Identification and characterization of important allergens from mugwort pollen by IEF, SDS-PAGE and immunoblotting

Mugwort (Artemisia vulgaris L.) pollen allergens, separated by SDS-PAGE or IEF, were identified after transfer to NCM by incubation with a panel of sera from 16 patients with clinical mugwort pollen allergy, followed by [125I]anti-IgE and autoradiography. Of the at least 23 components separated by SDS-PAGE in a 15% polyacrylamide gel, at least 15 components with mol. wts 12,000-100,000 bound IgE from the panel of patient sera. A component of mol. wt 22,000 bound IgE from at least 94% of the patient sera tested and for all but three sera this component also bound the greatest quantity of IgE. Five other components with mol. wts 12,000, 17,000, 29,000, 39,000 and 42,000 bound IgE from 75-94% of the patient sera. After separation by IEF, at least 28 protein bands were detected in the pI region 3.5-7.2 and at least seven bands were found in the region 8.6-9.3. At least 11 bands in the pI range 4.2-7.3 and at least five bands in the pI region 8.5-9.2 bound IgE from the panel of patient sera. The most intense radiostaining was observed with a component having a pI of 4.35, which bound IgE from 31% of the patient sera. Immunoblotting of the SDS-PAGE and IEF gels using specific rabbit antisera and human sera against three important mugwort pollen allergens, denoted Ag 9, Ag 12 and Ag 13, was performed to determine the mol. wt and pI of these allergens which had earlier only been identified in CIE/CRIE. The results revealed that Ag 13 had a mol. wt of 61,000 and a pI of 4.35, Ag 12 had a mol. wt of 22,000 and AG 9 had pIs in the region 4.55-5.55 (six isoforms). Ag 9 did not bind IgE after SDS-PAGE and was thus not identified in the SDS-PAGE pattern, and Ag 12 failed to be detected in the NCM after transfer from IEF gels. By crossed immunoelectrofocusing, Ag 12 was found to consist of several isoforms predominantly located in the pI region 3.5-5.1. The immunoblotting analysis also revealed that the glycoprotein allergen Art v II was not detected after transfer from either SDS-PAGE or IEF gels. In conclusion, immunoblotting analysis of SDS-PAGE and IEF gels are useful methods for characterization of mugwort pollen extract, but it should be noted that some important allergens which are easily identified in CIE/CRIE may fail to be detected by these methods.     

Anaphylactic reaction to young garlic

BACKGROUND: Garlic is well known to cause contact dermatitis and asthma. However, it is a very rare cause of food allergy. We present the case of a 23-year-old woman with previous history of allergy to pollen and dried fruit, and food-dependent, exercise-induced anaphylaxis for which no specific food could be identified as responsible, who experienced an anaphylactic reaction after eating young garlic. METHODS: Skin prick tests and specific IgE immunoassay with several pollens and foods were performed, as well as the prick-prick test with young garlic and SDS-PAGE followed by immunoblotting IgE to young garlic and other Liliaceae species, mustard, sesame, parsley, celery, hazelnut, almond, and pollen of birch and mugwort. RESULTS: Skin prick tests and specific IgE were mainly positive for grass, plane tree, and mugwort pollen; peanut; hazelnut; walnut; almond; and mustard. Prick-prick tests with young garlic and garlic were positive. Total IgE was 113 U/ml. SDS-PAGE immunoblotting showed IgE-binding bands at 12 kDa to young garlic, garlic, onion, and leek extracts. Similar bands could also be detected with mugwort pollen and hazelnut extract. CONCLUSIONS: We describe IgE-mediated reaction to young garlic in a patient sensitized to pollen and dried fruit.     

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.     

Hypersensitivity to mugwort (Artemisia vulgaris) in patients with peach allergy is due to a common lipid transfer protein allergen and is often without clinical expression

BACKGROUND: The observation of mugwort-specific IgE antibodies in patients with peach allergy suggests that mugwort sensitization might play a role in sensitization to peach. OBJECTIVE: We sought to study the clinical manifestations of mugwort hypersensitivity in patients with peach allergy, identify the common allergens, and evaluate their IgE crossreactivity. METHODS: Patients with oral allergy syndrome for peach and specific IgE antibodies to mugwort were investigated for respiratory symptoms during the mugwort season. Peach and mugwort allergens were identified by means of SDS-PAGE and IgE immunoblotting. Immunoblotting inhibition experiments were done to study cross-reactivity between peach and mugwort and other pollens. RESULTS: Seventeen patients were studied, 10 with no seasonal respiratory symptoms and 7 with clear late summer respiratory symptoms. In IgE immunoblotting the 10 asymptomatic patients reacted only to a 9-kd allergen of both mugwort and peach, whereas the 7 patients with pollinosis reacted to other allergens. Ten patients with mugwort allergy, no history of allergy to peach, and negative results for peach-specific IgE antibodies were also studied. The mugwort 9-kd protein was identified as a lipid transfer protein (LTP) homologous to peach LTP. Immunoblotting inhibition showed that IgE binding to the peach 9-kd band was totally inhibited by 4 microg of peach LTP but only by 400 microg of mugwort LTP, whereas 4 microg of both mugwort and peach LTP totally inhibited the mugwort immunoblotting. The results were similar with other pollens. CONCLUSIONS: Patients sensitized only to the 9-kd LTP of mugwort do not present hay fever symptoms, and this sensitization is a consequence of the peach sensitization.     

The Helix aspersa (brown garden snail) allergen repertoire

BACKGROUND: Ingestion of snails can induce strong asthmatic or anaphylactic responses, mainly in house-dust-mite-sensitized patients. The aim of this study was to identify the Helix aspersa (Hel a), Theba pisana (The p) and Otala lactea (Ota l) allergens and the extent of their cross-reactivity with the Dermatophagoides pteronyssinus (Der p) mite. PATIENTS AND METHODS: In 60 atopic patients, skin prick tests (SPT) to snail and D. pteronyssinus, total and specific IgE, specific IgE immunoblots, RAST and immunoblot inhibition assays were performed. RESULTS: Mean total IgE was >1,000 kU/l. Mean specific IgE (class 6 for Der p and class 2 for Hel a) SPT were positive in 44 patients for snail and in 56 for mite. Isoelectric focusing (IEF) and SDS-PAGE followed by immunoblotting of H. aspersa extract enabled the identification of 27 and 20 allergens, respectively. Myosin heavy chains from snails (molecular weight >208 kDa) disclosed two major allergens. Hel a and Der p RAST were strongly inhibited by their homologous extracts, with Hel a RAST being inhibited by the Der p extract to a much greater extent (72.6%) than the inverse (5.6%). A complete inhibition of the immunoblots by their homologous extract was obtained. However, Hel a extract did not inhibit Der p IEF separated recognition. On the other hand, mite extract extensively inhibited snail immunoblots from both IEF and SDS-PAGE separations. Immune detection on chicken, pig, rabbit, cow and horse myosins did not reveal any IgE cross recognition with snail. CONCLUSIONS: In most cases of snail allergy, mite appeared to be the sensitizing agent. Nevertheless, snails may also be able to induce sensitization by themselves. This hypothesis is supported by the finding of specific IgE to Hel a in 2 patients who did not show specific IgE to Der p, and one of them was suffering from asthma after snail ingestion.     

A study of allergens in celery with cross-sensitivity to mugwort and birch pollens

Sixty-one sera with positive RAST to mugwort pollen ( Artemisiae vulgaris ) were submitted to RASTs for birch pollen ( Betula verrucosa) and celery ( Apium graveolens ). In 36 cases RAST results were positive for celery. In addition, 23 sera presented specific IgE to birch pollen. The binding of specific IgE to individual allergens in celery, mugwort pollen and birch pollen was studied by the immunoblotting technique. This involved electrophoretic separation of allergenic extracts, electrotransfer of proteins onto nitrocellulose sheets and sensitive immunoenzymatic detection. Eighteen sera had specific IgE binding to two celery components of molecular weight around 15 kD. All these sera also detected a 15 kD allergen in mugwort and two allergens in birch of 14 kD and 16 kD molecular weight. The sera that did not detect the 15 kD bands in celery failed to react with both the 15 kD mugwort component and the 14 and 16 kD birch components. Specific cross-inhibitions of the detection of these allergens on immunoblots were obtained by pre-incubation of the sera with crude extract of the three species. These results strongly suggest that such allergens display some structural identity and that they could be at the origin of some cases of crossed hypersensitivity to celery, mugwort pollen and birch pollen.     

*Basidiomycete allergens: comparison of three Ganoderma species

High atmospheric concentrations of basidiospores occur in various parts of the world. Ganoderma basidiospores are distinctive, easily identifiable in aeroallergen surveys, and widely abundant. Previous studies showed that Ganoderma basidiospores cause respiratory allergies. Thus, we investigated various extracts (spore, cap, and/or mycelial) of G. meredithae, G. lucidum, and G. applanatum for allergen components. Analyses included radioallergosorbent test (RAST) inhibition and IgE blots from isoelectric focusing (IEF) and SDS-PAGE. RAST inhibition with spores and caps of G. meredithae and G. lucidum showed that spores inhibited caps better than caps inhibited spores. Species differences were minor. Coomassie blue (CB) staining of IEF gels detected at least 23 protein bands (pI 3.6-6.6) in caps of G. meredithae and G. lucidum. G. meredithae spore extracts contained 17 of these (pI 3.6-5.0, 6.6). Spores and caps of G. meredithae contained 13 and 11 allergen bands, respectively, on IEF blots. SDS-PAGE of G. meredithae spore and cap showed one and four bands, respectively, by CB staining, but IgE blots showed 13 bands in cap and 17 in spore. Culture mycelia of G. lucidum and G. applanatum attained significant and essentially constant RAST activity by day 4. Activity was also present in culture supernatant by day 4. Blots of mycelium and supernatant detected a single allergen in day-8 mycelia and subsequently six allergen bands in day-16 mycelia and eight in day-16 supernatant (one appeared as a doublet). These data show that Ganoderma extracts contain a complex mixture of allergens. Differences among species were minor; spores and mycelia are apparently better sources of allergens than caps.     

Primary sensitization to sweet bell pepper pollen in greenhouse workers with occupational allergy

BACKGROUND: In a previous investigation, a high prevalence of allergy to sweet bell pepper pollen was found among exposed horticulture workers. Allergy to plant-derived food is often the consequence of primary sensitization to common pollen allergens. OBJECTIVE: We therefore investigated the cross-reactivity between sweet bell pepper pollen and pollen from grass, birch or mugwort. METHOD: We selected 10 sera from greenhouse workers who had, besides specific IgE against sweet bell pepper pollen, also IgE to grass, birch or mugwort pollen. Cross-reactivity was tested by the inhibition of IgE binding to solid-phase coupled sweet bell pepper pollen extract. The 10 sera were also analysed for IgE binding to sweet bell pepper pollen by immunoblotting. RESULTS: With these sera, no or small inhibition of IgE binding to sweet bell pepper pollen extract was observed with grass, birch and mugwort pollen. With immunoblotting, major IgE-binding structures were seen at 14, 29 and 69 kDa in sweet bell pepper pollen extract. CONCLUSION: The results of our study demonstrate that sweet bell pepper pollen contains allergens that have no or limited cross-reactivity with common pollen allergens. With sera from the 10 patients tested, sensitization to sweet bell pepper pollen was not the consequence of primary sensitization to common pollen allergens.     

Crossreactivity between allergens in natural rubber latex and banana studied by immunoblot inhibition

Background An association between allergic reactions to natural rubber latex and to banana has been reported but the immunochemical properties of the putative crossreacting allergens remain unknown. Obfective To study extracts of banana and natural rubber latex and sera from latex-allergic patients for possible crossreacting allergens and IgE antibodies. Methods Sera from 22 latex-allergic patients and 22 control subjects with no evidence of allergy to latex or to banana were studied. All patients had positive and controls negative reactions in skin-prick testing using an eluate of latex gloves. IgE antibodies to natural rubber latex and to banana were evaluated by immunoblotting and by radioailergosorbent test (RAST) and crossreactivity between allergens in banana and natural rubber latex by immunoblot inhibition. Skin-prick testing was used to examine in vivo reactivity to banana. Results Ten of the 22 (45%) latex-allergic patients sera recognized altogether 14 allergens in banana by immunoblotting. The most frequently identified banana allergens were 23, 32, 36, 39 and 47kDa proteins. The banana skin-prick test was positive in 14 of 18 (78%) latex-allergic patients studied and banana RAST in 12 of 14 patient sera tested. Fourteen of 21 interviewed patients reported symptoms from eating or handling bananas. In immunoblot inhibition studies a dose-dependent inhibition of IgE binding to banana extract with natural rubber latex proteins was observed in all five patient sera tested and, likewise, the binding of IgE to natural rubber latex extract was inhibited with banana proteins in four of the five patient sera. Conclusions The present results confirm the existence of crossreacting allergens in natural rubber latex and banana and provide new information on the immunochemical nature and heterogeneity of these allergens.     

IgE-mediated sensitization to English plantain pollen in seasonal respiratory allergy: identification and partial characterisation of its allergenic components.

Characterisation by SDS-PAGE immunoblotting of plantain pollen extract showed that components of 16,000-20,000 M(r) were frequently reactive with IgE antibody in the sera of subjects with seasonal respiratory allergy. Other, more weakly IgE-binding allergens were seen in the range of 40,000-60,000 M(r). HPLC followed by RAST inhibition demonstrated that components of approximately 17,000 M(r) were also responsible for much of the IgE-binding activity of the extract. These components appeared to have pI values between 4.5 and 5.2. RAST inhibition showed that there were no common IgE-binding epitopes in grass pollen and plantain pollen extracts, indicating that skin test responses should not necessarily be interpreted in terms of cross-reaction. 82 subjects with a clinical history of seasonal, respiratory allergy were screened in a skin prick test survey. 28% were skin test positive to plantain pollen extract. The frequency of positive skin test reactions to plantain pollen extract was greater than that to Betula (23%) and Artemisa (16%), both which are considered to be important allergens. In a larger survey positive RAST scores to plantain pollen were given by 34% of sera from subjects with respiratory allergy. Plantain pollen sensitivity should therefore be considered during diagnosis of seasonal allergy.     

In vivo tests with pollen extracts previously investigated by means of direct RAST titration allergen assay

The allergenic potency of different birch, Timothy and mugwort pollen extracts was determined by means of a direct RAST titration allergen assay. For birch and Timothy allergens, the results of skin and provocation tests did not confirm the results of the in vitro determinations of allergenicity. There was a poor correlation between the results of skin tests and the results of Phadebas RAST for determination of specific IgE to mugwort, whereas the correlation between skin tests and RAST for other allergens was excellent. It is concluded that direct RAST titration allergen assay is not adequate for all kinds of allergen preparations and that the Phadebas RAST for mugwort is less sensitive than the RAST for other allergens. The diagnostic efficacy of the different allergen preparations could not be evaluated.     

IgE-mediated anaphylaxis to Thiomucase, a mucopolyssacharidase: allergens and cross-reactivity

BACKGROUND: Thiomucase is a mucopolysaccharidase obtained from ovine tissues mainly used to facilitate the diffusion of local anaesthetics and in the treatment of cellulitis. A patient with an anaphylaxis in relation to the intramuscular administration of Thiomucase is reported. OBJECTIVE: To investigate Thiomucase allergens and their possible relationship with dander allergens and animal albumins. MATERIAL AND METHODS: Skin prick tests (SPT) and serum-specific IgE were performed with Thiomucase and danders. Thiomucase SDS-PAGE immunoblotting was performed in order to study allergens. RAST/CAP inhibition and SDS-PAGE immunoblotting inhibition were carried out to study the cross-reactivity. RESULTS: Skin prick tests (SPT) were positive to Thiomucase, animal dander (cat, dog, sheep, other), bovine serum albumin (BSA), and echinococcus. Specific IgE was also positive to Thiomucase, animal dander (cat, dog, sheep, other), BSA and echinococcus. In the RAST-CAP inhibition assays BSA was nearly completely inhibited by Thiomucase, Thiomucase was partially inhibited by BSA and cat and Echinococcus granulosus was partially inhibited by sheep and Thiomucase. In the Thiomucase SDS-PAGE immunoblotting several proteins fixed IgE, ranging from 20 kDa to > 94 kDa, the strongest with 43 kDa. The IgE fixation to BSA, cat and sheep in the SDS-PAGE immunoblotting was completely inhibited by the preincubation of the serum with Thiomucase. CONCLUSIONS: An IgE-mediated anaphylaxis to Thiomucase is documented. Multiple allergens are recognized in Thiomucase by the patient serum, the main with 43 kDa. Partial cross-reactivity with BSA, cat dander and sheep dander is documented.     

Quality of timothy pollen (Phleum pratense) from different pollen seasons and different suppliers

We investigated seven batches of timothy (Phleum pratense) pollen from four different pollen seasons and three different manufacturers in the USA and Europe by several biochemical and immunochemical methods. By measuring the contents of hexoses, proteins and the total allergenic activity using RAST inhibition we could not find any significant differences in quality between pollen of different seasons, different geographical origin and different manufacturers. There were only slight differences in the staining of some protein bands and in peak height of some antigens in isoelectric focusing (IEF), SDS-PAGE and crossed immunoelectrophoresis. These differences in IEF and SDS-PAGE patterns seem to be specific for the pollen samples from the USA and Europe, respectively. We also observed minor differences in IEF immunoprint patterns, mainly for some basic proteins. The allergen patterns in crossed radioimmunoelectrophoresis were very similar, with only minor differences in concentration of the basic allergens. It appears that timothy pollen from different years can be produced in different geographical areas by different manufacturers with fairly constant allergenic activity.     

The immunological relationship of epitopes on major tree pollen allergens.

The major allergens of birch (Bet v I), alder (Aln g I), hazel (Cor a I) and hornbeam (Car b I) were investigated by means of high-resolution two-dimensional electrophoresis combined with immunoblotting. Eleven sera derived from patients allergic to birch pollen as well as mouse monoclonal antibodies BIP 1 and BIP 4, raised against Bet v I, were used as probes. Human IgE antibodies detected 10 spots in birch (Mr 17 kDa, pI 4.9-5.9); four spots in alder (Mr 18.5 kDa, pI 4.7-5.3); four spots in hazel (Mr 17 kDa, pI 5.0-5.8); and 12 + 7 spots in hornbeam (Mr 16.5 kDa, pI 4.9-6.6 and Mr 18 kDa, pI 5.2-6.7), respectively, representing major allergens. Each patient tested reacted in a similar fashion with the spot cluster(s) of a certain allergen. BIP 1 detected the same spot clusters as patients' IgE. BIP 4 reacted with the 17-, 18.5- and 18-kDa spots of birch, alder and hornbeam, but did not react with the 17-kDa spots of hazel and the 16.5-kDa spots of hornbeam. In inhibition experiments with birch pollen extract as inhibitor, IgE binding to Bet v I, as well as to Aln g I, Cor a I and Car b I was abolished, thus suggesting that IgE binding to major tree pollen allergens is confined to shared epitopes. These findings indicate that it might be sufficient to use only Bet v I for diagnostic procedures as well as for immunotherapy in patients with tree pollen allergy.     

Purification and characterization of Lep d I, a major allergen from the mite Lepidoglyphus destructor

A major allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been purified by affinity chromatography using an anti-Lep d I monoclonal antibody. The purity of the protein obtained by this procedure was assessed by reverse-phase HPLC. Lep d I displayed a molecular weight of 14 kD on SDS-PAGE under non-reducing conditions, and 16 kD in the presence of a reducing agent. Analytical IEF revealed a little charge microheterogeneity, showing three bands with pIs 7.6-7.8. Purified Lep d I retained IgE-binding ability, as proved by immunoblotting experiments after SDS-PAGE and RAST with individual sera from L. destructor-sensitive patients. Results from the latter technique demonstrated that 87% of L. destructor-allergic patients had specific IgE to Lep d I, and a good correlation between IgE reactivity with L. destructor extract and Lep d I was found. In addition, RAST inhibition experiments showed that IgE-binding sites on Lep d I are major L. destructor-allergenic determinants, since Lep d I could inhibit up to 75% the binding of specific IgE to L. destructor extract; on the other hand, Lep d I did not cross-react with D. pteronyssinus allergens.     

Allergenic cross-reactivity of olive pollen

BACKGROUND: Sera of patients allergic to olive (Olea europaea) pollen were used to analyze the IgE cross-reactivity between olive-pollen extract and other pollens obtained from phylogenetically unrelated species. METHODS: We used IgE immunostaining of pollen extracts blotted to nitrocellulose membranes after SDS-PAGE and inhibition analysis of this binding. RESULTS: A high inhibition of the IgE binding on olive-pollen extract was exhibited by birch, mugwort, pine, and cypress pollens, suggesting that these extracts contain proteins which share common epitopes and thus can be recognized by olive-allergic sera. IgE binding to Gramineae pollen extracts was not inhibited by olive-pollen extract, indicating a primary sensitization of the patients to these species. From the inhibition assays, the presence of an allergen of 45 kDa in the olive pollen, which has no homologous counterparts in other allergenic species, has been inferred. CONCLUSIONS: Olive pollen contains allergens which cross-react with pollens from unrelated species, a fact that could simplify the diagnosis and treatment of pollinosis.     

Determination and characterization of cross-reacting allergens in latex, avocado, banana, and kiwi fruit

Sera of 11 patients were used to characterize allergens in kiwi fruit, latex, avocado, and banana by SDS-PAGE/immunoblotting and to determine cross-reactions between these allergen extracts in EAST inhibition and immunoblot inhibition. By SDS-PAGE/immunoblotting. allergens with apparent molecular weights of 21, 38. 40. and 42 kDa were visualized in latex extract. In avocado extract. IgE-binding components of 27, 43, 52, 58, 65, 75, and 88 kDa were to be seen, whereas, in banana extract, a 40-kDa protein showed strong IgE binding. Furthermore, allergens of 52,58,88, and 94 kDa were detected in the extract of banana. Cross-reactions between these allergen extracts were determined by EAST inhibition. Immunoblot inhibition demonstrated that almost all IgE-reactive bands in nitrocellulose-blotted latex, avocado, and banana extracts and two components of 43 and 67 kDa in kiwi fruit shared common IgE epitopes.     

Type I allergy to elderberry (Sambucus nigra) is elicited by a 33.2 kDa allergen with significant homology to ribosomal inactivating proteins

BACKGROUND: Patients suffering from allergic rhinoconjunctivitis and dyspnoea during summer may exhibit these symptoms after contact with flowers or dietary products of the elderberry tree Sambucus nigra. OBJECTIVE: Patients with a history of summer hayfever were tested in a routine setting for sensitization to elderberry. Nine patients having allergic symptoms due to elderberry and specific sensitization were investigated in detail. We studied the responsible allergens in extracts from elderberry pollen, flowers and berries, and investigated cross-reactivity with allergens from birch, grass and mugwort. METHODS: Sera from patients were tested for IgE reactivity to elderberry proteins by one-dimensional (1D) and 2D electrophoresis/immunoblotting. Inhibition studies with defined allergens and elderberry-specific antibodies were used to evaluate cross-reactivity. The main elderberry allergen was purified by gel filtration and reversed-phase HPLC, and subjected to mass spectrometry. The in-gel-digested allergen was analysed by the MS/MS sequence analysis and peptide mapping. The N-terminal sequence of the predominant allergen was analysed. RESULTS: 0.6% of 3668 randomly tested patients showed positive skin prick test and/or RAST to elderberry. IgE in patients' sera detected a predominant allergen of 33.2 kDa in extracts from elderberry pollen, flowers and berries, with an isoelectric point at pH 7.0. Pre-incubation of sera with extracts from birch, mugwort or grass pollen rendered insignificant or no inhibition of IgE binding to blotted elderberry proteins. Specific mouse antisera reacted exclusively with proteins from elderberry. N-terminal sequence analysis, as well as MS/MS spectrometry of the purified elderberry allergen, indicated homology with ribosomal inactivating proteins (RIPs). CONCLUSION: We present evidence that the elderberry plant S. nigra harbours allergenic potency. Independent methodologies argue for a significant homology of the predominant 33.2 kDa elderberry allergen with homology to RIPs. We conclude that this protein is a candidate for a major elderberry allergen with designation Sam n 1.     

Occupational rhinitis and bronchial asthma due to artichoke (Cynara scolymus).

BACKGROUND: The artichoke is a perennial horticultural plant that belongs to the Compositae family. OBJECTIVE: To present case studies of 2 vegetable warehouse workers who developed occupational rhinitis and bronchial asthma by sensitization to artichoke. METHODS: Skin prick tests with common inhalants and foods were performed. Specific IgE to artichoke, Parietaria judaica pollen, and Olea europaea pollen extracts was measured by a specific IgE enzyme immunosorbent assay kit. Molecular mass of the allergens was studied by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting technique. Patients underwent a nasal challenge test, and one patient provided peak expiratory flow rate (PEFR) measurements in her workplace. RESULTS: In both patients, results of skin prick tests to artichoke were positive. Levels of specific IgE for artichoke were 0.68 kU/L in patient 1 and 2.14 kU/L in patient 2. The protein composition of the artichoke extract, studied by SDS-PAGE, showed that most bands ranged from 30 to 14 kDa. The IgE-binding bands with the serum samples of patient 1 showed apparent molecular masses of 56, 48, 38, 31, 27, 25, 16, and 15 kDa; however, the serum samples of patient 2 showed IgE bands of 21 and 19 kDa. Western blotting of artichoke extract showed a complete inhibition of IgE-binding bands when serum samples were preincubated with P. judaica pollen extract. Nasal challenge with artichoke extract triggered a peak nasal inspiratory flow decrease of 81% and 85% in patient 1 and patient 2, respectively. Finally, patient 1 recorded a PEFR decrease of up to 36% after exposure to artichoke in her workplace. CONCLUSIONS: SDS-PAGE immunoblotting inhibition performed for the artichoke extract showed a total disappearance of the specific IgE binding bands when serum samples were previously incubated with P. judaica pollen extract, thus establishing the existence of a serologic cross-reactivity between artichoke and P. judaica pollen.     

Anaphylaxis to camomile: clinical features and allergen cross-reactivity

BACKGROUND: Medicinal remedies of plant origin became very popular in recent years, and allergic reactions to these are on the rise, accordingly. Camomile has been reported as a potential trigger of severe anaphylaxis. The allergens responsible for camomile allergy have not been characterized as yet. OBJECTIVE: The present study aims at reviewing the clinical symptomatology of immediate-type reactions in a series of patients sensitized to camomile and at characterizing the responsible allergens. METHODS: Fourteen patients with a history of allergy either to camomile or to spices or weeds, and a positive skin prick test/RAST to camomile were investigated for related allergic reactions to food, pollen and others. IgE-binding patterns were determined by immunoblotting, inhibition tests and deglycosylation experiments. RESULTS: Ten of 14 patients had a clinical history of immediate-type reactions to camomile, in some cases life threatening. Eleven subjects were also sensitized to mugwort in prick or RAST, eight to birch tree pollen. Using a polyclonal rabbit anti-Bet v 1 antibody, a homologue of the major birch pollen allergen Bet v 1 was detected in two camomile blots. In four cases a group of higher molecular weight allergens (23-50 kDa) showed IgE-binding to camomile. All allergens proved heat stable. Binding was inhibited in variable degrees by extracts from celery roots, anize seeds and pollen from mugwort, birch and timothy grass. Deglycosylation experiments proved the presence of carbohydrate determinants in camomile which were not responsible for IgE-binding, though. Profilins (Bet v 2) were not detected in our camomile extracts. CONCLUSION: Incidence and risk of type I allergy to camomile may be underestimated. Concurrent sensitization to mugwort and birch pollen is not infrequent. Bet v 1 and noncarbohydrate higher molecular weight proteins were found to be eliciting allergens and are responsible for cross-reactivity with other foods and pollen.