Identification of the major allergen of Macrobrachium rosenbergii (giant freshwater prawn)

Objective To characterize the major allergens of Macrobrachium rosenbergii (giant freshwater prawn). Methods Raw and cooked extracts of the giant freshwater prawn were prepared. The IgE reactivity pattern was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technique with the sera of 20 skin prick test (SPT) positive patients. The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional (2-DE) electrophoresis, mass spectrometry and bioinformatics tools. Results SDS-PAGE of the raw extract showed 23 protein bands (15-250 kDa) but those ranging from 40 to 100 kDa were not found in the cooked extract. From immunoblotting experiments, raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins, respectively, with a molecular mass ranging from 15 to 155 kDa. A heat-resistant 36 kDa protein was identified as the major allergen of both extracts. In addition, a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract. The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots. Of these, 10 spots showed specific IgE reactivity with patients' sera. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis led to identification of 2 important allergens, tropomyosin and arginine kinase. Conclusions It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.

Identification of the major allergen of Macrobrachium rosenbergii (giant freshwater prawn)

Objective: To characterize the major allergens of Macrobrachium rosenbergii (giant freshwater prawn). Methods: Raw and cooked extracts of the giant freshwater prawn were prepared. The IgE reactivity pattern was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technique with the sera of 20 skin prick test (SPT) positive patients. The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional (2-DE) electrophoresis, mass spectrometry and bioinformatics tools. Results: SDS-PAGE of the raw extract showed 23 protein bands (15-250 kDa) but those ranging from 40 to 100 kDa were not found in the cooked extract. From immunoblotting experiments, raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins, respectively, with a molecular mass ranging from 15 to 155 kDa. A heat-resistant 36 kDa protein was identified as the major allergen of both extracts. In addition, a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract. The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots. Of these, 10 spots showed specific IgE reactivity with patients’ sera. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis led to identification of 2 important allergens, tropomyosin and arginine kinase. Conclusions: It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.

Identification of the major allergen of Macrobrachium rosenbergii (giant freshwater prawn)

<p><b>OBJECTIVE</b>To characterize the major allergens of Macrobrachium rosenbergii (giant freshwater prawn).</p><p><b>METHODS</b>Raw and cooked extracts of the giant freshwater prawn were prepared. The IgE reactivity pattern was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting technique with the sera of 20 skin prick test (SPT) positive patients. The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional (2-DE) electrophoresis, mass spectrometry and bioinformatics tools.</p><p><b>RESULTS</b>SDS-PAGE of the raw extract showed 23 protein bands (15-250 kDa) but those ranging from 40 to 100 kDa were not found in the cooked extract. From immunoblotting experiments, raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins, respectively, with a molecular mass ranging from 15 to 155 kDa. A heat-resistant 36 kDa protein was identified as the major allergen of both extracts. In addition, a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract. The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots. Of these, 10 spots showed specific IgE reactivity with patients' sera. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis led to identification of 2 important allergens, tropomyosin and arginine kinase.</p><p><b>CONCLUSIONS</b>It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.</p>

Buckwheat allergy in adults: comparison of specific IgE between homemade ELISA and CAP system, and identification of IgE-binding components / 천식및알레르기

BACKGROUND AND OBJECTIVE: Ingestion and inhalation of buckwheat flour can induce IgE-mediated bronchoconstriction and anaphylaxis in sensitized individuals, especially in childhood. The aim of this study was to determine the sensitization rate of buckwheat allergen, measure the level of specific IgE to buckwheat, and identify IgE-binding components in adult patients with various allergic diseases. METHODS: 1,738 allergy patients and 40 healthy controls were enrolled. Skin prick tests were performed using homemade buckwheat extract. The specific IgE level to homemade buckwheat allergen was measured by ELISA, and results were compared to those of the CAP system. ELISA inhibition tests were done to evaluate allergenic relationships with major food allergens and IgE binding components were identified using IgE immunoblot analysis. RESULTS: Among 1,738 patients tested, 60 patients (3.5%) showed more than a 2+ response on skin prick tests to buckwheat. The prevalence of serum specific IgE to buckwheat ranged from 24% in patients with a 2+ response to buckwheat skin prick test, to 50% in patients with a 4+ response. The mean absorbance value increased with skin reactivity although it was not statistically significant. However, CAP results were significantly correlated with skin reactivity (p<0.05). A significant correlation was noted between (the) results by homemade ELISA and CAP. IgE immunoblot demonstrated 20 IgE binding components ranging from 20 to 114 kDa, and 10 components were bound to IgE in more than 50% of the patients tested. CONCLUSION: Natural buckwheat allergens should be considered as one of the causative food allergens in exposed adults. Specific IgE results by homemade ELISA were comparable with those of CAP system. Twenty IgE binding components and 10 major allergens were noted within natural buckwheat allergen. Further studies will be needed to evaluate the allergenic relationships with other food allergens.

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.

Identification and characterization of buckwheat allergen / 천식및알레르기

BACKGROUND AND OBJECTIVE: Buckwheat is considered one of the most important food allergens in Korea. Although a very small amount is ingested or inhaled, it can cause serious allergic reactions. However, the major allergens of buckwheat still remain to be elucidated. The aim of our study was to identify and characterize the major allergen of buckwheat seed. MATERIAL AND METHOD: Dialysis membrane with a cut-off MW 1kD was used for the preparation of crude buckwheat seed allergen extract. SDS-PAGE under reducing conditions and IgE immunoblotting were performed using sera from 15 buckwheat sensitive subjects. Isoelectric focusing and lectin blotting assay were done. RESULT: Western blot analysis showed more than 15 IgE-reactive buckwheat proteins. Among them, a 24kD protein was shown to be the most frequently bound to sera from allergic subjects (54%). Isoelectric point of 24kD protein was around 5.9. In lectin blotting assay, 24kD protein did not bind to Con A nor five other lectins. CONCLUSION: A 24kD protein was the most frequently recognized allergenic component in buckwheat seed. Isoelectric point was around 5.9. Glycosylation was not detected in 24kD of buckwheat protein.

Identification of major allergens from the house dust mites, Dermatophagoides farinae and Dermatophagoides pteronyssinus, by electroblotting

The allergens were separated from the extracts of house dust mites by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by autoradiography. Over 30 protein bands of the whole body extract of Dermatophagoides farinae were apparent on 10-20% gradient SDS-PAGE, and 13 bands with MW between 93KD and 12KD bound with specific IgE antibodies in patients' sera sensitive to house dust mites. The major allergenic component of the whole body extract of D. farinae was the protein of MW 14-15KD, which was detected in 95.7% of 47 patients' sera sensitive to house dust mites. The extract of Dermatophagoides pteronyssinus supplied by Bencard Company, England was thought to contain feces enriched material as noted in a few broad protein bands on SDS-PAGE. Seven allergenic components were shown by autoradiography. The protein band of MW 14-15KD was one of the most frequently revealed allergens on autoradiography, which has appeared in 32.5% of 40 patients' sera sensitive to house dust mites. The electrobotting technique used in the present study was fast, convenient and highly useful for both the identification of allergen components and the screening of specific IgE antibody. The individual variations of IgE immune responses to the allergenic components of the two house dust mites were discussed.