Walnut allergens: molecular characterization,detection and clinical relevance

Food‐induced allergies have been regarded as an emergent problem of public health. Classified as important allergenic ingredients, the presence of walnut and other nuts as hidden allergens in processed foods constitutes a risk for sensitized individuals, being a real problem of allergen management. Attending to the increasing importance dedicated to walnut allergy, this review intends to provide the relevant and up‐to‐date information on main issues such as the prevalence of walnut allergy, the clinical threshold levels, the molecular characterization of walnut allergens and their clinical relevance, as well as the methodologies for walnut allergen detection in foods. As the walnut used in human diet comes from Juglans regia and Juglans nigra, the molecular characterization of the allergens from both species included in the prolamins (Jug r 1, Jug n 1 and Jug r 3), cupins (Jug r 2, Jug n 2 and Jug r 4) and profilins (Jug r 5), together with respective clinical relevance, were compiled in this review. The most recent progresses on walnut allergen detection techniques (protein‐ and DNA‐based) are described and critically compared, including the emergent multitarget approaches.     

Systemic allergic reaction to coconut (Cocos nucifera) in 2 subjects with hypersensitivity to tree nut and demonstration of cross-reactivity to legumin-like seed storage proteins: New coconut and walnut food allergens

BACKGROUND: Two patients with tree nut allergy manifested by life-threatening systemic reactions reported the subsequent onset of systemic reactions after the consumption of coconut. OBJECTIVE: Herein, the IgE-binding proteins from coconut are described, and in vitro cross-reactivity with other nuts is investigated. METHODS: The IgE-binding profile of coconut endosperm tissue extract was analyzed by SDS-PAGE followed by immunoblotting. Immunoblot inhibition studies with walnut, almond, peanut, and coconut were performed. RESULTS: Sera IgE from both patients recognized reduced coconut allergens with molecular weights of 35 and 36.5 kd. IgE from 1 patient also bound a 55-kd antigen. Preabsorption of sera with nut extracts suppressed IgE binding to coconut proteins. Preabsorption of sera with coconut caused the disappearance of IgE binding to protein bands at 35 and 36 kd on a reduced immunoblot of walnut protein extract in 1 patient and suppression of IgE binding to a protein at 36 kd in the other patient. CONCLUSION: The reduced coconut protein at 35 kd was previously shown to be immunologically similar to soy glycinin (legumin group of seed storage proteins). The clinical reactivity in these 2 patients is likely due to cross-reacting IgE antibodies primarily directed against walnut, the original clinical allergy reported, and most likely to a walnut legumin-like protein. Coconut allergy in patients with tree nut allergy is rare; these are the first 2 patients ever reported, and therefore there is no general indication to advise patients with tree nut allergy to avoid coconut.     

Identification and cloning of a complementary DNA encoding a vicilin-like proprotein, jug r 2, from english walnut kernel (Juglans regia), a major food allergen

BACKGROUND: Walnuts and other tree nuts are important food-allergen sources that have the potential to be associated with life-threatening, IgE-mediated systemic reactions in some individuals. OBJECTIVE: The purpose of this study was to characterize a complementary (c)DNA clone encoding one of the walnut food allergens. METHODS: A cDNA expression library prepared from walnut somatic embryo was screened for IgE reactivity with patient serum. A reactive clone of 2060 bp, which encoded a protein of 593 amino acids in length, was subcloned by excision into the pGEX expression vector. IgE-binding inhibition experiments were performed. RESULTS: A recombinant fusion protein was induced and shown to bind serum IgE from 9 of 15 patients tested, thus identifying a major allergen. This clone, named Jug r 2, exhibited significant homology with genes encoding the vicilin group of seed proteins. An IgE-binding inhibition experiment suggested that the encoded protein undergoes posttranslational modification into at least one major polypeptide (47 kd) and possibly several others, which is similar to the vicilin-like proteins characterized in cocoa bean (Theobroma cacao) and cottonseed (Gossypium hirsutum). N-terminal sequencing of the 47-kd band, Jug r 2, identified it as a mature protein obtained from the precursor. A second IgE-binding inhibition experiment showed that there is minimal or no cross-reactivity between Jug r 2 and pea vicilin, peanut proteins, or cacao proteins. CONCLUSION: Jug r 2 is the third vicilin food allergen identified in addition to vicilins from soy and peanut. The availability of recombinant food allergens should help advance studies on the immunopathogenesis and possible treatment of IgE-mediated food hypersensitivity.     

Identification and characterisation of the IgE-binding proteins 2S albumin and conglutin gamma in almond (Prunus dulcis) seeds

BACKGROUND: Almond proteins can cause severe anaphylactic reactions in susceptible individuals. The aim of this study was the identification of IgE-binding proteins in almonds and the characterisation of these proteins by N-terminal sequencing. METHODS: Five sera were selected from individuals with a positive reaction to food challenge. Sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting were performed on almond seed proteins. Purified IgE-binding proteins were tested for immunoblot inhibition with sera pre-incubated with extracts of hazelnut and walnut. RESULTS: N-terminal sequences of the 12-, 30- and 45-kD proteins were obtained. The 45- and 30-kD proteins shared the same N terminus, with 60% homology to the conglutin gamma heavy chain from lupine seed (Lupinus albus) and to basic 7S globulin from soybean (Glycine max). The sequences of the N-terminal 12-kD protein and of an internal peptide obtained by endoproteinase digestion showed good homology to 2S albumin from English walnut (Jug r 1). Immunoblot inhibition experiments were performed and IgE binding to almond 2S albumin and conglutin gamma was detected in the presence of cross-reacting walnut or hazelnut antigens. CONCLUSIONS: Two IgE-binding almond proteins were N-terminally sequenced and identified as almond 2S albumin and conglutin gamma. Localisation and conservation of IgE binding in a 6-kD peptide obtained by endoproteinase digestion of 2S albumin was shown.     

Cloning and sequencing of a gene encoding a 2S albumin seed storage protein precursor from English walnut (Juglans regia), a major food allergen

Background: Walnuts rank third in per capita consumption of tree nuts in the United States and can be associated with systemic IgE-mediated reactions in some individuals. Objective: The objectives of the study were to clone a gene encoding one of the major food allergens in the walnut kernel and to characterize the recombinant allergen. Methods: A cDNA expression library in the lambda vector Uni-ZAP, which was prepared from walnut somatic embryos, was screened by using a patient's sera that reacted with multiple protein bands on immunoblotting. Results: A cDNA clone containing an insert of 663 bp was identified and named Jug r 1. DNA sequence analysis of this clone revealed that it encoded a protein 142 amino acids in length. Comparison of the encoded protein sequences with protein databases revealed that this clone exhibits a 46.1% identity with the Brazil nut (Bertholletia excelsa) methionine-rich 2S albumin seed storage protein precursor, Ber e 1. Jug r 1 appears to be an important walnut food allergen; 12 of 16 sera from patients allergic to walnuts demonstrated IgE binding to the 2S albumin seed storage protein precursor fusion protein. An IgE-binding inhibition study suggests that the walnut 2S protein precursor undergoes posttranslational modification into a large and small subunit that is similar to castor seed, cottonseed, mustard seed, and Brazil nut 2S seed storage protein allergens. Interestingly, the gene encoding this allergenic protein in Brazil nuts has recently gained notoriety because of its experimental use as a transgene to enhance the nutritional quality of legumes. Conclusion: This is now the sixth definitive 2S albumin seed storage protein demonstrated to bind IgE, suggesting that this class of proteins is inherently allergenic. (J Allergy Clin Immunol 1998;101:807-814.)     

Identification and cloning of a complementary DNA encoding a vicilin-like proprotein, Jug r 2, from English walnut kernel (Juglans regia), a major food allergen

Background: Walnuts and other tree nuts are important food-allergen sources that have the potential to be associated with life-threatening, IgE-mediated systemic reactions in some individuals. Objective: The purpose of this study was to characterize a complementary (c)DNA clone encoding one of the walnut food allergens. Methods: A cDNA expression library prepared from walnut somatic embryo was screened for IgE reactivity with patient serum. A reactive clone of 2060 bp, which encoded a protein of 593 amino acids in length, was subcloned by excision into the pGEX expression vector. IgE-binding inhibition experiments were performed.Results: A recombinant fusion protein was induced and shown to bind serum IgE from 9 of 15 patients tested, thus identifying a major allergen. This clone, named Jug r 2, exhibited significant homology with genes encoding the vicilin group of seed proteins. An IgE-binding inhibition experiment suggested that the encoded protein undergoes posttranslational modification into at least one major polypeptide (47 kd) and possibly several others, which is similar to the vicilin-like proteins characterized in cocoa bean (Theobroma cacao) and cottonseed (Gossypium hirsutum ). N-terminal sequencing of the 47-kd band, Jug r 2, identified it as a mature protein obtained from the precursor. A second IgE-binding inhibition experiment showed that there is minimal or no cross-reactivity between Jug r 2 and pea vicilin, peanut proteins, or cacao proteins. Conclusion: Jug r 2 is the third vicilin food allergen identified in addition to vicilins from soy and peanut. The availability of recombinant food allergens should help advance studies on the immunopathogenesis and possible treatment of IgE-mediated food hypersensitivity. (J Allergy Clin Immunol 1999;1311-20.)     

Linear IgE epitope mapping of the English walnut (Juglans regia) major food allergen, Jug r 1.

BACKGROUND: Peanut and tree nut allergies can be life-threatening, and they appear to be growing in prevalence. Jug r 1, a 2S albumin seed storage protein, was previously characterized as a major English walnut food allergen. OBJECTIVE: We sought to identify the linear IgE-binding epitopes of Jug r 1 and to determine which, if any, amino acids are necessary for this binding to occur. METHODS: Pools of sera from walnut-allergic patients and overlapping peptides synthesized on an activated cellulose membrane were used to screen for IgE-binding epitopes. Mutational analysis of the immunodominant epitope was carried out through single and multisite amino acid substitutions. Inhibition assays were performed through use of affinity-purified IgE, soluble forms of the epitope peptide, and the recombinant 2S albumin, rJug r 1. RESULTS: One immunodominant linear epitope was identified. Amino acid mutations to the epitope demonstrated that the residues RGEE, at positions 36 through 39, were minimally required for IgE binding. Probing of this epitope with sera from each of 20 patients revealed 15 of the sera to be positive. Binding of patients' IgE to the epitope was inhibited with a soluble form of the peptide; however, soluble peptide did not completely inhibit the binding of IgE to the intact rJug r 1. CONCLUSION: One major linear IgE-reactive epitope and its critical core amino acid residues have been identified. Mutation of any of these core amino acids resulted in loss of IgE binding to the epitope, and this points toward the feasibility of reducing allergenicity in genetically modified walnuts. However, strong evidence for the existence of conformational epitopes was also obtained.     

Identification of sesame seed allergens by 2-dimensional proteomics and Edman sequencing: seed storage proteins as common food allergens

BACKGROUND: Sesame seed allergy is becoming increasingly prevalent, probably because of its use in international fast-food and bakery products. Despite this fact, few studies have focused on the identification of its major allergenic proteins. OBJECTIVE: The aim of this study was to identify allergenic proteins of sesame seeds (Sesamum indicum). METHODS: Extracted sesame seed proteins were separated by means of SDS-PAGE and 2-dimensional (2-D) PAGE. Immunolabeling was performed with individual patient sera from 20 patients with sesame seed allergy. Selected proteins were further analyzed by means of Edman sequencing. RESULTS: IgE-binding proteins were identified at 78, 52, 45, 34, 32, 29, 25, 20, 9, and 7 kd. Analyzing internal sequences, the protein at 45 kd, which was recognized by 75% of the patients, was found to be a 7S vicilin-type globulin, a seed storage protein of sesame and named Ses i 3. The protein at 7 kd was found to be a 2S albumin, another seed storage protein of sesame and named Ses i 2. Seed storage proteins are known food allergens in peanut, walnut, Brazil nut, and soybean. Interestingly, one known IgE-binding epitope of the peanut allergen Ara h 1 has 80% homology with the corresponding area of Ses i 3. The different amino acids were previously shown not to be critical for IgE binding in Ara h 1. In addition, the proteins at 78 and 34 kd were found to be homologous to the embryonic abundant protein and the seed maturation protein of soybeans, respectively. CONCLUSION: The identification of 4 sesame seed allergens is the first step toward generating recombinant allergens for use in future immunotherapeutic approaches. In addition, the detection of conserved IgE binding epitopes in common food allergens might be a useful tool for predicting cross-reactivity to certain foods.     

Homology modelling of the major peanut allergen Ara h 2 and surface mapping of IgE-binding epitopes

Three-dimensional models built for the peanut Ara h 2 allergen and other structurally-related 2S albumin allergens of dietary nuts exhibited an overall three-dimensional fold stabilized by disulphide bridges well conserved among all the members of the 2S albumin superfamily. Conformational analysis of the linear IgE-binding epitopes mapped on the molecular surface of Ara h 2 showed no structural homology with the corresponding regions of the walnut Jug r 1, the pecan nut Car i 1 or the Brazil nut Ber e 1 allergens. The absence of epitopic community does not support the allergenic cross-reactivity observed between peanut and walnut or Brazil nut, which presumably depends on other ubiquitous seed storage protein allergens, namely the vicilins. However, the major IgE-binding epitope identified on the molecular surface of the walnut Jug r 1 allergen shared a pronounced structural homology with the corresponding region of the pecan nut Car i 1 allergen. With the exception of peanut, 2S albumins could thus account for the IgE-binding cross-reactivity observed between some other dietary nuts, e.g. walnut and pecan nut.     

A soybean G2 glycinin allergen. 1. Identification and characterization

BACKGROUND: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, was shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 21 kD. These findings suggested that unique proteins in soybeans might be responsible for soybean allergic reactivity. The objective of the present study was to identify unique proteins in soybean extracts that bind to specific IgE from soybean-sensitive individuals, and to characterize the allergen using physicochemical methods and IgE binding. METHODS: Two-dimensional and preparative SDS-PAGE/IgE immunoblot analysis was used to identify a 22-kD soybean-specific allergen from crude soybean extracts. N-terminal sequence analysis was used to determine the identification of the protein binding IgE from soybean-sensitive individuals. RESULTS: IgE immunoblot and amino acid sequence analysis identified the 22-kD protein as a member of the G2 glycinin soybean protein family. Further investigation revealed that the IgEs reacted with basic chains from each member of the glycinin family of soybean storage proteins. CONCLUSIONS: Each of the subunits from glycinin, the storage protein that is the most prevalent component of soybean, are major allergens.     

Anaphylaxis to lemon soap: citrus seed and peanut allergen cross-reactivity.

BACKGROUND: Many individuals allergic to peanuts have multiple allergen sensitivity. OBJECTIVE: To report the first case, to our knowledge, of a peanut allergic patient who exhibited cosensitivity to citrus seeds and who had experienced anaphylaxis to lemon soap. METHODS: Extracts of peanut and seeds from different varieties of citrus fruit (orange, lemon, and mandarin) were prepared and resolved with 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Direct and inhibition immunoblotting of the patient's serum on the extracts was used to examine the pattern of IgE reactivity and the presence of cross-reactive allergens. RESULTS: Numerous IgE reactive proteins were demonstrated in each citrus seed extract and the peanut extract. Complete IgE cross-reactivity was demonstrated among the different citrus seed extracts. Partial cross-reactivity was demonstrated between the peanut and orange seed extracts. CONCLUSIONS: Citrus seeds contain numerous IgE reactive proteins that are completely cross-reactive among orange, lemon, and mandarin. When peanut allergy coexists with citrus seed allergy, IgE cross-reactivity between peanut and citrus seed proteins can be demonstrated, suggesting a basis to this cosensitivity.     

Identification of a 7S globulin as a novel coconut allergen.

BACKGROUND: Coconut (Cocos nucifera) is a monocotyledonous plant of the Arecaceae family. Allergy to coconut is infrequent, with only 5 cases reported so far in the medical literature. OBJECTIVE: To identify coconut allergens in 2 patients allergic to this food. METHODS: We describe 2 patients allergic to coconut: an adult pollen-allergic patient monosensitized to coconut who presented with severe oropharyngeal symptoms and a child with a previous allergy to walnut, not allergic to pollen, who developed anaphylaxis on coconut ingestion. Both patients had positive skin prick test results and serum specific IgE (CAP) to coconut. IgE sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting was performed to identify the allergens involved, and a strong IgE binding band detected in both patients was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Stability to pepsin digestion of the coconut extract and its cross-reactivity with tree nuts were studied. RESULTS: An immunoblot showed an almost identical profile of IgE binding proteins in the coconut extract in both patients who reacted strongly to a band of approximately 29 kDa. The peptide analysis by MALDI-TOF MS of this band obtained the sequence GHGKREDPEKR. The protein with the highest correlation with this peptide was found to be a 7S globulin from Elaeis guineensis, another oil palm species also belonging to the Arecaceae family. The 29-kDa band was digested by pepsin in less than 1 minute. Cross-reactivity among coconut, walnut, and hazelnut was demonstrated by CAP inhibition in patient 2. CONCLUSION: We have identified a 7S storage protein as a novel coconut allergen.     

Ses i 6, the sesame 11S globulin, can activate basophils and shows cross-reactivity with walnut in vitro

BACKGROUND: Sesame allergy is increasingly being reported, and multi-sensitization to peanut and tree nuts has been described. The clinical relevance and cross-reactivity of many sesame proteins, such as Ses i 6, are unknown. OBJECTIVE: The aims of this study were to perform a preliminary examination of the cross-reactivity of Ses i 6 in vitro, examine the ability of Ses i 6 to activate basophils in a modified basophil activation test (mBAT), and assess whether such an assay may help to distinguish between potentially relevant and irrelevant IgE reactivity towards 11S globulin proteins. METHODS: Inhibition immunoblotting and chicken anti-rJug r 4 antibodies were used to determine the cross-reactivity of rSes i 6. Basophils from atopic donors were stripped of resident IgE before passive sensitization with food-allergic sera and challenged with protein extracts or recombinant protein. Basophil activation was measured using two activation markers, CD203c and CD63, via flow cytometry. RESULTS: IgE immunoblotting showed cross-reactivity between rJug r 4 and rSes i 6 using sera from two human donors and chicken IgY. Additionally, rSes i 6 activated basophils passively sensitized with sesame-allergic sera. Cross-reactive serum from a sesame-allergic but walnut-tolerant donor was not able to activate basophils when challenged by walnut extract despite IgE reactivity to walnut determined by immunoblotting. CONCLUSIONS: The sesame 11S globulin shows partial immunological cross-reactivity with walnut, and although it is classified as a minor allergen, activated basophils sensitized with serum from seven out of eleven sesame-allergic donors. Additionally, the mBAT may help distinguish between clinically relevant and irrelevant in vitro IgE cross-reactivity of seed storage proteins in nuts and seeds and thus warrants use in further studies.     

IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries

BACKGROUND: Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. OBJECTIVE: We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. METHODS: Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. RESULTS: IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. CONCLUSION: The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.     

Homology modelling and conformational analysis of IgE-binding epitopes of Ara h 3 and other legumin allergens with a cupin fold from tree nuts

Linear IgE-binding epitopes identified in legumin allergens of peanut (Ara h 3) and other allergenic tree nuts (Jug r 4 of walnut, Cor a 9 of hazelnut, Ana o 2 cashew nut) were mapped on three-dimensional models of the proteins built up by homology modelling. A conformational analysis revealed that consensual surface-exposed IgE-binding epitopes exhibited some structural homology susceptible to account for the IgE-binding cross-reactivity observed among peanut and tree nut allergens. This structurally related cross-reactivity seems irrespective of the botanical origin of the allergens and thus demands that persons allergic to peanut avoid other three nuts to prevent possible allergic reactions. IgE-binding epitopes similar to those found in 11S globulin allergens do not apparently occur in other vicilin allergens with the cupin fold from peanut (Ara h 1) or tree nuts (Jug r 2 of walnut, Cor a 1 of hazel nut, Ana o 3 of cashew nut).     

The atopic dog as a model of peanut and tree nut food allergy

BACKGROUND: Animal models are needed that mimic human IgE-mediated peanut and tree nut allergy. Atopic dogs have been previously used in a model of food allergy to cow's milk, beef, wheat, and soy, with the demonstration of specific IgE production and positive oral challenges similar to those seen in human subjects. OBJECTIVE: We sought to sensitize dogs to peanut, walnut, and Brazil nut and to assess whether sensitization is accompanied by clinical reactions and whether there is cross-reactivity among the different preparations. METHODS: Eleven dogs were sensitized subcutaneously by using an established protocol with 1 microg each of peanut, English walnut, or Brazil nut protein extracts in alum first at birth and then after modified live virus vaccinations at 3, 7, and 11 weeks of age. The dogs were sensitized to other allergens, including soy and either wheat or barley. Intradermal skin tests, IgE immunoblotting to nut proteins, and oral challenges were performed with ground nut preparations. RESULTS: At 6 months of age, the dogs' intradermal skin test responses were positive to the nut extracts. IgE immunoblotting to peanut, walnut, and Brazil nut showed strong recognition of proteins in the aqueous preparations. Each of the 4 peanut- and the 3 Brazil nut-sensitized dogs and 3 of the 4 walnut-sensitized dogs reacted on oral challenge with the corresponding primary immunogen at age 2 years. None of the peanut-sensitized dogs reacted clinically with walnut or Brazil nut challenges. One of the walnut-sensitized dogs had delayed (overnight) vomiting to Brazil nut. CONCLUSIONS: On the basis of measurements of the mean amount of allergen eliciting a skin test response in dogs, the hierarchy of reactivity by skin testing is similar to the clinical experience in human subjects (peanut > tree nuts > wheat > soy > barley). Cross-reactivity, which was not apparent between soy and peanut or tree nuts or between peanut and tree nuts, was slight between walnut and Brazil nut. The results give further support to the dog as a model of human food allergy.     

Immunoglobulin E (IgE)-mediated cross-reactivity between mesquite pollen proteins and lima bean, an edible legume

Immunoglobulin E (IgE)-mediated food allergy often develops as a consequence of allergic sensitization to pollen proteins. Mesquite (Prosopis juliflora) tree pollen is reported to be cross-reactive with other pollen species, but little has been reported on its cross-reactivity with plant-derived foods belonging to the same/different families. The present study investigates the in vitro cross-reactivity of mesquite pollen and lima bean (Phaseolus lunatus), an edible seed belonging to the Leguminosae family. Of 110 patients (asthma, rhinitis or both) tested intradermally, 20 showed marked positive reactions with Prosopis pollen extract. Of these, 12 patients showed elevated specific IgE to Prosopis pollen extract alone and four to both Phaseolus and pollen extract. In vitro cross-reactivity was investigated using inhibition assays [enzyme-linked immunosorbent assay (ELISA) inhibition, immunoblot inhibition], histamine release and lymphoproliferation. P. lunatus extract could inhibit IgE binding to P. juliflora in a dose-dependent manner, requiring 400 ng of protein for 50% inhibition in ELISA assay. Immunoblot and immunoblot inhibition demonstrated the presence of 20, 26, 35, 66 and 72 kDa as shared IgE binding components between the two extracts. Histamine release, peripheral blood mononuclear cells proliferation and interleukin (IL)-4 levels also suggested allergenic cross-reactivity. In conclusion, there is humoral and cellular cross-reactivity between Prosopis pollen and Phaseolus seed allergens.     

Engineering, characterization and in vitro efficacy of the major peanut allergens for use in immunotherapy

BACKGROUND: Numerous strategies have been proposed for the treatment of peanut allergies, but despite the steady advancement in our understanding of atopic immune responses and the increasing number of deaths each year from peanut anaphylaxis, there is still no safe, effective, specific therapy for the peanut-sensitive individual. Immunotherapy would be safer and more effective if the allergens could be altered to reduce their ability to initiate an allergic reaction without altering their ability to desensitize the allergic patient. METHODS: The cDNA clones for three major peanut allergens, Ara h 1, Ara h 2, and Ara h 3, have been cloned and characterized. The IgE-binding epitopes of each of these allergens have been determined and amino acids critical to each epitope identified. Site-directed mutagenesis of the allergen cDNA clones, followed by recombinant production of the modified allergen, provided the reagents necessary to test our hypothesis that hypoallergenic proteins are effective immunotherapeutic reagents for treating peanut-sensitive patients. Modified peanut allergens were subjected to immunoblot analysis using peanut-positive patient sera IgE, T cell proliferation assays, and tested in a murine model of peanut anaphylaxis. RESULTS: In general, the modified allergens were poor competitors for binding of peanut-specific IgE when compared to their wild-type counterpart. The modified allergens demonstrated a greatly reduced IgE-binding capacity when individual patient serum IgE was compared to the binding capacity of the wild-type allergens. In addition, while there was considerable variability between patients, the modified allergens retained the ability to stimulate T cell proliferation. CONCLUSIONS: These modified allergen genes and proteins should provide a safe immunotherapeutic agent for the treatment of peanut allergy.     

Potential allergens of green gram (Vigna radiata L. Millsp) identified as members of cupin superfamily and seed albumin

Background No systematic study on allergenicity of green gram seed proteins have been performed so far, although incidences of IgE‐mediated reaction to green gram seedlings have been reported. Objective We sought to investigate the allergenic potential of green gram, followed by identification and characterization of its relevant allergens using proteomic approaches. Methods BALB/c mice were sensitized intraperitoneally with green gram proteins, and levels of specific Igs, Th2 cytokines, histamine, anaphylactic symptoms and histopathological responses were studied. Twelve naso‐bronchial allergic patients with a history of sensitization to green gram were selected on the basis of positive skin prick test and elevated specific IgE levels. Green gram allergens were identified and characterized by their ability to endure pepsin, by IgE immunoblot of two‐dimensional (2D) gels in combination with mass spectrometry and by bioinformatics approaches. Results Increased specific IgE, IgG1, Th2 cytokine and histamine levels, high anaphylactic scores and histological changes in lungs and spleen of green gram crude protein extract‐treated mice are indicative of its sensitization ability. Four proteins (molecular weights: 52, 50, 30 and 18 kDa) showed pepsin resistance and IgE‐binding capability with sensitized human and mice sera. The four proteins tentatively named as Vig r2 (52 kDa, pI 5.7), Vig r3 (50 kDa, pI 5.8), Vig r4 (30 kDa, pI 6.6) and Vig r5 (18 kDa, pI 5.5) showed significant sequence similarity with known allergens of soybean, lentil, pea, lupin, etc. Mass spectrometric analysis identified Vig r2 as 8S globulin β‐isoform precursor, Vig r3 as 8S globulin α‐isoform precursor and Vig r4 as seed albumin. Conclusion and Clinical Relevance Green gram seeds contain at least four clinically relevant allergenic proteins, namely Vig r2, Vig r3, Vig r4 and Vig r5 that were capable of inducing strong IgE‐mediated reactions. One of the most important steps towards diagnostic and therapeutic approaches to deal effectively with food allergy is continued identification of newer food allergens and their characterization. The significance of this study can be enormous as the data generated may work as basic biology data in developing a green gram species modified genetically that may have reduced allergenicity. Cite this as: A. Misra, R. Kumar, V. Mishra, B. P. Chaudhari, S. Raisuddin, M. Das and P. D. Dwivedi, Clinical & Experimental Allergy, 2011 (41) 1157–1168.     

Detection of immunoglobulin antibodies in the sera of patients using purified latex allergens

BACKGROUND: Latex allergy is largely an occupational allergy due to sensitization to natural rubber latex allergens present in a number of health care and household products. Although several purified allergens are currently available for study, information on the usefulness of these purified, native or recombinant allergens in the demonstration of specific immunoglobulin (Ig) E in the sera of patients is lacking. OBJECTIVE: To evaluate the purified latex allergens and to demonstrate specific IgE antibody in the sera of health care workers and spina bifida patients with clinical latex allergy. METHODS: Two radioallergosorbent and an enzyme-linked immunosorbent assay (ELISA) using latex proteins Hev b 1, 2, 3, 4, 6 and 7 along with two glove extracts and Malaysian nonammoniated latex (MNA) were evaluated to demonstrate IgE in the sera of health care workers and spina bifida with latex allergy and controls with no history of latex allergy. RESULTS: ELISA using the purified latex allergens demonstrated specific IgE in 32-65% health care workers and 54-100% of spina bifida patients with latex allergy. The corresponding figures for RAST were 13-48 and 23-85 for RAST-1 and 19-61 and 36-57 for RAST-2. These results were comparable with the results obtained with glove extracts and crude rubber latex proteins. CONCLUSIONS: When used simultaneously, latex proteins Hev b 2 and Hev b 7 reacted significantly with specific serum IgE in 80% of health care workers and 92% of spina bifida patients with latex allergy by ELISA technique, while this combination gave lower positivity when the RASTs were used. By the addition of Hev b 3, specific IgE was detected in all spina bifida patients with latex allergy. Both RASTs failed to show specific IgE in the control subjects, while the ELISA showed significant latex-specific IgE in 22% of controls.