IgE response to fur animal allergens and domestic animal allergens in fur farmers and fur garment workers

Objective The aim of this study was to compare the IgE response to the most commonly farmed fur animals with that to domestic animals. Methods IgE-immunoblotting and RAST-inhibition analyses were performed using RAST-positive sera from fur workers sensitized to fur allergens and sera from patients sensitized to domestic animal allergens. Results The urine extracts of mink, blue fox, silver fox, racoon dog and fitchew contained more protein bands than the fur extracts did. Allergens with the same molecular weight were found in all of the fur and urine extracts. The most prominent allergenic bands had molecular weights of 62–67 kDa, 23–25 kDa and 18–19 kDa. With crossreacting sera the reciprocal RAST inhibition with all five animal extracts indicated common IgE-binding epitopes, probably common allergens (especially the 62–67 kDa bands). Urine and fur contain common allergens, since urine allergens strongly inhibited the IgE-binding to fur allergens. The IgE binding to allergenic bands of fur animal extracts was also observed in immunoblotting when dog and cat RAST-positive sera were used, but not for cow RAST- positive sera. RAST inhibition of dog-positive sera with fur animal extracts and fur-positive sera with dog extract confirmed the crossreactivity of these IgE antibodies. No such inhibition was seen with cow extract. Conclusion The results of the RAST inhibition and immunoblotting suggest that fur animals have IgE binding epitopes or allergens in common with cat and dog – possibly albumin but not with cow.     

Identification of orchard grass (Dactylis glomerata) pollen allergens following electrophoretic transfer to nitrocellulose

Orchard grass (cocksfoot) pollen extracts, fractionated by polyacrylamide gradient electrophoresis or SDS gel electrophoresis were electroblotted onto nitrocellulose membranes and probed with sera from orchard grass pollen-allergic patients and 125I-anti-human IgE. The IgE-binding components of the pollen were detected by autoradiography. Elution studies showed that allergens could be extracted immediately and continuously over a 3-hour period. Two fractions of MWs 28,000 and 30,000 could be detected only after 20 min extraction. SDS-PAGE separations gave the better resolution revealing 19 electrophoretically-separate components, 13 of which bound human IgE. All of the IgE-binding components had MWs in the range 14,000 - 70,000. Three of the bands bound IgE from more than 85% of the serum samples. Following gradient gel electrophoresis, IgE binding was exhibited by 10 bands in the range MW 5,000 to greater than 669,000. The technique used allows one to quantitatively examine patients' sera for allergen-specific IgE antibodies and to identify the clinically important allergens. Results revealed numerous allergenic components over a wide MW range while patterns of IgE binding with different patients' sera demonstrated a great diversity of IgE antibody responses. This study demonstrates the suitability of the electroblotting technique combined with autoradiography for the investigation of allergenic components of grass pollen extracts and hence has application to extract standardization and immunotherapy. Such studies can be carried out rapidly, economically and with a high degree of sensitivity.     

Altered antigenicity of M-177, a 177-kDa allergen from the house dust mite Dermatophagoides farinae, in stored extract

BACKGROUND: A high molecular weight allergen, M-177 (177 kDa) was isolated from Dermatophagoides farinae using a specific antibody raised to an allergenic clone Mag 3, which was obtained by immunoscreening a mite cDNA library. The potent IgE reactivity of M-177 is comparable with that of Der f 2. OBJECTIVE: The aim of this study was to analyse the molecular characteristics and the allergenic activity of M-177 in stored mite extracts. METHODS: Antigens were analysed by immunoblotting and enzyme-linked immunosorbent assay (ELISA; inhibition). Allergenic activity was estimated from IgE reactivity and the results of a histamine release assay. RESULTS: The intact M-177 molecule was present in high concentrations in fresh extract obtained from purified mite bodies, but was only detected in small amounts in stored extracts. Instead of the intact molecule, anti-Mag 3 antibody detected various cross-reactive antigens in the stored preparations. Studies of a stored liquid extract showed that these cross-reactive antigens were produced by the degradation of M-177, and that this change was suppressed by the addition of protease inhibitors. Interestingly, the allergenic activity of the fragmented M-177 (sM-177) isolated from the stored extracts was greater than that of the intact antigen. Specific IgE reacted with sM-177 in 84.2% of 38 sera samples from patients allergic to mites, while 65.8% were positive for M-177-specific IgE. Similarly, the histamine release test showed that sM-177 had greater allergenic activity in vitro. ELISA inhibition indicated that the increased allergenic activity resulted from alteration of the antigenicity with the degradation of M-177. CONCLUSIONS: M-177 is a protease-sensitive allergen. The breakdown products of M-177 provoked higher allergenic activity than the intact allergen.     

Gelatin prepared from tuna skin: a risk factor for fish allergy or sensitization?

BACKGROUND: Although fish gelatin may represent a useful alternative to bovine or porcine gelatin, the clearly recognized high prevalence of fish allergy could increase the risk of anaphylaxis to gelatin. The rationale for investigating tuna gelatin rather than gelatin from more allergenic fishes is the availability of an industrial gelatin under development. The infrequent occurrence of tuna allergy should influence the safety of a derived product. The present study investigated IgE antibodies to tuna-skin-derived gelatin in adults and children with documented fish allergy or sensitization. METHODS: Serum samples were taken from 100 consecutive patients with fish allergy or sensitization and tested for IgE antibodies against hydrolyzed or nonhydrolyzed gelatin extracted from tuna skin as compared to extracts from tuna flesh, tuna skin as well as bovine or porcine gelatin. Patients with tuna allergies or sensitization were sensitive to the same tuna species (yellowfin) as that from which the gelatin was obtained. IgE antibodies to these various extracts were analyzed using SDS-PAGE and immunoblotting. RESULTS: Only 3 of the 100 serum samples tested gave evidence of reactivity to gelatin extracted from tuna skin. Cross-reactivity between bovine/porcine and fish gelatin was not observed. CONCLUSION: The risk of adverse reactions to tuna skin gelatin seems to be significantly lower than the risk of fish allergy. Fish gelatin may represent a valuable alternative to bovine or porcine gelatin.     

Occupational allergy in an entomological research center. II. Identification of IgE-binding proteins from developmental stages of the blowfly Lucilia cuprina and other species of adult flies

Sera from 30 workers in an entomological research institute and from five Sydney asthmatics, all with serum IgE antibodies to sheep blowfly (Lucilia cuprina) antigens, were studied with the aim of identifying individual IgE-binding proteins in extracts of L. cuprina adult flies, larvae and eggs, and in extracts of related species of flies from the order Diptera. Using protein blotting, 21, 18 and nine different IgE-binding components were identified in extracts of L. cuprina adult flies, larvae and eggs, respectively. A component(s) of MW 67 kD found in all three developmental stages, showed the highest frequency of IgE-binding; reacting, for example, with 70% of the sera tested with the adult fly extract. Some components were detected in only one of the three developmental stages. Investigations of possible allergenic cross-reactivity between L. cuprina and extracts from six other related species of flies revealed IgE-binding bands in each of the extracts as well as in an extract of Agrotis infusa, a cutworm not belonging to the order Diptera. One strongly reacting component of MW 20 kD was detected in extracts of five different fly species as well as in the A. infusa extract. The results suggested that allergenic cross-reactivity between some fly species exists, and may extend to taxonomically unrelated insect species.     

Allergenicity and cross-reactivity of rye grass pollen extracts revealed by monoclonal antibodies

Using monoclonal antibodies the immunogenic and allergenic characteristics of rye Group I were redefined by SDS-PAGE analysis and immunoblotting. The purified rye Group I from NIH possesses a major component of approximately 34000 Da against which most of the sera from grass-sensitive patients and none from non-atopic volunteers contain specific IgE antibodies. Monoclonal antibodies to rye Group I were raised and used to purify the antigen and to verify the cross-reactivity between grass extracts. The 3 monoclonal antibodies studied recognized different components of timothy grass and 2 of them recognized kentucky june grass but none recognized components of ragweed extracts.     

The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum

BACKGROUND: For the detection of allergen-specific IgE in serum, IgE-binding assays such as the radioallergosorbent assay (RAST) are commonly used. In this study, the applicability and sensitivity of the stripped basophil histamine release bioassay was investigated and compared to the RAST. METHODS: Basophils were stripped of their IgE by an acidic buffer, sensitized by human serum and stimulated by allergen with or without interleukin (IL)-3. The histamine release was determined by fluorometric analysis. RESULTS: We showed that for enhancement of the maximal histamine release and the sensitivity of the stripped basophil assay, the priming cytokine IL-3 can be added to the basophils simultaneously to the stimulus. Preincubation of the cells with IL-3, as described in other studies, was not necessary. The bioassay can be used to study the specificity of IgE-mediated reactions. Basophils sensitized by serum absorbed to a particular allergen did not respond to this allergen anymore. This method is very suitable to study cross-reactivity between allergens. The results obtained in the bioassay were comparable to those obtained in the RAST. Using the RAST, lower concentrations of allergen-specific IgE were detected than in the bioassay. However, sera containing IgE against minor allergenic components were negative in the RAST, but strongly positive in the basophil assay. CONCLUSIONS: The stripped basophil histamine release bioassay is useful to complement and extend serological detection of allergen-specific IgE. Especially with sera containing IgE against minor components, this assay is more suitable than the RAST. Furthermore, in this assay, the dependency of IgE and of allergen-specific IgE in reactions can be studied in more detail.     

Allergenic components of Candida albicans identified by immunoblot analysis

Allergenic components of Candida albicans fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes were identified using sera from 30 asthmatic patients who showed positive skin test and RAST (radio-allergosorbent test) to C. albicans. The IgE-binding yeast components in the complex antigen preparation were then detected by reaction with enzyme-labelled anti-human IgE antibodies. They were confirmed by Coomassie blue R-250 staining of the membrane to visualize all protein bands after reaction with the enzyme substrate. The IgE-binding patterns of the sera tested were heterogeneous, displaying a total of 16 identifiable components with molecular weights ranging from 20 to 94 kD. A 40 kD component showed the highest IgE-binding frequency, being recognized by 23 (77%) of the 30 sera examined. The other 15 allergenic components identified were recognized by less than 25% of the sera tested. Only two of the 30 serum samples contained IgE antibodies reactive with seven to eight allergenic components. Ten of the 30 sera reacted with only one allergenic component, and the remaining serum samples recognized two to five of the 16 identified allergens. Results described in this study are applicable to allergen standardization work and provide a basis for further study on the role of C. albicans in clinical allergy.     

Detection of IgE antibodies to a wide range of insect species in subjects with suspected inhalant allergies to insects

Sera obtained from subjects diagnosed on the basis of case history and skin tests as having inhalant allergies to insects, were tested for the presence of IgE antibodies to antigens from the house fly Musca domestica, a blowfly Calliphora stygia, the common clothes moth Tineola bisselliella, warehouse moth Ephestia cautella, cockroach Blattella germanica, carpet beetle Anthrenus verbasci and silverfish Ctenolepisma longicaudata. Approximately one-third of the sera reacted with extracts from all seven species, over half the sera reacted with four of the extracts and only 3 sera proved negative to all of the extracts. Twenty-six of the sera also contained IgE antibodies that reacted with the house dust mite Dermatophagoides farinae. Tests with a further eleven species of flies from the order Diptera and with extracts of the grain borer Rhyzopertha dominica, locust Chortoicetes terminifera and Bogong moth Agrotis infusa revealed strong IgE antibody responses to all of the species. Electroblotting studies revealed up to 15 IgE-binding components in extracts from 11 different insect species including 6 species of flies. Some IgE-binding insect components with similar electrophoretic mobilities indicated the possible presence of common allergens in extracts from different species. In particular, blots of each of the fly extracts showed a dense IgE-binding component of MW approximately 37,000 daltons. 'Pan allergy' to insects may occur in subjects who have been sensitized to one or a few insects and allergenic similarities may extend to at least some other noninsect members of the phylum Arthropoda.     

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.     

IgE-mediated hypersensitivity to taugeh (sprouted small green beans)

This report describes a case of allergy toward taugeh (sprouted small green beans), an important constituent of eggroll. With use of the skin prick test, RAST, and histamine release test, IgE antibodies against taugeh extract could be demonstrated. A partial cross-reactivity was observed between IgE-binding components from taugeh and peanuts. Peanut allergic patients could also experience adverse reactions after ingestion of taugeh.     

Evaluation of IgE-sensitization to fungi in HIV-positive patients with eczematous skin reactions.

BACKGROUND: Human immunodeficiency virus infection is associated with declining immune function and polyclonal B-cell activation leading to elevated IgE-levels. In selected patient categories, increased total IgE may be associated with allergic diseases. Furthermore, a significant number of patients with low CD4+ cell numbers have various skin manifestations, eg, eczema and dermatophytosis. Patients with chronic fungal infections and a tendency to produce increased levels of specific IgE may become allergic and IgE-mediated mechanism may contribute to inflammatory reactions in the skin. OBJECTIVE: This study investigates IgE-sensitization of patients infected with human immunodeficiency virus to a panel of fungal extracts of Candida albicans, Fusarium moniliforme, Penicillium notatum, Pityrosporum ovale, and Trichophyton rubrum. METHODS: Fifteen HIV-positive patients with eczematous skin manifestations and five non-atopic healthy controls were evaluated by basophil histamine release and skin prick test with fungal extracts. The extracts were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions and analyzed by IgE-immunoblotting with sera from the patients and controls. RESULTS: Thirteen of 15 patients (87%) released histamine to one or more of the fungi. Skin prick test was positive to one or more fungi in 7 (47%) patients. Patient sera revealed binding to a wide range of IgE-binding components present in the fungal extracts. The IgE response was most often directed against a 46-kD main protein in the Candida albicans extract. There was no correlation between total serum IgE, CD4+ cell counts, and frequency of IgE-sensitization to fungi. CONCLUSION: The human IgE response in HIV-infected patients appears to be polyspecific and may be directed against various fungi of which Candida albicans may be an important allergen. It is possible that the sensitization is due to frequent infections with Candida albicans in this patient population. No unspecific fungal reactions were noted among control patients. These results suggest that allergen-specific IgE-mediated mechanism may contribute to the pathogenesis of the eczematous skin reaction in HIV-infected patients.     

Immunoblot analysis of components of Penicillium notatum recognized by human IgE antibodies

Components of the crude extract of Penicillium notatum recognized by human IgE antibodies (Abs) were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The allergenic components were identified with sera from 19 allergic patients and 20 blood donors. The allergen-Ab complexes were visualized by 125I-labeled monoclonal antihuman IgE and autoradiography. A total of 11 allergenic components, ranging in molecular weights (MWs) from 94,000 to 20,000 daltons, were identified. Heterogeneity in the IgE-binding patterns of the serum samples tested was also observed. However, the major allergen appears to be the component with an MW of about 68,000 daltons that was recognized by IgE Abs in 56% of the 39 sera analyzed. Furthermore, the component with an MW of about 64,000 daltons that was recognized by IgE Abs in 46% of the sera analyzed was also considered as an important allergen. Results obtained from this study will be useful in additional characterization of allergens of P. notatum and related fungal species.     

Allergologic exploration of germins and germin-like proteins,a new class of plant allergens

BACKGROUND: Germins and the related germin-like proteins (GLPs) are glycoproteins expressed in many plants in response to biotic and abiotic stress. To test the potential impact of germins and GLPs, recombinant germin from Triticum aestivum (tGermin) and GLPs from Arabidopsis thaliana (tGLP), both produced in transformed tobacco plants, were used. METHODS: Sera from 82 patients with type I allergy to birch, grass or mugwort pollen and/or wheat were tested in immunoblot for IgE binding to tGermin and tGLP, and the IgE reactivity after chemical and enzymatic deglycosylation was analysed. The biological activity of tGermin and tGLP was determined in a histamine release assay and in skin prick testing (SPT). RESULTS: In an immunoblotting assay, 24 out of 82 tested sera (29.26%) from allergic patients showed IgE-binding to tGermin, and 18 of these sera (21.95%) displayed also IgE-binding to tGLP. The deglycosylation experiments indicated that glycan moieties contribute significantly to the IgE-binding of tGermin and tGLP. Both tGermins and tGLP induced specifically histamine release in an in vitro assay as well as in SPT. CONCLUSION: Our in vitro and in vivo findings demonstrate that germin and GLPs are capable to bind IgE most likely via carbohydrate determinants, and represent allergenic molecules.     

Identification and partial purification of pollen allergens from Artemisia princeps

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.     

Evaluation by double-blind placebo-controlled oral challenge of the clinical relevance of IgE antibodies against plant glycans

Background:  The clinical relevance of immunoglobulin E (IgE) to plant glycans is a longstanding debate. We sought to evaluate their clinical reactivity using the human glycoprotein lactoferrin expressed in rice.
Methods:  Allergic patients with IgE antibodies against plant glycans were analyzed for the presence of IgE against rice-produced lactoferrin. The potency of IgE to induce mediator release was assessed by basophil histamine release and skin prick tests (SPTs). Clinical relevance was evaluated by double-blind placebo-controlled oral challenge (DBPCOC).
Results:  Twenty-four of 29 sera (82.7%) with IgE antibodies against plant glycans demonstrated IgE binding to transgenic lactoferrin. In three of five cases transgenic lactoferrin induced histamine release. Compared to a control major grass pollen allergen lactoferrin concentrations needed for biological activity of IgE were 5–6 orders of magnitude higher. Skin prick test and DBPCOC were negative in five patients with potential clinical reactivity that volunteered to undergo these in vivo challenges.
Conclusions:  Poor or no biological activity and lack of clinical relevance of IgE-binding plant glycans (five out of five) was demonstrated using human lactoferrin expressed in rice as a model.     

*IgE antibodies against midge and moth found in Japanese asthmatic subjects and comparison of allergenicity between these insects

The specific IgE antibodies to moth (Bombyx mori) and midge (Chironomus yoshimatsui) were measured by the Pharmacia CAP system in 51 house-dust-mite-sensitive asthma patients. None of these patients had definite histories of exposure to these insects or apparent evidence of insect-induced asthma symptoms. The RAST-inhibition assay was performed to investigate cross-allergenicity between these two insects. Furthermore, IgE immunoblotting was done to study the IgE-binding components in moth and midge extracts. Thirty (59%) of these patients showed positive IgE antibodies to moth, while 25 (49%) showed positive IgE antibodies to midge. Those frequencies of positivity were similar to that for Japanese cedar pollen, which is well known to cause allergy. A significant correlation (r=0.863) was observed between IgE antibody liters of these two insects. The results from the RAST-inhibition assay indicated cross-allergenicity between these insects and also the existence of species-specific allergens. Fifteen IgE-binding components in moth extract were observed. The most frequent IgE-binding protein was the 79-kDa (84.2%), followed by the 72-kDa (78.9%), the 82-kDa (57.9%), and the 76-kDa (57.9%) proteins. Those were considered to be major allergens in moth. Twenty-four IgE-binding components in midge extract were observed. However, no IgE-binding protein to which over 50% of patient sera reacted was observed. These results suggest that these two insects may be considered to bear important allergens and that there is cross-allergenicity between these insects as well as species-specific allergens.     

Group V allergens in grass pollens. II. Investigation of group V allergens in pollens from 10 grasses

In an earlier study an allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated and physicochemically characterized. In this study Phl p V and immunochemically similar components from other grass pollens (group V allergens) have been investigated using immunoelectrophoretic techniques. To study the allergenic importance of the group V allergens, the allergenic compositions of 10 grass pollen extracts were investigated in crossed radioimmunoelectrophoresis (CRIE) using 20 sera from grass pollen-allergic donors. Group V allergens were identified using monospecific rabbit antibodies raised against Phl p V, anti-Phl p V, which react with other group V allergens usually producing dense precipitates in immunoelectrophoresis. In this way group V allergens were identified in eight extracts, and when present the precipitate corresponding to the group V allergen was the dominant IgE binding precipitate. All identified group V allergens bound IgE in at least 17 of the 20 investigated sera. Monospecific rabbit antibodies raised against the group I allergen of Lolium perenne (rye grass), anti-Lol p I, do not precipitate group V allergens, indicating that there are no immunochemical similarities between group I and group V allergens. In SDS-PAGE anti-Phl p V identifies IgE-binding components with molecular weights between 26 and 33 kD. In contrast, anti-Lol p I binds to components of slightly higher molecular weight. Apparently, the group V components are allergens that are physicochemically and immunochemically distinct from group I allergens.     

Occupational asthma caused by fish inhalation

Occupational asthma (OA) due to fish inhalation, confirmed by specific bronchial challenge (SBC), has not been described as yet in medical literature, as far as we know. We describe two patients whose asthma was induced by occupational exposure to fish and confirmed by serial measurements of PEFR and SBC. Two fish-processing workers reported asthma symptoms related to their workplace. They were skin tested with fish extracts and their sera assayed for IgE antibodies to various fish species. Nonspecific bronchial reactivity was assessed by methacholine challenge. The occupational relationship was confirmed by PEFR monitoring in working and off-work periods. SBC with fish extracts was carried out to confirm the diagnosis of OA. Skin tests with raw and cooked plaice, salmon, hake, and tuna in patient 1 and anchovy, sardine, trout, salmon, Atlantic pomfret, and sole in patient 2 were positive. Specific IgE serum antibodies were found to salmon in patient 1 and to trout, anchovy, and salmon in patient 2. PEFR measurements differed significantly (P<0.001) between work and off-work periods for both patients. A bronchial challenge with methacholine was positive in patient 1. SBC with raw hake, salmon, plaice, and tuna extracts in patient 1 and raw salmon extract in patient 2 were all positive with an immediate response. SBC with Dermatophagoides pteronyssinus extract was entirely negative in both patients. In three asthmatic, non-fish-allergic controls, SBC with tuna, hake, salmon, and plaice were all negative. These results suggest that fish inhalation can elicit IgE-mediated occupational asthma.