IgE Immunoblotting Research Articles

Increased allergen production in turnip ( Brassica rapa) by treatments activating defense mechanisms

Background: Practical applications to enhance the productivity of agriculture by using plants with improved resistance to pathogens are expected to increase in the near future. Defense proteins play an important role in pathogen resistance, and some defense-related proteins are significant cross-reacting allergens. For example, cross-allergies are common among patients allergic to natural rubber latex (NRL), which contains many defense-related proteins. Objective: Using a model plant (ie, turnip), we studied whether allergen contents increase after treatments activating defense mechanisms of the plants. Methods: Whole or wounded turnips treated with salicylic acid, ethephon, or water were incubated for 2, 4, or 8 days. Allergen content was investigated by IgE immunoblotting with sera from patients allergic to NRL. An induced protein that bound IgE most intensively was purified and further characterized by mass analysis, amino acid sequencing, IgE-ELISA, and skin prick tests. Results: In immunoblotting, clear IgE-binding bands were discernible only in samples from chemically treated plants. IgE was bound most intensively to a protein with an apparent molecular weight of 25 kd in SDS-PAGE and with a determined molecular weight of 18.7 kd. Sequenced peptides of the 18.7-kd protein showed over 70% homology to prohevein, a major allergen of NRL, and to many other prohevein-like defense proteins. In ELISA, sera from 30 of 34 (88%) adults and 21 of 26 (81%) children previously shown to have IgE against prohevein bound to the purified protein. In skin prick testing with the protein, 4 of 6 patients allergic to NRL had positive reactions. Conclusion: These results demonstrate that activating defense mechanisms of plants may considerably increase their allergen content. (J Allergy Clin Immunol 1999;104:194-201.)

Characterization of allergens from Penicillium oxalicum and P. notatum by immunoblotting and N-terminal amino acid sequence analysis.

Penicillium species are important causative agents of extrinsic bronchial asthma. However, little is known about the allergens of these ubiquitous fungal species. Objective The object was to analyse the composition, the allergenic cross-reactivity and the N-terminal sequences of allergens from two prevalent airborne Penicillium species, P. oxalicum and P. notatum. The allergenic composition and the immunoglobulin (Ig)E cross-reactivity were analysed by immunoblot and immunoblot inhibition, respectively, using sera from asthmatic patients. The N-terminal amino acid sequences of major allergens were determined by Edman degradation. Allergens identified were also characterized by immunoblotting using monoclonal antibody (MoAb) PCM39 against the alkaline serine proteinase major allergen of P. citrinum. Among the 70 asthmatic sera tested, 18 (26%) and 17 (24%) had IgE immunoblot reactivity towards components of P. oxalicum and P. notatum, respectively. Major allergens (> 80% frequency of IgE-binding) from both species are the 34 and 30 kDa proteins of P. oxalicum and the 34 and 32 kDa proteins of P. notatum. IgE cross-reactivity among these major allergens and the 33 kDa major allergen of P. citrinum can be detected by immunoblot inhibition studies. The N-terminal amino acid sequences of the 34 kDa allergen of P. oxalicum and of the 32 and the 28 kDa allergens of P. notatum share homology with sequences of the vacuolar serine proteinase from Aspergillus fumigatus. The N-terminal amino acid sequence of the 34 kDa allergen of P. notatum shows sequence homology with that of alkaline serine proteinase from P. citrinum. Results obtained from immunoblotting showed that MoAb PCM39 reacted with the 34, 30 and 16 kDa IgE-binding components of P. oxalicum, and with the 34, 32 and 28 kDa IgE-binding components of P. notatum. Results obtained suggest that the 34 kDa major allergen of P. oxalicum may be a vacuolar serine proteinase. The 34 and the 32 kDa major allergens of P. notatum may be the alkaline and the vacuolar serine proteinases of P. notatum, respectively. The 30 and 16 kDa IgE-binding components of P. oxalicum and the 28 kDa IgE-binding component of P. notatum may be breakdown products of the 34 and the 32 kDa major vacuolar serine proteinase allergens of P. oxalicum and P. notatum, respectively.

Hevein-like protein domains as a possible cause for allergen cross-reactivity between latex and banana

Background: Patients allergic to natural rubber latex (NRL) frequently exhibit immediate hypersensitivity reactions to banana, but the molecular basis of these putative cross-reactions is unknown. Objective: We wanted to examine whether proteins resembling hevein, a major NRL allergen, exist in banana and whether coexisting allergy to NRL and banana can be explained by IgE cross-reactivity to these proteins. Methods: Allergens in banana cross-reacting with hevein were identified by IgE immunoblot inhibition. The cross-reacting proteins were purified by reversed-phase chromatography and characterized by amino acid sequencing. Allergen cross-reactivity was further assessed by IgE ELISA, IgE ELISA inhibition, and skin prick testing. Results: In immunoblotting, 9 of 15 sera from patients allergic to NRL with IgE to hevein showed IgE binding to 32- and 33-kd banana proteins. These 2 protein bands were also targets to IgG from a hevein-immunized rabbit. IgE binding to both 32- and 33-kd protein bands was totally inhibited by hevein (10 ng/mL) in all 5 sera from patients allergic to NRL sera studied. N-terminal sequencing of the purified 32- and 33-kd proteins revealed 80% identity to the N-terminus of hevein. An internal peptide (19 amino acids) of the 33-kd protein gave over 90% identity to endochitinases of several plants. In ELISA all 15 sera from patients allergic to NRL had IgE to the purified 32-kd banana protein. In ELISA inhibition hevein (10 ng/mL) inhibited approximately 50% of IgE binding to the solid-phase 32-kd banana protein in a pool of sera from patients allergic to NRL. Conclusion: These results suggest that the commonly occurring hypersensitivity to banana in patients allergic to NRL could be explained by cross-reacting IgE antibodies binding to epitopes in hevein and in a hevein-like domain of a previously undescribed endochitinase in banana. (J Allergy Clin Immunol 1998;102:1005-12.)

Recombinant birch pollen allergens (rBet v 1 and rBet v 2) contain most of the IgE epitopes present in birch, alder, hornbeam, hazel, and oak pollen: A quantitative IgE inhibition study with sera from different populations

Background: Pollen from trees of the order Fagales are important allergen sources in most parts of the world. Clinical, immunochemical, and molecular biology studies indicate that they contain cross-reactive allergens. The major birch pollen allergen, Bet v 1, and birch profilin, Bet v 2, a highly cross-reactive allergen, have been cloned and expressed in Escherichia coli. Objective: The purpose of this study was to demonstrate the presence of allergens in Fagales pollens that share IgE epitopes with recombinant Bet v 1 and Bet v 2 and to determine the percentage of birch, alder, hornbeam, hazel, and oak pollen–specific IgE that can be preabsorbed with rBet v 1 and rBet v 2 from 102 sera of different populations of subjects allergic to Fagales tree pollen. Methods: The presence of rBet v 1– and rBet v 2–homologous allergens in tree pollen extracts was investigated by IgE immunoblot inhibition experiments, and the percentage of tree (birch, alder, hornbeam, hazel, and oak) pollen–specific IgE that was bound by a mixture of rBet v 1 and rBet v 2 was determined by RAST-based quantitative IgE inhibition experiments. The clinical significance of IgE antibody cross-reactivity was studied by skin prick testing with rBet v 1, rBet v 2, and Fagales pollen extracts. Results: Natural birch, alder, hornbeam, hazel, and oak pollen contain allergens that share IgE epitopes with rBet v 1 and rBet v 2. A combination of rBet v 1 and rBet v 2 accounted for 82% of tree pollen–specific IgE on average. Most of the tree pollen–specific IgE was directed against rBet v 1. Conclusion: rBet v 1 and rBet v 2 contain most of the Fagales pollen–specific IgE epitopes and may therefore substitute natural tree pollen extracts not only for diagnosis but also for patient-tailored immunotherapy of tree pollen allergy. (J Allergy Clin Immunol 1998;102:579-91.)

Cellular and Molecular Characterization of a Major Soybean Allergen

Soybean proteins share a large number of cross–reacting allergens with other members of the legume family; however, soy–allergic patients rarely react clinically to other members of the legume family. Gly m Bd 30K, an IgE–binding protein with a molecular weight of 30 kD, was identified in soybean extracts by Western IgE–immunoblot analysis. This monomeric allergen was shown to have an N–terminal amino acid sequence and amino acid composition identical to that of the seed 34–kD protein, P34, a thiol protease of the papain family. Electron–microscopic immunolocalization of P34 monoclonal antibodies and IgE binding to sections of soybean seeds showed dense staining throughout the vacuolar bodies, localizing the allergens in protein storage vacuoles of seed cotyledons. We used pooled serum from soybean–sensitive patients to determine the linear IgE–specific epitopes in the 34–kD allergen amino acid sequence. B–cell epitope mapping revealed 10 regions of IgE–binding activity using an overlapping peptide strategy of 15–mers offset by 8 amino acids throughout the P34 sequence. Smaller overlapping peptides, 10–mers offset by 2 amino acids, revealed 16 distinct linear epitopes, 9 of which were mapped to the mature protein. No obvious amino acid sequence motifs could be identified by the smaller IgE–binding epitopes. Using individual patient serum, 5 immunodominant epitopes were identified in this allergen.

Molecular Cloning of IgE–Binding Fragments of Alternaria alternata Allergens

Exposure to the hyphomycete Alternaria alternata is recognized as an important risk factor for asthma. IgE immunoblotting has been used to catalogue the number and M_r of allergens in A. alternata extracts, with estimates ranging from 10 to 30, although few are present in nearly all extracts studied. Several A. alternata allergens have been cloned, including a subunit of the major allergen Alt a 1, ribosomal P2 phosphoprotein, aldehyde dehydrogenase and a yeast YCP4 homolog. We have cloned two sequences encoding IgE–binding fragments of allergens from an A. alternata λgt11 cDNA library using pooled atopic sera from A. alternata–sensitive individuals. One is homologous to a region near the C–terminus of hsp70 from Cladosporium herbarum; a near–complete isoallergen variant of A. alternata ribosomal P2 protein was also cloned. Their lacZ fusion proteins had reactivities of 5 and 14%, respectively, with individual atopic sera, indicating that the corresponding allergens are both minor. This study describes one new A. alternata allergen candidate and implicates ribosomal P2 protein as an allergen thtat is stable between independently isolated clones.

Immunoglobulin G (IgG) subclass and IgE responses in human paragonimiases caused by three different species.

In 40 cases of human paragonimiases caused by Paragonimus westermani (20 cases), P. miyazakii (10 cases), and P. skrjabini (10 cases), responses of serum immunoglobulin G (IgG), IgG subclasses, and IgE were analyzed by immunoblotting with crude antigens prepared from egg, 4-week-old juvenile, and adult forms of P. westermani. The 32- and 35-kDa proteins in the adult extracts showed specific reactions regardless of the causative species (39 of 40 cases; 98%). Sera of patients infected with P. westermani and P. miyazakii reacted strongly with the 28-, 46-, and 94-kDa proteins of egg extracts, while those from patients infected with P. skrjabini reacted faintly. No sera from patients with other trematodiases (0 of 15 cases), cestodiases (0 of 20 cases), or lung cancer (0 of 5 cases) or from healthy controls (0 of 10 individuals) showed positive reactions. Analysis by IgG subclass revealed that IgG4 (33 of 40 cases; 83%) and IgG1 (29 of 40 cases; 73%) antibodies in the patient sera recognized the 32- and 35-kDa proteins predominantly. IgG3 reaction was found in 50% (10 of 20 cases) and 30% (3 of 10 cases) of the sera of patients infected with P. westermani and P. miyazakii, respectively. In an IgE immunoblot, 83% (33 of 40 cases) of the sera from paragonimiasis patients reacted with the 32- and 35-kDa proteins while no sera from patients with heterologous diseases and healthy controls showed a positive reaction. Both 32- and 35-kDa proteins in adult extracts of P. westermani were highly reliable for serodiagnosis of human paragonimiases.

Biochemical Characterization of Pru a 2, a 23-kD Thaumatin-Like Protein Representing a Potential Major Allergen in Cherry (Prunus avium)

Background: The prevalence of allergy to fruits and vegetables increased with pollinosis over the last 10 years. So far, clusters of hypersensitivity have been established and corroborated by the molecular characterization of individual cross-reacting allergens. Several case studies demonstrated the existence of allergic reactions to fruits of the subfamily Prunoideae (apricots, cherries, plums and peaches). Here, we present the characterization of a major allergen in cherry. Methods: Characterization was performed using IgE immunoblotting and immunoblot inhibition, N-terminal sequencing, mass spectroscopy analysis and PCR-based cDNA cloning. Results: A 23-kD protein was identified as IgE-binding component. As all cherry-extract-reactive sera displayed IgE-binding to this band, it was designated a major allergen from Prunus avium (Pru a 2). Sequencing the corresponding cDNA identified Pru a 2 as a thaumatin-like protein belonging to the group 5 of pathogenesis-related proteins. Conclusions: A thaumatin-like protein in cherry has been identified as a major allergen (Pru a 2). Homologous proteins from the thaumatin family share sequence similarities and should therefore be checked for the capability to elicit an IgE-mediated allergic reaction.

IgE, IgA, and IgG responses to common yeasts in atopic patients.

This study was undertaken to analyze the differences in exposure and sensitization to five common environmental yeasts. The responses of IgG, IgA, and IgE to Candida albicans, C. utilis, Cryptococcus albidus, Rhodotorula rubra, and Saccharomyces cerevisiae and purified S. cerevisiae enolase were analyzed by immunoblotting (IgE-IB), and the cross-reactivity of their IgE-binding components by IgE-IB inhibition. Twenty atopic subjects, with asthma, allergic rhinitis, or atopic dermatitis were included. In skin prick tests (SPT), 12 of the patients showed simultaneous reactivity to at least two of the five yeasts, four reacted to one of the yeasts, and four had no responses. Antigens run in SDS-PAGE and transferred to nitrocellulose were probed with enzyme-labeled IgA-, IgG-, and IgE-specific antibodies. The IgE immunoblotting revealed most IgE-binding bands in C. albicans (11 bands) followed by C. utilis (eight bands), S. cerevisiae (five bands), R. rubra (five bands), and Cr. albidus (four bands). Six of the IgE-binding bands of C. albicans and C. utilis shared molecular weight, and only two bands shared molecular weight with other yeasts. These were the 46-kDa band, shared by all five yeasts, and a 13-kDa band shared by four yeasts. Prominent IgE binding was seen to a 46-kDa band of C. albicans (seven patients), C. utilis (five patients), and S. cerevisiae (one patient) and to corresponding weak bands of Cr. albidus and R. rubra (one patient). The possible cross-reactivity of the 46-kDa band was analyzed by IgE-IB inhibition and densitometry, revealing clear C. albicans inhibition of C. utilis (80%) and enolase (98%) (autoinhibition 100%). The strongest IgG responses were seen against S. cerevisiae and C. albicans. The responses were mainly against mannans of C. albicans and S. cerevisiae, suggesting that most of the exposure is to these yeasts. Yeasts with different types of exposure, from saprophytic growth on human mucous membranes to exposure by air and food, were shown to cross-react at the allergenic level. Atopic patients primarily sensitized by C. albicans and S. cerevisiae may develop allergic symptoms by exposure to other environmental yeasts due to cross-reacting IgE antibodies.

IgE antibodies to recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) account for a high percentage of grass pollen–specific IgE

Background: Pollen from different grass species are some of the most potent elicitors of Type I allergy worldwide. The characterization of antigenic structures and IgE epitopes common to different grass species is relevant to define reagents for diagnosis and specific therapy of grass pollen allergy. Objective: The purpose of this study was to estimate the percentage of IgE directed to common, cross-reactive, or both types of epitopes shared by recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) and natural pollen extracts from nine different monocots ( Anthoxanthum odoratum, Avena sativa, Cynodon dactylon, Lolium perenne, Phragmites australis, Poa pratensis, Secale cereale, Triticum sativum, Zea mays) by using sera from different populations. Methods: Natural pollen extracts from nine different monocot species were characterized regarding their allergen contents by using specific antibodies and by IgE immunoblot inhibition with recombinant allergens. The percentage of grass pollen–specific IgE that was preabsorbed with a combination of recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) and recombinant birch profilin (Bet v 2) was determined by ELISA in sera from 193 European, American, and Asian subjects. Results: IgE to recombinant pollen allergens accounted for a mean 59% of grass pollen–specific IgE. A lower inhibition of IgE binding to certain natural extracts ( C. dactylon and Z. mays) could be attributed to the absence of immunologically detectable group 5 and group 2 allergens in these species. Conclusion: We define four recombinant pollen allergens that account for a substantial proportion of grass pollen–specific IgE. The recombinant pollen allergens characterized may represent candidates not only for diagnosis but also for patient-tailored immunotherapy of grass pollen allergy.

Identification of common allergenic structures in mugwort and ragweed pollen

Background: Despite the rare occurrence of ragweed in Middle Europe, a surprisingly high number of patients allergic to mugwort, a frequently encountered weed, display IgE reactivity against ragweed pollen allergens. Objective: The aim of this study was to investigate whether the high prevalence of IgE reactivity against ragweed in patients allergic to mugwort is caused by the presence of common allergenic determinants. We also sought to characterize any cross-reactive allergens. Methods: Common allergenic structures in mugwort and ragweed pollen were characterized by qualitative IgE immunoblot inhibition experiments performed with natural allergen extracts and recombinant allergens. The degree of cross-reactivity was estimated by quantitative CAP-FEIA competitions. The clinical significance of cross-reactive IgE antibodies was studied with histamine release experiments and nasal provocation tests. Results: Mugwort and ragweed RAST values were significantly correlated in a population of 82 Austrian patients allergic to mugwort. IgE antibodies cross-reacted with allergens of comparable molecular weight that were present in both extracts. By using recombinant birch profilin and specific antisera for IgE inhibition experiments, profilin was identified as one of the cross-reactive components in mugwort and ragweed pollen. Preincubation of sera from patients allergic to mugwort with mugwort extract inhibited IgE binding to ragweed pollen extract greater than 80%. Mugwort and ragweed pollen extract induced comparable histamine release and reduction of nasal air flow in a patient with IgE reactivity against the major mugwort allergen Art v 1. Conclusion: In addition to profilin, mugwort and ragweed pollen contain a number of cross-reactive allergens, among them the major mugwort allergen Art v 1. Cross-reactive IgE antibodies can lead to clinically significant allergic reactions. (J Allergy Clin Immunol 1998;101:196-206.)

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.

Parvalbumin, a cross-reactive fish allergen, contains IgE-binding epitopes sensitive to periodate treatment and Ca 2+ depletion

Background: Type I allergy to fish is a severe health problem in countries in which a large percentage of the population derive income from fishing. Objective: The aim of the study was to characterize cross-reactive IgE-binding components in six different fish species (cod, tuna, salmon, perch, carp, and eel). The effect of reducing extraction conditions, periodate treatment, and depletion of Ca 2+ on binding of IgE to the allergens was investigated. Methods: Extracts were prepared under nonreducing and reducing conditions. IgE-binding components were characterized by IgE immunoblotting, and cross-reactive epitopes were studied by IgE-immunoblot inhibition experiments. To reveal calcium-sensitive or carbohydrate-containing epitopes, nitrocellulose-blotted extracts were exposed to ethylene glycol bis(β-aminoethyl ether)-N,N,N ́,N ́-tetraacetic acid (EGTA) and periodate. Results: Sera from all patients allergic to fish ( n = 30) displayed IgE reactivity to parvalbumin, a 12 kd protein present in fish extracts from six different species. Reducing extraction conditions had no effect on IgE binding to parvalbumins, whereas periodate treatment and depletion of protein-bound calcium led to a substantial reduction of IgE binding. Parvalbumins from six different species contained cross-reactive IgE epitopes. Conclusion: Parvalbumin represents a cross-reactive fish allergen. It contains IgE epitopes that are sensitive to periodate treatment and Ca 2+-depletion.

Identification of Tyrophagus putrescentiae allergens and evaluation of cross-reactivity with Dermatophagoides pteronyssinus.

House dust mites are the most common cause of allergic sensitization in respiratory allergic patients in the world. Tyrophagus putrescentiae (TP), which was followed by Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus (DP), has been reported as the third most common house mite in Korea. We previously reported that many respiratory allergic patients had become concomitantly sensitized to DP, DF and TP. The aims of this study were to identify the allergic components of TP and to evaluate the cross-reactivity between TP and DP. The allergenic components of TP and DP extracts were determined with SDS-PAGE and IgE immunoblotting analysis. The cross-reactivity as evaluated by ELISA inhibition and inhibitory immunoblotting experiments. According the SDS-PAGE, the protein components of the two extracts were somewhat different, although a few components displayed identical molecular weights. The 18 kD protein of TP was the most prevalent allergen in the sera of patients sensitized to TP and DP. Both of the maximum inhibition percentages of optic densities of TP-specific IgE in ELISA with TP and DP extract were 100%, respectively and the 50% inhibitory dose (ID50) of TP extract and DP extract were 0.01 micrograms/ml and 0.02 micrograms/ml, respectively. Maximum inhibition of optic densities of DP-specific IgE in ELISA with TP and DP extracts were 29% and 100%, respectively and the ID50 of DP extract was 0.007 micrograms/ml. On inhibitory immunoblotting of DP specific IgE, 5 micrograms/ml of TP extract completely inhibited 16 kD without inhibiting the other allergic component. Whereas, on inhibitory immunoblotting of TP-specific IgE, 5 micrograms/ml of DP extract completely inhibited all the IgE binding components of TP. These results suggested that the major allergen of TP may be the 18 kD component and we also concluded that TP allergens have a strong cross-reactivity with DP extracts, but that DP allergens only have partial cross-reactivity with TP extracts.

Occupational asthma induced by garlic dust

Background: Garlic dust has not been a frequently encountered cause of IgE-mediated disease. Objective: We report on 12 patients (all of them garlic workers) with the clinical criteria for occupational asthma. Methods: Skin prick tests and serum-specific IgE determinations were performed with common inhalants, garlic, and other members of the Liliaceae family (onion, leek, and asparagus). Bronchial challenge test with garlic powder was performed in all patients. Garlic and onion extract proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis. Immunoblot and IgE immunoblot inhibition analyses were performed with patients' sera on extracts of garlic, onion, and pollens of Phleum pratense and Chenopodium album. Results: Garlic sensitization was demonstrated by bronchial challenge test in seven patients (group 1) and ruled out in the remaining five (group 2). Clinical data were similar in both groups. The patients with garlic allergy had a mean age of 27 years, and all of them had pollen allergy; sensitization to other members of the Liliaceae family was also common. Electrophoresis of garlic extract revealed two major protein bands at approximately 12 and 54 kd. During IgE immunoblotting, the pool of sera reacted with garlic proteins mainly at 54 kd. Preincubation with onion, Phleum, and Chenopodium partially abolished the IgE binding to several allergens of garlic. Conclusion: We report on seven patients in whom an occupational garlic allergy was demonstrated. Garlic allergy is relatively rare but seems to affect young subjects with pollen allergy, and sensitization to other members of the Liliaceae family is common. The results of this study confirm the presence of some structurally similar allergens in garlic, onion, and certain pollens. (J Allergy Clin Immunol 1997;100:734-8.)

Effects of air pollution and other environmental factors on birch pollen allergens.

To determine the effects of anthropogenic pollution on water-soluble proteins and specifically allergens in birch (Betula pendula and B. pubescens) pollen, we analyzed extracts of pollen from the pollution gradient around a factory complex (emitting sulfur oxides and heavy metals) by sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis (PAGE) and IgE immunoblotting. In addition, tree density-associated shading of the tree habitat, and quantity and quality of proteins and allergens in pollen of the two birch species were studied. The two studied birch species gave identical allergen profiles even though their protein profiles differed. Distance from the factory did not affect the amount of birch pollen major allergen, Bet v 1 (17 kDa), or of two other strong allergens (23 and 36 kDa). Trees growing in shaded places had significantly stronger responses to Bet v 1 and to the 23-kDa allergen than trees growing in open or half-open environments. Thus, we propose that combined heavy metal and sulfur dioxide pollution does not have an important effect on birch pollen allergens. Instead, other factors, e.g., shading and soil properties of the tree habitat, as well as the genetic background of the tree, may have a stronger influence on the quantity and relative composition of allergens.

Evidence of Hop Japanese pollinosis in Korea: IgE sensitization and identification of allergenic components

Background: Hop Japanese (Hop J) pollens are abundant in the air of Korea during the autumn season. Their significance as a source of allergic sensitization is still unclear. Objectives: We sought to detect the sensitization rate to Hop J pollen as an inhalant allergen and to identify its allergenic components. Method: We carried out skin prick tests with Hop J pollen extract in 1287 patients with respiratory allergy who visited our hospital over the course of 1 year. The serum-specific IgE antibody to Hop J pollen antigen was detected by ELISA, and its binding specificity was confirmed by the ELISA inhibition test. To confirm the respiratory sensitization, bronchoprovocation tests were performed in 16 asthmatic patients sensitive to this pollen. To characterize allergenic components, Hop J pollen extract was analyzed by means of sodium dodecylsulfate–polyacrylamide gel electrophoresis followed by IgE immunoblotting. Results: A positive result on the skin prick test (G2+ of the antigen to histamine ratio) was noted in 79 (6.1%) patients. The serum-specific IgE antibody was detected in 18 (41.9%) patients among 43 positive reactors tested. The ELISA inhibition test with the addition of Hop J pollen extract showed a dose-dependent response. Minimal inhibitions were noted with addition of ragweed and mugwort pollen extracts. Nine asthmatic patients showed significant bronchoconstriction after inhalation of the Hop J pollen extract (five early and four dual asthmatic responders), and all of them had high specific IgE binding. Immunoblot analysis revealed 12 IgE-binding components ranging from 13 to 89 kd. Three bands (13 kd, 74 kd, and 80 kd) were bound to the IgE among the sera tested from more than 50% of the patients. Conclusion: We believe that the Hop J pollen should be considered as a relevant allergen during the autumn season and thus included in skin test batteries in Korea. Some patients diagnosed as having “intrinsic” asthma or rhinitis might be sensitized to this pollen and other previously unknown allergens. (J Allergy Clin Immunol 1997;100:475-9.)

Periodate treatment of Anisakis simplex allergens.

Anaphylactic reactions after parasitized fish consumption are mediated by an IgE response. However, positive skin tests and specific IgE can also be found in many asymptomatic subjects who recognize a single medium-mol.-wt. antigen by IgE immunoblot. The study aimed to find out whether this unspecificity was due to the carbohydrate moieties of parasite antigens. Sixty-two patients with suspected parasite allergy, 51 blood donors, 18 bakers, and 38 atopic patients were studied by blotting. Parasite proteins were treated with periodate. Several selected sera were inhibited with a crude wheat extract and fungal amylase. Twelve patients (19%), eight donors (16%), six bakers (33%), and one atopic patient (3%) recognized a single medium-mol.-wt. band in blotting and should be considered false-positive. This band was periodate-sensitive, but specific IgE to this allergen could not be inhibited by a wheat extract nor by fungal amylase and was clinically irrelevant. Diagnosis of Anisakis simplex hypersensitivity by skin tests and/ or specific IgE values should always be confirmed by specific IgE immunoblotting in order to detect the presence of clinically unrelated antibodies directed to periodate-sensitive allergens. These allergens are probably not a carbohydrate moiety of a parasite glycoprotein.

The use of IgE immunoblotting as a diagnostic tool in Anisakis simplex allergy

Background: The fish parasite Anisakis simplex is the etiologic agent of anisakiasis and induces IgE-mediated reactions. Skin prick tests (SPTs) and the measurement of specific IgE to A. simplex were, in our experience, not valid tools with which to discriminate between allergic and nonallergic patients because many control subjects also had positive results. Objective: The study was carried out to assess the usefulness of IgE immunoblotting in the diagnosis of allergy to A. simplex. Methods: We have studied 61 patients with acute symptoms of urticaria, angioedema, or anaphylaxis and positive specific IgE to A. simplex. According to the anamnesis, time interval between ingestion of fish and clinical onset of symptoms, and exclusion of other causes of allergy, three different groups of patients were established: group A (allergic), group NA (nonallergic), and group D (doubtful). Fifty-one healthy donors were included as control subjects (group C). IgE immunoblotting with A. simplex whole-body extract was performed in all patients and control subjects. Results: Four patterns of immunoblotting were observed: type 1, with a group of several bands of medium molecular weight and others of low molecular weight; type 2, two or more bands of medium molecular weight; type 3, only one band of medium molecular weight; type 4, without any band. There was a significant predominance of blotting type 1 in group A and type 4 in group C. Conclusion: These data suggest that IgE immunoblotting is the most useful approach to A. simplex allergy diagnosis. (J Allergy Clin Immunol 1997;99:497-501.)

Wheat α-amylase inhibitor: A second route of allergic sensitization

Background: Low molecular weight allergens may be responsible for hypersensitivity reactions after the ingestion of wheat. Objective: The purpose of this investigation was to identify relevant, low molecular weight allergens after the ingestion of wheat protein. Methods: Serum samples were collected from seven children with wheat allergy and one adult with baker's asthma. Control serum samples were collected from wheat-tolerant patients. Wheat extracts were prepared and separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% gels revealing numerous protein bands. IgE immunoblot analysis of crude wheat extracts identified multiple IgE-binding proteins. Wheat proteins were separated further with two-dimensional gel electrophoresis, which was followed by IgE immunoblotting investigations. Results: Immunoblot analysis identified a 15 kd wheat protein that bound IgE from all five children with wheat allergy who were evaluated. No IgE binding to this wheat protein was demonstrated in any of the control subjects. Samples representing the 15 kd wheat protein (isoelective point, 5.85) were selected. The N-terminal peptide sequence of this protein (residues 1 to 20) matched to a wheat α-amylase inhibitor. Conclusion: These data demonstrate that wheat α-amylase inhibitor is a relevant allergen in patients experiencing hypersensitivity reactions after the ingestion of wheat protein. This wheat protein, which has been implicated as an important allergen in patients with baker's asthma, represents a sensitizing allergen after both ingestion and inhalation. (J Allergy Clin Immunol 1997;99:239-44.)