the grasses. Predicted from their primary structures, these allergens are polygalacturonases. This class of enzymes is already known from microorganisms, and these enzymes are recognized as potential inducers of asthma. Our studies indicate that the group 13 allergens show a considerable microheterogeneity and degradation, especially after depletion of low-molecular-mass components. One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.

Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen.

The knowledge of IgE-binding epitopes on allergen molecules is important for better understanding allergen-antibody interactions and, thus, for developing new strategies for immunotherapy. Our purpose was to more precisely define the number and structure of IgE-binding epitopes of a paradigmatic major grass pollen allergen. We performed an IgE-binding epitope mapping of rHol l 5, a group V pollen allergen of velvet grass (Holcus lanatus), with overlapping fragments (length between 15 and 186 amino acids), which were expressed in E. coli as MBP fusion proteins. Using sera of 65 grass pollen allergic patients, the fragments were analysed by immunoblotting for IgE reactivity. Specificity of antibody binding was confirmed by competitive blot inhibition assays. At least four different continuous IgE-binding epitopes were identified on small fragments (about 30 amino acids), and at least five different discontinuous IgE-binding epitopes on larger fragments, which were destroyed by further fragmentation. The fragments were differentially recognized by individual patients' sera. By investigating IgE-binding to one of the small fragments in more detail, we found further epitope regions on this fragment. It was noteworthy that IgE reactivity to small fragments was weak compared to large fragments or to the complete molecule. Competitive blot inhibition experiments showed that binding of IgE antibodies to the small fragments was specific but with lower avidity than to the complete rHol l 5. rHol l 5 harbours multiple discontinuous as well as continuous IgE-binding epitopes spread over the whole molecule, which were individually recognized by IgE antibodies from different patients. Low avidity of IgE antibodies to small fragments suggests that the continuous epitope regions do not represent the complete epitope and are most probably parts of discontinuous epitopes.

IgE binding to unique hazelnut allergens: identification of non pollen-related and heat-stable hazelnut allergens eliciting severe allergic reactions.

BACKGROUND: Usually hazelnut allergic patients suffer from the tree pollen associated oral allergy syndrome (OAS) caused by cross-reactive structures. Anaphylactic reactions elicited by hazelnuts happen rarely but are of high clinical significance. Considering that hazelnuts are ingredients in processed foods, hazelnuts may play an important role as hidden allergens for these high risk patients. Therefore, we analyzed the IgE reactivity of a young woman with severe allergic reactions after ingestion of hazelnuts without any association to tree pollen allergy. AIM OF THE STUDY: The aim of this study was to identify and characterize these potent hazelnut-specific allergens. We compared these allergens to structures displayed by sera from patients with a completely or partially non pollen-related hazelnut allergy and with birch pollen-related hazelnut allergy. None of the sera had a clinical history of anaphylaxis. Special emphasis was placed on the heat stability and cross-reactivity of these allergens. METHODS/RESULTS: Using Western blotting with extract from birch pollen and EAST inhibition techniques we were able to show that the allergens in the serum sample of the young woman were not cross-reactive with birch pollen. Immunoblot experiments with extracts from native and heated hazelnuts and EAST inhibition tests further characterized these allergens to be heat-stable. Unlike the IgE binding pattern of the sera from the patients with pollen-related hazelnut allergy, low molecular weight proteins below 10 kDa were identified by the sera from the patients without pollinosis. CONCLUSIONS: Since the binding pattern of the serum sample of the young woman was different from that of the sera from patients without pollen allergy but less severe symptoms, we assume an association between single non pollen-dependent hazelnut allergens in the low molecular range and severe allergic reactions. These results enable us to approach a subgroup of hazelnut allergens which we believe to be responsible for anaphylactic reactions in hazelnut allergic patients after ingestion of heat-stable hazelnut structures in processed food stuff, independent of pollinosis.

Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage.

We previously cloned a panel of peanut allergens by phage display technology. Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene. To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)(10)-tagged fusion proteins in conventional E. coli BL21(DE3) cells. The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8-10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells. Hence, by using modified BL21(DE3) E. coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E. coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage. IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E. coli hosts. The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)(10)-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells. This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells.

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen phl p 13 from timothy grass pollen (Phleum pratense).

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.

Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology.

BACKGROUND: Peanut kernels contain many allergens able to elicit IgE-mediated type 1 allergic reactions in sensitized individuals. Sera from sensitized patients recognize variable patterns of IgE-binding proteins. The identification of the IgE-binding proteins of peanut extract would faciliate improvement of diagnostic and immunotherapeutic approaches as well as development of sensitive test systems for the detection of hidden peanut allergens present as additives in various industrial food products and the investigation of their stability during processing of food products. METHODS: We applied the pJuFo cloning system based on the phage surface display of functional cDNA expression products to clone cDNAs encoding peanut allergens. Sera (n = 40) of peanut-allergic individuals were selected according to case history, radioallergosorbent test and immunoblot analysis to demonstrate IgE binding towards the newly identified recombinant allergens. RESULTS: In addition to the known allergens Ara h 1 and Ara h 2 we were able to identify four allergens with estimated molecular weights of 36, 16, 14.5 and 14 kDa. Three of them formally termed Ara h 4, Ara h6 and Ara h 7 show significant sequence similarities to the family of seed storage proteins and the fourth (Ara h 5) corresponds to the well-known plant allergen profilin. Immunoblotting of the six expressed recombinant allergens with 40 patients sera shows 14 individual recognition patterns and the following frequency of specific IgE binding: Ara h 1 was recognized by 65%, Ara h 2 by 85%, Ara h 4 by 53%, Ara h 5 by 13%, Ara h 6 by 38% and Ara h 7 by 43% of the selected sera. CONCLUSIONS: All of the selected peanut-positive sera can detect at least one of the six identified recombinant allergens which can be used to establish individual patients' reactivity profiles. A comparison of these profiles with the clinical data will possibly allow a further insight into the relationship between clinical severity of the symptoms and specific IgE levels towards the six peanut allergens.

Grass group I allergens (beta-expansins) are novel, papain-related proteinases.

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.