Survey of IgE Reactivity to Nonbiting Midges in Korea and Identification of IgE-Binding Protein

PURPOSE: Chironomids (nonbiting midges) are widely and abundantly distributed near ponds, rivers, and artificially dammed pools used for irrigation. Chironomids contain allergens and cause airway allergy in humans. In this study, we aimed to examine the allergic potential of chironomids in inhabitants living near artificially dammed pools. METHODS: We examined immunoglobulin E (IgE) reactivity to chironomid extracts in the sera of residents living around installed dams and assessed the correlations of IgE responses between chironomids (Chironomus flaviplumus, Chironomus kiiensis, Cricotopus bicinctus) and house dust mites (Dermatophagoides farinae). In addition, we identified potential IgE binding proteins specific for adult C. bicinctus, a popular species in Korea. Specific IgE antibodies in sera collected from the participants against the extracts were tested using enzyme-linked immunosorbent assay (ELISA). RESULTS: The average IgE-positive rates were 10.4%, 8.1%, and 8.2% in C. bicinctus, C. flaviplumus, and C. kiiensis, respectively. The IgE-positive rate and IgE titer of C. bicinctus antigen were higher in residents living around installed dams than in those who lived other places (P = 0.013). Western blotting using sera having high IgE titers to C. bicinctus in ELISA showed the presence of a protein of approximately 42 kDa that was homologous to the actin protein isoform in C. bicinctus extracts as demonstrated using mass spectrometry. CONCLUSIONS: Our results showed that people living near installed dams were more sensitized to C. bicinctus and that the 42 kDa IgE-binding protein could be useful for further studies on chironomid allergic disease and clinical applications.

Effect of Amino Acid Polymorphisms of House Dust Mite Der p 2 Variants on Allergic Sensitization

PURPOSE: The sequence variations of the Der p 2 allergen of Dermatophagoides pteronyssinus diverge along 2 pathways with particular amino acid substitutions at positions 40,47,111, and 114. The environmental prevalence and IgE binding to Der p 2 variants differ among regions. To compare IgE binding to Der p 2 variants between sera from Bangkok, Thailand and Perth, Western Australia with different variants and to determine the variant-specificity of antibodies induced by vaccination with recombinant variants. METHODS: The structures of recombinant variants produced in yeast were compared by circular dichroism and 1-anilinonaphthalene 8-sulfonic acid staining of their lipid-binding cavity. Sera from subjects in Bangkok and Perth where different variants are found were compared by the affinity (IC50) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2.0109 was measured. RESULTS: The secondary structures of the recombinant variants resembled the natural allergen but with differences in ANS binding. The IC50 of Der p 2.0101 required 7-fold higher concentrations to inhibit IgE binding to the high-IgE-binding Der p 2.0104 than for homologous inhibition in sera from Bangkok where it is absent, while in sera from Perth that have both variants the IC50 was the same and low. Reciprocal results were obtained for Der p 2.0110 not found in Perth. Direct binding revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to other variants. CONCLUSIONS: The affinity of IgE antibody cross-reactivity, the direct IgE binding, and the specificities of antibodies induced by vaccination show that measures of allergic sensitization and therapeutic strategies could be optimized with knowledge of Der p 2 variants.

Comparison of IgE-binding components between 2 house dust mites in adult allergic patients

PURPOSE: This study investigated the differences in the profile of IgE-binding components between Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farina (Df) in respiratory allergic patients sensitized to Dp/Df. METHODS: Eighteen patients with respiratory allergic diseases having higher levels of serum specific IgE to Df compared to those to Dp (>twice) were enrolled. IgE-immunoblot analysis using Dp and Df extracts were used to compare the IgE binding components. Study subjects were classified into 2 groups according to the results of IgE-immunoblot analysis: 6 subjects having IgE-binding components to group 1 and 2 allergens (group B) and 12 subjects not having them (group A). RESULTS: Group A subjects were older (47.92±8.51 vs. 35.50±11.10, P=0.039) and males were dominant (75% vs. 0% P=0.009). IgE-immunoblot analysis demonstrated that all the group B subjects had IgE bindings to 2 major components, 14 and 25 kDa, while group A subjects had IgE bindings to high-molecular weight components ranging from 60-98 kDa. The enzyme-linked immunosorbent assay inhibition test showed a significant inhibition with additions of Df, not with Dp in group B subjects. Serum specific IgE levels to Dp and Df were significantly higher in group B than in group A, while its ratio (Df to Dp) was significantly higher in group A. No differences were noted in clinical parameters, total IgE, or eosinophil cationic protein levels. CONCLUSION: Heterogeneity of IgE binding patterns to Dp and Df extracts was noted according to the ratio of serum specific IgE (Df/Dp).

IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2

PURPOSE: Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach. METHODS: The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry. RESULTS: Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces. CONCLUSIONS: Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy.

Immunoglobulin E-binding reactivities of natural pollen grain extracts from selected grass species in the Philippines

BACKGROUND: Pollen grains have been reported to be present in the Philippine atmosphere but studies regarding their allergenicity are limited. OBJECTIVE: The present study aimed to profile the sensitization of allergic individuals to selected grass pollen species and to characterize the pollen proteins that may be responsible for this allergenic response. METHODS: The protein profile of the grass pollen extracts from Cynodon dactylon, Saccharum spontaneum, Sporobulus indicus, Chloris barbata, Oryza sativa, Imperata cylindrica, and Zea mays was analyzed by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis. The specific-IgE profile of the allergic individuals and the allergenic potential of the pollen extracts were evaluated through Enzyme-linked Immunosorbent Assay and IgE immunoblotting. RESULTS: Sensitization of the allergic individuals to the pollen extracts was detected with I. cylindrica and O. sativa to be the most frequently recognized with more that 92% reactivity, whereas for C. dactylon and Z. mays, were found to have less than 25% reactivity. CONCLUSION: Multiple IgE-binding proteins from S. indicus, S. spontaneum and C. barbata that were detected may be responsible for the allergic reactions among Filipino subjects.

Measurement of specific IgE to abalone(Haliotis discus hannai) and identification of IgE binding components / 천식및알레르기

BACKGROUND: Abalone, which is a member of the shellfish family, can often induce severe allergic reactions in sensitized individuals, but there have been only a few studies ofn its allergenic components. A recent study has identified two major allergens with molecular weights of 38 and 49 kDa in South African abalone. OBJECTIVES: The aim of this study is to evaluate skin test prevalence and IgE sensitization to northern disk abalone (Haliotis discus hannai) which is one of the major abalones in this country, and to identify its allergenic components. METHODS: Skin prick tests were performed with 62 home-made extracts of domestic foods including abalone, turban shell, triton shell, shrimp etc. in 1,738 patients with various allergic diseases. Serum specific IgE antibodies to abalone were determined by ELISA in 81 positive responders on skin prick tests to abalone extract and 40 non-atopic healthy controls. ELISA inhibition tests were performed to evaluate cross-allergenecity between abalone and other sea foods(turban shell, triton shell, shrimp and house dust mite). Allergenic components of Haliotis discus hannai were identified by SDS-PAGE and IgE-immunoblot analysis. RESULTS: The positive response rate(A/H ratio> or=2+) to abalone on skin prick test was 4.7% in patients with various allergic diseases. Serum spcecific IgE to abalone was detected in 23(34.5%) of 67 patients. Serum specific IgE levels to abalone tended to increase according to skin test reactivity without statistical significance(p>0.05). ELISA inhibition tests showed significant dose-dependent inhibitions with addition of turban shell, triton shell and shrimp extracts IgE immunoblot analysis showed ten allergenic components (33, 37, 40, 60, 63, 71, 76, 86, 92, 111 kDa), of which seven allergens (40, 60, 63, 71, 76, 86, 92 kd) were bound to IgE in more than 50% of the sera tested. CONCLUSION: The sensitization rate to abalone was 4.7% in allergy patients. Serum specific IgE to abalone was detected by ELISA, and 7 major allergens within abalone were identified. Further studies will be needed to elucidate their clinical significances.

Sensitization rate of Trichophyton spp. allergen in various allergic diseases and identification of its allergens with immunoblotting / 천식및알레르기

BACKGROUND: Trichophyton is one of the most common genera of dermatophytes. It has been reported that Trichophyton spp. might be one of the causative allergens in patients with asthma, rhinitis, urticaria and angioedema. OBJECTIVES: To analyze the sensitization rate of Trichophyton, to determine serum specific IgE antibody, and to confirm Trichophyton as a causative antigen in patients with allergic diseases. METHODS: A total of 1,806 patients were enrolled in this study. Skin prick test was performed with 50 common inhalant allergens and 20 food allergens. Serum specific IgE antibodies were determined by ELISA using Trichophyton mentagrophytes antigen in 60 patients among positive skin responders to Trichophyton antigens and in 20 controls. For evaluation of cross-reactivity between Trichophyton and other fungal species, competitive ELISA inhibition test was performed. SDS-PAGE and IgE-immunoblot analysis using T. mentagrophytes antigen were applied in 7 patients with high specific IgE titers. RESULTS: 102 patients (5.7%) showed positive response to T. mentagrophytes on skin prick test, and six patients showed isolated positive responses. Serum specific IgE increased according to skin reactivity (p<0.05). SDS-PAGE and IgE-immunoblot showed 10 IgE-binding components (11, 17, 27, 32, 35, 38, 42, 48, 49, 51 kDa) within Trichophyton extracts. Trichophyton-ELISA inhibition test showed dose-dependent inhibitions with additions of Trichophyton antigens, while minimal inhibitions were noted with additions of Fusarium, Alternaria, Aspergillus and Clados- porium. CONCLUSIONS: Trichophyton could induce IgE sensitization in allergy patients. The sensitization rate on skin prick test was 5.7%. Trichophyton antigen should be included in skin prick test battery to screen causative agents for allergy patients.

Skin reactivity and specific IgE sensitization to Tetranychus urticae and identification of IgE binding components / 천식및알레르기

BACKGROUND AND OBJECTIVES: Tetranychus urticae(TU) is a widely distributed parasitic mite found on fruit trees and green house flowers. A recent investigation demonstrated that TU inhalation causes allergic asthma even in non-farmers. We tried to evaluate skin reactivity and specific IgE sensitization to TU, identify IgE binding components, and evaluate allergenic rela- tionship with house dust mite(HDM). MATERIALS AND METHODS: We carried out skin prick test with TU in 1806 respiratory allergy patients over 1 year living in urban and rural areas. ELISA was performed for detection of specific IgE antibody. To evaluate the cross allergenicity between TU and HDM, ELISA inhibition test was carried out with two kinds of pooled sera ; serum pool A included patients' sera sensitized to both TU and HDM, and serum pool B included sera sensitized only to TU. To identify IgE binding components, SDS-PAGE followed by IgE-immunoblot were applied. RESULTS: 358 patients(19.8%) showed positive response(A/H > or = 2+) on skin prick test. Twelve patients showed isolated positive response to TU. Specific IgE was detected in sixty patients(54.5%) out of 110 sensitized patients. ELISA inhibition test using two sera pools (A and B) showed significant inhibitions by TU with minimal inhibitions by HDM. SDS-PAGE and IgE-immunoblot with patients' individual sera sensitized to both TU and HDM showed 10 IgE binding components (67kD, 29kD, 27kD, 10kD, 14kD, 39kD, 46kD, 35kD, 72kD, 77kD) and two(67kD and 29kD) were bound to IgE in more than 50% of sera tested. In patients' sera sensitized only to TU, nine IgE binding components(67kD, 10kD, 14kD, 29kD, 39kD, 46kD, 72kD, 77kD, 9kD) were found and two(67kD and 10kD) were bound to IgE in more than 50%. CONCLUSION: Of allergy patients visiting the Allergy Clinic, 19.8% were sensitized to TU and specific IgE was detected in 54.5% of them. No cross allergenicity was noted between TU and HDM. Eleven IgE binding components and three (67kD, 10kD and 29kD) major allergens were identified.

IgE binding patterns to German cockroach whole body extract in Korean atopic asthmatic children

It is widely known that the cockroach is an inhalant allergen in atopic asthma and allergic rhinitis. Even though Bla g I and Bla g II are considered as the major allergens, several relatively high-molecular weight (MW) cockroach allergens have also been recently identified by IgE-immunoblot in western countries. However, the environmental control and diagnostic tests mainly focussed on Bla g I and Bla g II. Furthermore there is no data about major IgE-binding cockroach antigens in Korea. We performed this study to identify the major German cockroach allergens in Korean atopic children. By the results of allergy skin tests, 14 children with atopic asthma (9 were cockroach-sensitive and 5 were cockroach-nonsensitive atopics) were enrolled in this study. We conducted IgE immunoblot and autoradiographic analysis using Yonsei-extract of German cockroach antigen produced in our laboratory, individual sera from 9 cockroach- sensitive children, and the pooled sera of 5 house-dust-mites-only-sensitive children. We performed an allergic skin test to cockroach mix, and a radioallergosorbent test (RAST) using German cockroach crude extract on all subjects. German cockroach-specific IgE was detected in 6 out of 9 subjects by RAST. We identified at least 15 IgE-binding protein bands, and among them, the components of MWs of 76, 64, 50, 38, and <14 kilodaltons (kDa) were the major German cockroach allergens in study subjects. Therefore, Bla g I (25-30 kDa) and Bla g II (36 kDa) could not be the absolute indicators of German cockroach sensitization and parameters of environmental control.

IgE Binding Pattern to Dermatophagoides Pteronyssinus in Younger and Older Asthmatic Children: An IgE Immunoblot Analysis / 소아알레르기및호흡기학회지

House dust mite is the most important causative allergens of childhood asthma and its positive rate of allergy skin test reaches up to 80% in atopic asthmatic children in Korea. Although the mite sensitization is more significant in older children(>4 years), it is also important in some younger(<4 years) asthmatic children. To identify the major allergens in Korean atopic asthmatic children and find the variations of IgE binding pattern to Dermatophagoides pteronyssinus(Dp) in younger and older age groups, we conducted IgE immunoblot. Sera were collected in 25 children with Dp-sensitive atopic asthma : GroupI(younger children aged 1-3), Goup molder children aged 442). Using our own-made crude Dp extract, we conducted IgE-immunoblot (biotin-avidine-phos-phatase system). In the group I, the 15KD protein was significantly bound by 69.2%(9/13) of sera and 98KD, 59KD, 311KD, 25KD components were bound by 8-16% of sera, respectively. In the group II, the 15KD and 25KD proteins were significant1y bound Immunob1ot by 100% and 67% of sera, and 18KD, 94KD by 33%, 98KD by 25%, 45KD by 8% of sera, respectively. In conclusion, the 15KD protein is the most prevalent IgE-binding antigen in all age groups, on the other hands, IgE-sensitization to 25KD component is confirmed mainly in the cases of older age group in this study.