IgE-binding Components Research Articles

Identification of Epicoccum purpurascens allergens by two-dimensional immunoblotting and mass spectrometry

Sensitization to allergens of Epicoccum purpurascens is reported from many places world over. Previously, the IgE binding proteins of Epicoccum were studied by one-dimensional immunoblotting (1-DE). However, the allergens recognized by 1-DE may be a mixture of proteins appearing at the same molecular weight. In the present study, IgE-binding components of E. purpurascens were detected using two-dimensional immunoblotting and mass spectrometry. Approximately 250 protein spots were identified by Coomassie staining in the range of 14–110 kDa and pI 3.5–9.5. Of these, 147 showed specific IgE reactivity with pooled patients’ sera whereas 16 protein components reacted with more than 50% of the individual patients’ sera. The frequency of IgE reactivity was highest (87.5%) for a 25-kDa protein (pI-7.85). Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of 16 protein components led to identification of 6 important allergens as phospho-glycerate kinase (PGK), RNA-dependent RNA polymerase, pyrroline-5-carboxylate dehydrogenase, 40S ribosomal protein and two proteins of unknown functions. The major allergens identified may be assessed for cross reactivity with other fungi. In conclusion, 2-DE followed by immunoblotting allowed characterization of E purpurascens allergens. Six new allergens have been identified using this technique and mass spectrometry. The availability of such relevant allergens would help in component-based diagnosis and therapy of fungal allergy.

Mala s 12 is a major allergen in patients with atopic eczema and has sequence similarities to the GMC oxidoreductase family*

Atopic eczema (AE) is a chronic inflammatory skin disorder, characterized by impaired skin barrier and itch. The yeast Malassezia belongs to the normal human skin microflora and can induce IgE- and T-cell-mediated allergic reactions in AE patients. Previously, we have identified several IgE-binding components in Malassezia sympodialis extract. Here, we report cloning, production and characterization of a M. sympodialis 67-kDa allergen. The sequence of the 67-kDa protein, termed Mala s 12, showed sequence similarity to the glucose-methanol-choline (GMC) oxidoreductase enzyme superfamily and was expressed as a recombinant protein in Escherichia coli. The purified protein bound flavin adenine dinucleotide with 1:1 stoichiometry per monomer of protein. The protein-bound flavin showed an extinction coefficient at 451 nm of 11.3 mM(-1)cm(-1). The recombinant 67-kDa protein did not show any enzymatic activity when tested as oxidase or dehydrogenase using choline, glucose, myo-inositol, methanol, ethanol, 1-pentanol, benzyl alcohol, 2-phenylethanol, cholesterol or lauryl alcohol as possible substrates. Recombinant Mala s 12 was recognized by serum IgE from 13 of 21 (62%) M. sympodialis-sensitized AE patients indicating that the 67-kDa component is a major allergen. The data show that Mala s 12 has sequence similarity to the GMC oxidoreductase family and is a major allergen in AE patients.

Allergic Asthma and Rhinitis Caused by Household Rabbit Exposure: Identification of Serum-Specific IgE and Its Allergens

Although rabbits are common domestic pets, severe respiratory allergic reactions to rabbits in households are unusual. Ory c 1, a 17-kDa glycoprotein found in saliva and fur, has previously been identified as a major rabbit allergen. In this report, we describe the cases of three patients with rabbit allergy who presented with asthma and/or rhinitis while living in households with detectable levels of serum-specific IgE and major IgE binding components. Three patients with rabbit allergy and 18 unexposed nonatopic healthy controls were enrolled. Enzyme-linked immunosorbent assays (ELISA) for serum-specific IgE and IgG4 to rabbit epithelium and inhibition ELISA were performed followed by sodium dodecye sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE immunoblotting. All three patients with rabbit allergy had high serum-specific IgE antibody levels compared with controls. The results of the inhibition ELISA showed significant inhibition with the addition of rabbit epithelium, whereas no significant inhibition was noted with the addition of cat and dog epithelia. Two IgE-binding components with molecular weights of 16 kDa and 67.5 kDa were identified by IgE immunoblotting. In conclusion, rabbit exposure may induce IgE-mediated bronchial asthma and/or rhinitis in domestic settings.

Allergic Asthma and Rhinitis Caused by Household Rabbit Exposure: Identification of Seum-Specific IgE and Its Allergens

RATIONALE: Although rabbits are common domestic pets, severe respiratory allergic reactions to rabbits in households are unusual. Ory c 1, a 17-kDa glycoprotein found in saliva and fur, has previously been identified as a major rabbit allergen. In this report, we describe the cases of three patients with rabbit allergy who presented with asthma and/or rhinitis while living in households with detectable levels of serum-specific IgE and major IgE binding components.

Rice ( Oryza sativa) allergy in rhinitis and asthma patients: A clinico-immunological study

Sensitization to foods varies in different countries reflecting a possible interaction of genetic factors, cultural and dietary habits. Rice is a major food consumed world wide and needs evaluation for IgE mediated reactions. The present study was carried out to identify rice allergy in patients of rhinitis and asthma and identify the allergenic proteins in raw and cooked rice. Of 1200 patients screened using standard questionnaire, 165 presented with history of rice allergy. Of these, 20 (12.1%) patients demonstrated marked positive skin prick test (SPT) and 13 showed significantly raised specific IgE to rice compared to normal controls. Double blind placebo controlled food challenge (DBPCFC) confirmed rice allergy in 6/10 patients. Immunoblot with hypersensitive individual patients’ sera showed 14–16, 33, 56 and 60 kDa proteins as major IgE-binding components in rice. Boiled rice retained four IgE reactive proteins of 16, 23, 33 and 53 kDa. In summary, IgE-mediated rice allergy affects 0.8% [(0.42–1.58) at 95% CI] of asthma and rhinitis cases. The subjects with severe SPT reactions (4 mm or above) and specific IgE, 6.9 ng/ml to rice demonstrated positive blinded food challenge with clinical symptoms.

Natural rubber latex and hymenoptera venoms share ImmunoglobinE‐epitopes accounting for cross‐reactive carbohydrate determinants

Epidemiological data on the prevalence and risk factors of latex sensitization have suggested a significant association between latex sensitization and the presence of one or more positive skin prick test responses to aeroallergens, food allergens and to one or more insect venoms. Xylose and core 3-fucose are typical complex glycans in plants and are foreign to mammals. Plant N-glycans and insect N-glycans may cross-react in humans. The aim of our study was to investigate whether there are cross-reactive IgE-binding structures in natural rubber latex (NRL) and hymenoptera venoms and to examine their nature. Hundred and twenty-five consecutive patients with insect venom allergy were screened for coincidental latex-specific IgE. IgE-binding components in the venoms from Apis mellifera and/or vespula species and in NRL extracts were characterized by IgE-immunoblotting to the natural allergen sources and determination of specific IgE to recombinant allergens. Cross-reactive components were investigated by inhibition experiments. The involvement of carbohydrates in the constitution of cross-reactive IgE-epitopes was further examined by specific IgE-binding to cross-reactive carbohydrate determinants (CCD) in bromelain and horseradish peroxidase as well as by periodate treatment. NRL glove extracts inhibited patients' serum IgE-binding to venom allergens. Vice versa, the IgE-binding to latex glove extracts could be inhibited by pre-incubation with the insect venoms. Specific IgE-binding to recombinant latex allergens was absent, whereas the cross-reactive IgE-epitopes were sensitive to periodate treatment and specific IgE to CCD (MMXF and MUXF type) could be detected. Insect venoms and NRL share IgE-binding CCD that may be responsible for positive serological test results to NRL in patients with insect venom allergy. This copositivity occurs frequently (13.6%) among venom-allergic individuals and did not elicit clinical symptoms upon contact to latex in the patients examined. In contrast, true cosensitization to insect venoms and NRL allergens can occur and may not be missed.

Biopotency and identification of allergenic proteins in Periplaneta americana extract for clinical applications

Commercial cockroach extracts for diagnosis and therapy show batch-to-batch variation. This study aimed to standardize Periplaneta americana extract based on major IgE binding components using hypersensitive patients' sera. Extracts were prepared in phosphate buffered saline (PBS) or NH 4HCO 3, from freeze-dried or 37 °C dried material and compared with commercial extracts by immunobiochemical methods. Cockroach positive patients' sera were collected after intradermal tests and specific IgE enzyme linked immunosorbent assay (ELISA). Allergenic proteins were identified by western-blotting and potency of extracts determined by ELISA-inhibition. Adult P. americana extract from freeze dried source material in PBS (PA extract) resolved into 45 protein bands and showed 22 IgE binding components with pooled patients' sera. It required 9–12 ng self-proteins for 50% ELISA-inhibition. Individual patients' sera identified 23, 28, 35, 38, 40, 49, 72, 78 and 97 kDa as major IgE binding components in PA extract. Nymph extract exhibited similar potency and protein profile to PA extract with 72 and 78 kDa proteins present in high intensities. Commercial extracts exhibited only 6–11 IgE reactive bands compared to PA extract and required 40 folds or more protein for 50% ELISA-inhibition. PA extract from freeze-dried source material seems a potent allergen preparation with 9-major IgE binding components. It can be referred to upgrade the quality of commercial extracts exhibiting low potencies due to poor quality source material, inadequate extraction procedures and improper storage.

An amplified sandwich EIA for the measurement of soy aeroallergens

Soy hull low-molecular-weight (SHLMW) allergens were responsible for the soy asthma epidemics in Barcelona, with one 7.5 kDa protein (Gly m 1) being the main IgE-binding component. The aims of this study were to develop a sensitive sandwich enzyme immunoassay (EIA) using rabbit polyclonal antibodies to measure low levels of SHLMW allergens, and to compare this method with the previously described human IgE EIA-inhibition technique. IgG was isolated from serum of rabbits immunized with a chromatographically purified SHLMW extract (SHLMWE). Antibody-binding profiles were compared with those of human IgE anti-soy protein antibodies by Western blot analysis. An amplified sandwich EIA was developed using the purified SHLMWE as a calibration standard. Results were expressed in nanograms per millilitre. To compare the two assays, 54 air samples were analysed by both methods. SDS-PAGE of the SHLMWE revealed four bands of 6, 8, 15 and 17 kDa. Gly m 1 in the SHLMWE was identified by fingerprinting. The detection limit of the assay was 40 pg/mL. The two methods correlated well (r=0.89; P<0.001). The allergen concentration was detected in all 54 (100%) samples by the sandwich EIA but in only 37 (68.5%) by the EIA inhibition. The amplified sandwich EIA for SHLMW components has a high sensitivity and appeared to be a useful tool for the measurement of airborne SHLMW allergens, even at relatively low concentrations. Moreover, the method uses rabbit antibodies at high dilutions and does not require human sera, with limited availability and quantitative and qualitative pool-to-pool variability.

Sensitization due to Gum Arabic (Acacia senegal): The Cause of Occupational Allergic Asthma or Crossreaction to Carbohydrates?

Background: A pharmaceutical industry worker was exposed to dust of gum arabic in the tablet coating plant and complained of work-related shortness of breath, chest tightness, runny nose, itching and redness of the eyes. This case was investigated for allergy to gum arabic and compared with a control group. The aim of the study was to identify the IgE-binding components responsible for the work-related symptoms. Methods: Skin prick tests (SPTs)and specific IgE (sIgE) measurements with environmental and occupational allergens, spirometry and a specific bronchial challenge with gum arabic were performed. One hundred and nineteen control subjects underwent SPT with gum arabic and 43 controls were tested for sIgE. Crossreactivity between gum arabic and horse radish peroxidase was investigated by IgE CAP inhibition. A combined procedure of immunoblotting and periodate treatment was applied to identify the epitope nature of gum arabic. Results: Allergy to gum arabic was shown by SPT, presence of sIgE and a positive bronchial challenge with gum arabic. Sensitization to gum arabic was demonstrated by SPT or sIgE in 7 and 5 controls, respectively. The results of inhibition with horse radish peroxidase, immunoblotting and periodate treatment suggest that gum arabic sIgE of the patient and 1 SPT-positive control subject were directed to the polypeptide chains of gum arabic. In contrast, gum arabic sIgE of the other controls reacted to carbohydrate components. Conclusions: Sensitization to gum arabic carbohydrate structures occurs casually in atopic patients with pollen sensitization without obvious exposure to gum arabic. This study suggests that allergy to gum arabic is mediated preferentially by IgE antibodies directed to polypeptide chains of gum arabic.

Higher pH level, corresponding to that on the skin of patients with atopic eczema, stimulates the release of Malassezia sympodialis allergens

The opportunistic yeast Malassezia is a trigger factor in atopic eczema (AE). Around 30-80% of patients with AE have an IgE and/or T-cell reactivity to the yeast. Several IgE-binding components have been identified in Malassezia extracts and 11 allergens have been cloned and sequenced. The pH of the skin surface in patients with AE is higher than that of normal healthy skin. We here investigate whether different pH conditions mimicking those of AE skin and healthy skin can influence the production and release of Malassezia allergens. Malassezia sympodialis (ATCC strain 42132) was cultured in Dixon broth at pH 6.1 to 5.0 for 1-15 days. Culture supernatants were analysed for the presence of IgE-binding components by immunoblotting. The M. sympodialis cells were analysed for allergen expression and production with immunocytochemistry and quantitative polymerase chain reaction. We found that M. sympodialis cells produce, express and release allergens to a greater extent when cultured at the higher pH. This was particularly true of a 67-kDa major allergen designated Mala s 12. The data suggest that the skin barrier in AE patients provides an environment that can enhance the release of allergens from M. sympodialis, which can contribute to the inflammation.

Evaluation of the sensitization rates and identification of IgE-binding components in wild and genetically modified potatoes in patients with allergic disorders

BackgroundThe potato is one of the most common types of genetically modified (GM) food. However, there are no published data evaluating the impact of genetic manipulations on the allergenicity of GM potatoes. To compare the allergenicity of GM potatoes with that of wild-type potatoes using in vivo and in vitro methods in adult allergy patients sensitized to potatoes.MethodsA total of 1886 patients with various allergic diseases and 38 healthy controls participated in the study. Skin-prick testing and IgE-ELISA were carried out with extracts prepared from wild-type and GM potatoes. An ELISA inhibition test was used to confirm the binding specificity. IgE-binding components in extracts from the two types of potato were identified by SDS-PAGE and IgE-immunoblotting. The effects of digestive enzymes and heat on the allergenicity of the extracts was evaluated by preincubating the potatoes with or without simulated gastric and intestinal fluids in the absence or presence of heat.ResultsPositive responses (ratio of the wheal size induced by the allergen to that induced by histamine (A/H) ≥ 2+) to wild-type or GM potato extracts, as demonstrated by the skin-prick test, were observed in 108 patients (5.7%). Serum-specific IgE was detected in 0–88% of subjects who tested positively. ELISA inhibition tests indicated significant inhibition when extract from each type of potato was added. IgE-immunoblot analysis demonstrated the presence of 14 IgE-binding components within the wild-type potato and 9 within the GM potato. Furthermore, a common 45-kDa binding component that yielded similar IgE-binding patterns was noted in more than 80% of the reactions using sera from patients sensitized to wild-type or GM potato. Exposure to simulated gastric fluid and heat treatment similarly inhibited IgE binding by extracts from wild-type and GM potatoes, whereas minimal changes were obtained following exposure of the extracts to simulated intestinal fluid.ConclusionOur results strongly suggest that genetic manipulation of potatoes does not increase their allergenic risk. The sensitization rate of adult allergy patients to both types of extract was 5.7%, and a common major allergen (45 kDa) was identified.

Analysis of IgE binding proteins of mesquite ( Prosopis juliflora) pollen and cross-reactivity with predominant tree pollens

Pollen from the mesquite tree, Prosopis juliflora, is an important source of respiratory allergy in tropical countries. Our aim was to partially characterize the IgE binding proteins of P. juliflora pollen extract and study cross-reactivity with prevalent tree pollen allergens. Intradermal tests with P. juliflora and five other tree pollen extracts were performed on respiratory allergy patients from Bikaner (arid) and Delhi (semi arid). Prosopis extract elicited positive skin reactions in 71/220 of the patients. Sera were collected from 38 of these 71 patients and all demonstrated elevated specific IgE to P. juliflora. Immunoblotting with pooled patients’ sera demonstrated 16 IgE binding components, with components of 24, 26, 29, 31, 35, 52, 58, 66 and 95 kDa recognized by more than 80% of individual patients’ sera. P. juliflora extract is allergenically potent requiring 73 ng of self-protein for 50% inhibition of IgE binding in ELISA inhibition. Cross-inhibition assays showed close relationship among P. juliflora, Ailanthus excelsa, Cassia siamea and Salvadora persica. IgE binding components of 14, 41, 52 and 66 kDa were shared allergens whereas 26 and 29 kDa were specific to P. juliflora. The findings suggest that purification of cross-reactive allergens will be helpful for diagnosis and immunotherapy of tree pollen allergic patients.

Sensitization to Empynase®(pronase B) in exposed hospital personnel and identification of the Empynase® allergen

Empynase is a proteolytic enzyme that is widely used as an anti-inflammatory drug in Korea. We evaluated the prevalence of sensitization to Empynase in association with respiratory allergy symptoms in exposed hospital personnel, and identified the IgE-binding components in the Empynase extract, using sera with high levels of specific IgE antibodies. A total of 154 hospital personnel (135 nurses and 19 pharmacists) who worked in a university hospital and 123 unexposed healthy control subjects were enrolled. A questionnaire was administered that addressed demographics, job category, history of atopic diseases, diverse symptoms including nasal and lower respiratory symptoms, and the association of symptoms with work. Skin prick tests (SPTs) to common aeroallergens and Empynase extract were performed. Empynase-specific IgE antibody was detected by ELISA, and ELISA inhibition tests were conducted. IgE-binding components were identified by SDS-PAGE and IgE immunoblotting. Forty-two subjects (27.3%) complained of work-related respiratory symptoms (WRRS). Five nurses (3.7%) and one pharmacist (5.3%) had work-related asthma symptoms, and 34 nurses (25.2%) and six pharmacists (31.6%) had work-related rhinitis symptoms. The prevalence of sensitization to Empynase on SPTs was 20.1%, and tended to be higher in pharmacists (31.6%) than in nurses (18.5%). It was estimated that 3.9-8.4% of hospital personnel had WRRS attributable to Empynase. The duration of exposure was longer in positive SPT responders than in negative responders (51.9+/-27.5 vs. 39.2+/-27.3 months, respectively; P<0.05), and the prevalence of Empynase-positive SPTs was significantly higher in subjects with asthma than in those without asthma (57.1% vs. 18.4%, respectively; P<0.05). The levels of Empynase-specific IgE antibodies were significantly higher in pharmacists (76.1+/-83.4 OD units) and nurses (56.3+/-103.0 OD units) than in normal controls (39.8+/-12.7 OD units; P<0.05). Seven subjects (two pharmacists and five nurses) had high serum levels of Empynase-specific IgE antibodies; six of these subjects had WRRS. ELISA inhibition tests were performed with the sera of these six subjects, revealing significant inhibition only with the addition of Empynase. Four strongly staining protein bands (sizes: 36, 33, 16, and 10 kDa) from Empynase extract were observed to bind to the IgE antibodies of sensitized subjects. Conclusion Exposure to Empynase powder may cause rhinitis and asthma in hospital personnel, and the pathogenic mechanism appears to be IgE mediated.

A case of occupational asthma and rhinitis caused by Sanyak and Korean ginseng dusts

Sanyak has been used as a food and herbal material for various symptoms in oriental medicine. There have been few reports of occupational asthma induced by herb materials (1–6). Until now there has been no account of occupational asthma caused by Korean ginseng. We describe a case of occupational asthma and rhinitis caused by Sanyak (Dioscorea batatas) and Korean ginseng (Panax ginseng), as confirmed by specific inhalation challenge tests. A 29-year-old female patient was presented to the emergency department (ED) of Eulji University Hospital in Daejeon, Korea, for the treatment of dyspnea, wheezing, and cough. The patient had been incidentally exposed to airborne Sanyak dust, during the process of grinding dried Sanyak into powder, 5 min before the onset of symptoms. She had been a merchant of herbal materials for the previous 26 months. Twelve months before her visit, she had been admitted to another hospital and diagnosed with bronchial asthma after the sudden onset of dyspnea following an exposure to airborne ginseng dust. The patient had been suffering from nasal itching, sneezing, rhinorrhea, and nasal obstruction during the spring season for 6 years and had also experienced itching and swelling of the lips, tongue, and throat after ingesting fresh chestnut, sweet potato, and ginseng. The patient was a non-smoker and had no family history of allergic diseases. The patient appeared acutely ill and had tachypnea. Diffuse expiratory wheezes were noted over both lung fields. Arterial blood gas measurements at the ED indicated a pH of 7.414, PaCO2 of 30.8 mmHg, PaO2 of 70.4 mmHg and SaO2 94.6%. The total IgE level was 663.0 IU/ml. After pharmacological treatment, her symptoms resolved in a day. Proteins were extracted from Ginseng and Sanyak, and used for skin-prick tests, inhalation challenge tests, and laboratory studies. The skin-prick testing was expressed as mean wheal diameter/ mean erythema diameter (in millimeters): Dermatophagoides pteronyssinus 6.5/23.5 (Allergopharma, Reinbek, Germany); alder tree pollen 5.5/33.5 (Allergopharma); birch tree pollen 3.5/17 (Allergopharma); ginseng (1 : 100 w/v) 3/12; Sanyak (1 : 100 w/v) 3.75/19; histamine 4/17.5; and saline 0/0. Nonspecific and specific challenge tests were performed during her stable state. The methacholine bronchial challenge test revealed a 20% decline of forced expiratory volume in 1 s[FEV1] at the concentration of 0.75 mg/ml. The bronchoprovocation tests showed early asthmatic responses to both Sanyak and ginseng extracts (1 : 1000 and 1 : 100 w/ v, respectively). Serum-specific IgE and IgG4 antibodies to Sanyak were detected by enzyme linked immunosorbent assay[ELISA], but there was no specific antibody binding to ginseng. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis[SDS-PAGE] and an IgEimmunoblot, a 35-kDa IgE-binding component was detected in the Sanyak extracts (Fig. 1), but no IgE-binding component was noted in the ginseng extract. The ELISA inhibition test, specific IgE binding, and IgG4 binding to Sanyak demonstrated specific, dosedependent inhibition by Sanyak extracts but not by other control agents. The patient in this study showed the occurrence of occupational asthma induced by Sanyak and Korean ginseng. Although there is a report of occupational asthma being induced by Brazilian ginseng (1) no cases of bronchial asthma caused by Korean ginseng have been previously reported. Brazilian ginseng and Korean ginseng are different plants in terms of taxonomic classification. Brazilian ginseng (Pfaffia paniculata) belongs to the Amaranthaceae family; in contrast, the Korean ginseng (Panax ginseng) belongs to the Araliaceae family (1). This study, it is suggested that Sanyakderived allergen can induce IgE-mediated allergic reactions. We were unable to demonstrate specific IgE and IgG4 antibodies to Korean ginseng extract despite the fact that the patient showed a positive bronchial provocation and positive responses to a skin-prick test. Further studies are needed to investigate the pathogenic mechanism of occupational asthma caused by Korean ginseng. This study was funded by the Korean government (A0505171).

Evaluating the Allergic Risk of Genetically Modified Soybean

Genetically modified (GM) soybean (carrying the EPSPS transgene) is the most common GM food in Korea. In order to assess whether genetic modification increases the allergenic risk of soybeans, the allergenicity and IgE-reactive components of wild-type and GM soybean extracts were compared in allergic adults who had been sensitized to soybeans. We enrolled 1,716 adult allergy patients and 40 healthy, non-atopic controls. Skin prick tests and IgE enzyme linked immunosorbent assays (ELISAs) were performed using wild-type and GM soybean extracts, along with other common inhaled allergens. The specificities of serum IgE antibodies from allergic patients and the identities of the IgE-reactive components of the soybean extracts were compared using ELISA inhibition testing, 2-dimensional gel electrophoresis, and IgE immunoblotting. To evaluate the effects of digestive enzymes and heat treatment, the soybean extracts were heated or pre- incubated with or without simulated gastric and intestinal fluids. The IgE sensitization rates to wild-type and GM soybeans were identical (3.8% of allergic adults), and circulating IgE antibodies specific for the two extracts were comparable. The results of the ELISA inhibition test, SDS-PAGE, and IgE immunoblotting showed a similar composition of IgE-binding components within the wild-type and GM extracts, which was confirmed using two-dimensional gel electrophoresis, IgE immunoblotting, and amino acid sequencing. None of the subjects had a positive response to purified EPSPS protein in the skin prick test, ELISA, or IgE immunoblot analysis. These findings suggest that the IgE sensitization rate to GM soybean extracts is identical to that of wild-type soybean extracts in adult allergy patients. In addition, based on both in vivo and in vitro methods, the allergenicity of wild type and GM soybean extracts was identical.

Sensitization to blackgram in patients with bronchial asthma and rhinitis: clinical evaluation and characterization of allergens

Legumes are important causative agents of type I hypersensitivity in south Asia and Europe but such studies are lacking in Indian population. The present study investigates blackgram sensitization in asthma and rhinitis patients and identifies immunoglobulin E (IgE)-binding proteins. Respiratory allergy patients were evaluated using standard questionnaire and skin prick tests (SPT) with common foods and aeroallergens. Blackgram-specific IgE level was estimated by enzyme-linked immunosorbent assay (ELISA) and sensitization was established by a double-blind, placebo-controlled food challenge (DBPCFC). The cross-reactivity of blackgram with other legumes was studied by immunobiochemical methods. Of 816 patients, 35 gave history of blackgram hypersensitivity. From these, 16 patients were SPT positive and 14 showed elevated specific IgE (three times of negative control) to blackgram. DBPCFC established blackgram sensitivity in four of 14 patients. Immunoblotting with individual patient's sera recognized eight most prevalent allergens of 78, 56, 47, 43, 40, 30, 28 and 16 kDa. Roasted blackgram showed six major allergens whereas 47, 43 and 28 kDa proteins retained IgE reactivity upon boiling. Blackgram extract required 14 ng of self protein for 50% ELISA inhibition whereas roasted and boiled blackgram required 16 and 120 ng protein. ELISA and immunoblot inhibition show partial inhibition to blackgram proteins by lentil, limabean and pea. Blackgram induces IgE-mediated reactions in 1.7% of asthma and rhinitis patients and contains eight major IgE-binding components, of which six retained IgE reactivity after roasting. Blackgram shares allergenicity with lentil and limabean.

Sequence homology: A poor predictive value for profilins cross-reactivity

BackgroundProfilins are highly cross-reactive allergens which bind IgE antibodies of almost 20% of plant-allergic patients. This study is aimed at investigating cross-reactivity of melon profilin with other plant profilins and the role of the linear and conformational epitopes in human IgE cross-reactivity.MethodsSeventeen patients with melon allergy were selected based on clinical history and a positive skin prick test to melon extract. Melon profilin has been cloned and expressed in E. coli. The IgE binding and cross-reactivity of the recombinant profilin were measured by ELISA and inhibition ELISA. The amino acid sequence of melon profilin was compared with other profilin sequences. A combination of chemical cleavage and immunoblotting techniques were used to define the role of conformational and linear epitopes in IgE binding. Comparative modeling was used to construct three-dimensional models of profilins and to assess theoretical impact of amino acid differences on conformational structure.ResultsProfilin was identified as a major IgE-binding component of melon. Alignment of amino acid sequences of melon profilin with other profilins showed the most identity with watermelon profilin. This melon profilin showed substantial cross-reactivity with the tomato, peach, grape and Cynodon dactylon (Bermuda grass) pollen profilins. Cantaloupe, watermelon, banana and Poa pratensis (Kentucky blue grass) displayed no notable inhibition. Our experiments also indicated human IgE only react with complete melon profilin. Immunoblotting analysis with rabbit polyclonal antibody shows the reaction of the antibody to the fragmented and complete melon profilin. Although, the well-known linear epitope of profilins were identical in melon and watermelon, comparison of three-dimensional models of watermelon and melon profilins indicated amino acid differences influence the electric potential and accessibility of the solvent-accessible surface of profilins that may markedly affect conformational epitopes.ConclusionHuman IgE reactivity to melon profilin strongly depends on the highly conserved conformational structure, rather than a high degree of amino acid sequence identity or even linear epitopes identity.

Coconut allergy in two children of Asian origins and identification of the IgE binding components

Coconut allergy in two children of Asian origins and identification of the IgE binding components

Respiratory allergy to the indoor ant ( Monomorium pharaonis) not related to sting allergy

Many studies are available on systemic reactions to ant sting, but few have described the direct role of ants in respiratory allergy. The nonstinging house ant, Monomorium pharaonis (pharaoh ant), is a highly infesting species in indoor environments. To determine whether the pharaoh ant is an indoor source of aeroallergens. Two patients with asthma who lived in homes with ant infestation were enrolled. Pharaoh ants were collected at the patients' homes, and crude extracts were prepared. Skin prick tests with ant extracts were performed. Specific IgE to pharaoh ant was measured by enzyme-linked immunosorbent assay (ELISA), and the allergenic components were determined by using immunoblot analysis. Cross-reactivity among pharaoh ant, imported fire ant, Pachycondyla chinensis ant, and other indoor allergens was evaluated by ELISA inhibition tests. Specific bronchial challenge testing was performed using pharaoh ant extracts. Both patients had positive skin test reactions to pharaoh ant extract and high levels of specific IgE antibodies to pharaoh ant. The ELISA inhibition test results demonstrated significant inhibition by pharaoh ant; however, P. chinensis, cockroach, and house dust mite showed no inhibition of the IgE binding to pharaoh ant. Two important IgE-binding components, 9.4 and 34 kDa, were identified by using immunoblot analysis. Pharaoh ant bronchial challenge test results showed typical early asthmatic reactions in 1 patient and dual asthmatic reactions in the other patient. Ants can induce IgE-mediated bronchoconstriction regardless of sting in sensitized patients. Ants should be taken into consideration as a cause of respiratory allergy in patients living in homes with visual evidence of infestation.

Chestnut as a Food Allergen: Identification of Major Allergens

To evaluate the clinical significance of chestnut as a food allergen in Korea, skin prick test and ELISA were done in 1,738 patients with respiratory allergies. To identify the IgE binding components, IgE-immunoblotting, 2D IgE-immunoblotting and MALDI-TOF were performed. To observe the effects of digestive enzymes and a boiling treatment, simulated gastric fluid (SGF) and simulated intestinal fluids (SIF) were incubated with chestnut extracts, and IgE-immunoblotting were then repeated. Skin prick test revealed that 56 (3.2%) patients showed more than 2+ of allergen to histamine ratio to chestnut. Among the 21 IgE binding components, 9 bands were found in more than 50% of the sera tested and the 24 kDa protein had the highest binding intensity. The amino acid sequence of the 24 kDa protein (pI 6.3) had homology with legume protein of oak tree. SGF, SIF and boiling treatment were able to suppress the IgE binding components. In conclusion, chestnut ingestion was shown to induce IgE mediated responses with a 3.2% sensitization rate. Twenty one IgE binding components and one new allergen (the 24 kDa protein) were identified. Digestive enzymes and boiling treatment were able to decrease the allergenic potency.