Further characterization of IgE-binding antigens in horse dander, with particular emphasis on glycoprotein allergens

IgE-binding components in an extract of horse dander were analyzed, especially with regard to the glycoprotein allergens. After SDS-PAGE under reducing conditions and blotting, several of the glycoprotein IgE-binding components, including two distinct bands of 27 and 31 kDa, were detected. Together with several other bands, they were shown to bind to the lectins Sambucus nigra agglutinin (SNA) and Datura stramonium agglutinin (DSA), indicating terminal sialic acid linked α2→6 to galactose, and galactose linked (β1→4 to N-acetylglucosamine, respectively. Carbohydrate analysis after SDS-PAGE and blotting showed the presence of mannose, galactose, 7V-acetylglucosamine, and N-acetyl neuraminic acid in the 27-kDa band. The 14.4-kDa glycoprotein component lost its IgE-binding activity after periodate oxidation. Minor differences in allergenic activity in the other glycoprotein allergenic components were also detected, indicating that the carbohydrate part of the molecule seems to play a role in the IgE-binding activity. Two-dimensional electrophoresis and subsequent immunoblotting were performed to estimate the approximate number, molecular mass, and pi of IgE-binding components. This resulted in a cluster of such components on the blot membrane.     

Immunotherapy in patients allergic to cat and dog dander

Twenty-four asthmatics allergic to cat and/or dog dander were included in a study to examine the efficacy and safety of immunotherapy (IT) with partially purified, standardized extracts of cat or dog dander. In the first placebo controlled, double-blind part of the study, 10 patients were treated with extracts of both cat and dog, 12 with cat extracts and 2 with dog extracts. Fifteen patients received active IT and 9 placebo injections. Patients treated with both extracts received active extracts only, or placebo only. Bronchial allergen challenge after 5 months demonstrated a significant fall in sensitivity to cat (P = 0.04) in patients treated with cat extracts. No significant changes were found in sensitivity to dog after treatment with dog dander extract or in the placebo groups. During this period, bronchial sensitivity to histamine did not change significantly in any of the groups. To examine the effect of more prolonged IT, 19 patients allergic to cat (17) and/or dog (9) were treated for 12 months. Bronchial sensitivity to cat decreased further (P = 0.003), while no significant change was found in dog extract-treated patients. In cat extract-treated patients a significant decrease in bronchial histamine sensitivity developed (P = 0.02). Systemic side effects were few, but in some cases, local side effects were a dose-limiting factor. This study demonstrated that IT with cat extract may benefit cat-allergic asthmatics, whereas no influence of IT with dog extract was detected in dog-sensitive asthmatics.     

Comparison of IgE-binding antigens in horse dander and a mixture of horse hair and skin scrapings

Extracts of horse dander (HD) and horse hair and skin scrapings (HHSS) have been compared with respect to their content of proteins and carbohydrates. The protein content of HD is more than double that of HHSS, while the carbohydrate content is of the same order. SDS-PAGE and IEE, both combined with immunoblotting, and CIE/CRIE showed the IgE-binding ability of the proteins/glycoproteins present in the two extracts. SDS-PAGE/immunoblotting showed the presence of mainly the same IgE-binding bands in the two extracts. Nine were detected in HD, and seven in HHSS. Four of these were glycoproteins. The most important allergen was a glycoprotein with molecular mass of 27 kDa showing IgE-binding ability to the 16 patient sera available. Another important allergen with molecular mass of 67 kDa is probably horse albumin, IEF/immunoblotting showed the presence of a glycoprotein with IgE-binding ability at pH 3.2 in both extracts. Tandem CRIE showed that both extracts contained the same four allergens as major allergens. In addition, as we observed when analyzing both extracts in CRIE against 16 different patient sera, each extract contained allergens not present in the other extract. We may conclude from this investigation that commercial allergen extracts to be used in diagnosis and immunotherapy should include both horse dander and horse hair and skin scrapings in the starting material for the preparation of extracts. If not, allergens which may be of importance for some patients will be missing.     

Dog and cat allergens: urinary proteins or dander proteins?

Allergens prepared from male dog and cat urines can be used for RAST diagnosis of dog and cat allergy. Only one out of 15 patients positive to epithelia or danders was negative to the urine. The urine antigens appear to be more specific than either epithelia or danders.     

Characterization of IgE-binding epitopes on Candida albicans enolase

Candida albicans enolase is one of the important allergens in Candida allergy. We isolated and purified 46 kDa C. albicans enolase (CAE) from C. albicans and characterized epitopes for IgE antibody by lectin-blotting and enzymatic digestion followed by sodium dodecyl sulfale polyacrylamide gel electrophoresis (SDS-PAGE) and immunobiotting. Lectin blotting and deglycozilation indicated that this protein did not contain polysaccharide side chains. The purified CAE and recombinant fusion protein produced from CAE gene possessed common epitopes for IgE antibody. We estimated IgE binding epitopes on the basis of reported amino acid sequences from the analysis of cDNA encoding CAE. V8 protease digestion of CAE gave six polypeptide fragments (A-F). The N-termini of each fragment were confirmed by amino acid sequence and the C-termini were estimated by molecular weights of each fragment and the specific cutting site of V8 protease. Fragment C (25.0 kDa; F-171-I-399) reacted to 90% IgE antibodies examined, whereas fragments D (21.0 kDa; F-171-1-360), E (16.2kDa: F-171-D-317) and F (13.0kDa; A-47-E-170) showed no IgE binding. Our results suggest that epitopes for IgE antibodies exist near the C-terminal of the protein.     

Haemagglutinating antibodies to cat dander in relation to exposure and respiratory allergy

Cat epithelial extract was coupled with chromic chloride to red cells and used as an antigen in a haemagglutination assay. Agglutinins coexisted with IgE type antibodies in 44% of patients with respiratory allergy and sensitivity to cat dander but occurred in only 1% of asthmatic patients sensitized to other allergens or in controls. The agglutinins were detected in six out of 104 cat fanciers, and three of those with agglutinins had asthma. The agglutinins were of IgG type and were heterogeneous in specificity.     

Clinical efficacy of specific immunotherapy to cat dander: a double-blind placebo-controlled trial

Objectives To assess the efficacy of specific immunotherapy with standardized cat dander extract, using objective endpoints and simulated ‘natural’ exposure to cats. Design Double-blind, randomized, placebo-controlled study carried out at a UK Allergy research clinic. Subjects Twenty-eight patients with moderate to severe allergic rhinoconjunctivitis with asthma due to cat allergy. Subjects were stratified for cat sensitivity, cat ownership and asthma, and the groups were well matched for all relevant parameters. Main outcome measures Symptom scores and peak flow rate during and after exposure to cats in a cat-room. Skin tests and conjunctival provocation thresholds. Results The actively treated group showed a marked reduction in symptoms during the cat exposure (mean score 61.6–17.1; P < 0.001) with no change in the placebo group (64.7 vs 62.1). The active group also showed a reduced peak flow response to cat exposure (mean fall of 85L/min pretreatment, 29L/min after treatment, P < 0.005) as well as reductions in conjunctival provocation sensitivity, skin sensitivity to cat extract and skin sensitivity to house dust mite (D. pteronyssinus). Skin reactivity to histamine and codeine were unaltered. No significant adverse reactions were encountered. Conclusions Specific immunotherapy seems to be an effective treatment for cat allergy. Allergy to cats is common and often poorly controlled on conventional pharmacotherapy. Although cat allergy has not traditionally been considered as a valid indication for immunotherapy in the UK, it should now be considered as a legitimate treatment, especially for those who are unable to avoid exposure.     

Cross-reactivity of IgE-binding components between boiled Atlantic shrimp and German cockroach

IgE-antibody reactivity to boiled Atlantic shrimp ( Pandalus borealis ) and German cockroach ( Blattella germanica ) of sera from 89 patients, sensitive to one or the other, was investigated with an enzymatic immunoassay for specific IgE detection (CAP-FEIA System, Pharmacia, Sweden). IgE serum levels to both antishrimp and anticockroach allergens were found to be positive in 76 of the 89 (85.4%) tested sera. A positive anticockroach IgE was very rare in the absence of detectable antishrimp IgE (five of 89 sera). Linear regression analysis on antishrimp and anti-German cockroach IgE levels-log plot revealed correlation coefficient (r) of 0.73. Inhibition experiments showed that boiled Atlantic shrimp extract inhibited CAP with German cockroach, and vice versa. Immunoblotting showed the strongest IgE binding for both allergenic extracts between 30 and 43 kDa. By blot inhibition, the binding capacity of German cockroach was totally-abolished by Atlantic shrimp extract, while German cockroach extract only partially IgE binding to Atlantic shrimp. Cross-reactivity exists between shrimp, an important food allergen, and German cockroach, which has an increasing role in allergic asthma. It could be important to determine the clinical significance of cross-allergy to both allergens, in which exposures occur in different ways.     

Characterization of allergens from deer: cross-reactivity with allergens from cow dander

Background Animal hair/dander proteins frequently cause Type I hypersensitivities. Species-specific and broadly cross-reacting allergens have been characterized in the past. Methods Sera from eight individuals suffering from symptoms due to exposure to deer and deer-derived products were investigated by immunoblotting. Extracts from deer, dog, cat, horse, rabbit and cow, respectively, were tested for IgE-binding. To reveal cross-reactivities patients' sera were preadsorbed with these extracts prior to testing with deer extract. Results Deer allergens with the molecular mass of 22 and 25 kD (major allergens), as well as 60 kD were identified. The 22 and 25 kD allergens are cross-reactive with the corresponding cow allergens. Conclusions Deer allergy is a rare sensitization mainly affecting persons exposed to deer, who displayed an atopie disposition. From our results it can be assumed that this hypersensitivity is partly associated with allergy to cow dander.     

Distribution of allergen activity and identification of the main IgE-binding glycopeptide of Parietaria judaica pollen

Parietaria judaica pollen extract was separated by preparative isoelectric focusing and showed two allergenic components (A1 and A2) heterogeneously distributed within the pH gradient (pH 3-10). By fused-rocket immunoelectrophoresis, A1 was detected from pH 3 to 10, whereas A2 was only found at pH values below 8. Both components, A1 and A2, showed IgE-binding ability throughout their pH ranges, as determined by crossed-radio immunoelectrophoresis. In addition, all the preparative isoelectric focusing fractions contained the main allergenic polypeptide of P. judaica (Pj10) when analyzed by SDS-PAGE. This polypeptide was shown to be a glycopeptide or to be bound to a glycoprotein with alpha-D-mannopyranoside or alpha-D-glycopyranoside residues. Implication of charged sugar residues in the wide distribution of P. judaica allergens in a pH gradient is discussed.     

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited. OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1. METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies. RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds. CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.     

Standardization of allergenic extracts of Aspergillus fumigatus. Liberation of IgE-binding components during cultivation

During cultivation of Aspergillus fumigatus a rapid liberation of IgE-binding components was found reaching maximum values during the logarithmic phase of growth (phase I). After a fall in IgE-binding titers during phase II, appearance of additional IgE-binding components was noted during the period of lysis of the microorganism (phase III). These latter allergenic components are different from the phase I IgE-binding components, as was shown by crossed-inhibition studies. The number of precipitating antigenic components was not related with the corresponding IgE-binding titers and showed an increase during all phases of growth. The rapid changes in both IgE- and IgG-binding properties and the discrepancies between precipitating properties and IgE binding are discussed in relation to standardization and quality control of aspergillus extracts.     

Allergy to pigeon tick (Argas reflexus): demonstration of specific IgE-binding components

BACKGROUND: The European tick, Argas reflexus, is an urban pest parasitizing urban pigeons and may cause a wide range of allergic reactions. METHODS: Specific IgE to A. reflexus, SDS-PAGE and IgE immunoblotting, performed with tick extract, were carried out in the sera of 6 patients who reported allergic reactions after tick bite. RESULTS: Specific IgE to A. reflexus (RAST class ranging from 1 to 3) were detected in the sera of 6 patients who reported allergic reactions (urticaria and angioedema in 2 and anaphylaxis in the other 4 patients) after tick bite. IgE reactivity to two bands of 22 and 40 kDa were identified in the patient sera. CONCLUSIONS: Allergy to A. reflexus has to be considered in allergic patients living in buildings where pigeons have their nests. The powerful sensitizing property of tick allergen is underlined by the observation that none of our patients was atopic.     

Characterization of recombinant cat albumin

BACKGROUND: Indoor allergens derived from animals and mites often contribute to exacerbation of skin manifestations in atopic dermatitis (AD) patients. OBJECTIVE: To produce and characterize recombinant cat albumin, a cross-reactive animal allergen. METHODS: A complete cDNA coding for cat albumin was obtained by RT-PCR amplification from cat liver RNA. Recombinant cat albumin was expressed in Escherichia coli as hexahistidine-tagged protein, purified by nickel affinity chromatography and studied for IgE reactivity with sera from cat-allergic patients by ELISA and immunoblotting. Furthermore, CD203c expression of basophils from cat-allergic patients upon exposure to recombinant cat albumin was analysed. RESULTS: Recombinant cat albumin, a cross-reactive animal allergen sharing most IgE epitopes with its natural counterpart, was produced in E. coli. It was recognized preferentially by IgE from AD patients and elicited IgE-dependent basophil activation in sensitized patients. CONCLUSIONS: Recombinant cat albumin may be used as a paradigmatic tool to analyse mechanisms of allergen-triggered exacerbation of AD, for diagnostic and, perhaps for therapeutic purposes.     

Extraction of IgE-binding components of Helicobacter pylori by immunoblotting analysis in chronic urticaria patients.

BACKGROUND: An association between Helicobacter pylori and chronic urticaria has been suspected previously. An IgE-mediated pathway might be a possible link between H. pylori infection and chronic urticaria, and therefore we wanted to prepare an optimal H. pylori antigen to detect H. pylori-specific IgE antibodies in chronic urticaria patients. METHODS: H. pylori antigen extracts were prepared in different ways to find the optimal antigen extract to be used in the assays. Immunoblotting was used to detect IgE-binding bands. The results were applied in an H. pylori RAST assay for specific H. pylori IgE antibodies in patient sera. RESULTS: In immunoblotting, the largest number of IgE-stained bands were visualized in the washing fluids and sonicated extracts, while strong heating and denaturing treatments destroyed the epitopes for IgE binding, suggesting that they belonged to the flagellar structures of H. pylori. However, in H. pylori-specific RAST analysis, specific IgE was found only in 1 of 25 H. pylori-infected patients. CONCLUSIONS: Our findings suggest that although IgE-binding epitopes were found in H. pylori, H. pylori-specific IgE antibodies are not common in chronic urticaria, and the clinical significance of the IgE response is unclear.     

Anaphylaxis caused by the new ant, Pachycondyla chinensis: demonstration of specific IgE and IgE-binding components

BACKGROUND: There have been no reports dealing with the pathogenic mechanism and IgE-binding components in patients with anaphylaxis caused by a sting from Pachycondyla chinensis. OBJECTIVES: This study was conducted to observe the clinical features of patients with P chinensis -induced anaphylaxis. The roles of specific (s) IgE and sIgG4 antibodies were evaluated, and IgE-binding components were identified. METHODS: Seven patients with P chinensis -induced anaphylaxis and 15 unexposed control subjects were enrolled. P chinensis ants were collected at the patients' homes, and venom was prepared as P chinensis extract. Five patients complained of bee venom-induced anaphylaxis and had positive sIgE levels to yellow jacket venom, wasp venom, or both as well. Serum sIgE and sIgG4 were detected by means of ELISA. To identify IgE-binding components within P chinensis extracts, 12% SDS-PAGE with immunoblot analysis was applied. RESULTS: All patients had positive skin prick test responses to P chinensis antigen and positive sIgE levels. Five (71%) patients had positive sIgG4 levels. Eight IgE-binding components (58, 46, 3l, 29, 27, 25, 22, and 12 kd) were noted, and the component at 12 kd was the most frequently found allergen (85%). IgE ELISA inhibition tests were performed on 2 groups of sera: one from patients with anaphylaxis induced by both P chinensis and bee venom (group A) and the other from patients with anaphylaxis induced by P chinensis venom alone without bee venom allergy (group B). ELISA inhibition tests with serum from group A showed significant inhibitions with addition of P chinensis extract, partial inhibitions with yellow jacket antigen, and minimal inhibitions with wasp or imported fire ant antigens. However, ELISA inhibition tests with serum from group B showed significant inhibitions with P chinensis antigen but no inhibition with wasp, yellow jacket, or imported fire ant antigens. CONCLUSIONS: IgE-mediated reactions contributed to the development of P chinensis -induced anaphylaxis. Eight IgE-binding components and one major allergen (12 kd) were identified. Further studies will be needed to clarify the role of sIgG4 and to identify allergenic relationships with major bee and wasp allergens.     

The relative risks of sensitivity to grass pollen, house dust mite and cat dander in the development of childhood asthma

The associations between skin sensitivity to various common allergens and the development of childhood asthma were ascertained in a longitudinal study of a birth cohort of New Zealand children up to the age of 13 years. Of 714 children skin-tested, 45.8% were sensitive to at least one of 11 allergens, the most common responses being to rye grass pollen (32.5%), house dust mite (30.1%) and cat dander (13.3%). Allergen-specific relative risk analysis, controlled for the effect of sensitivity to other allergens, demonstrated that sensitivity to house dust mite and to cat dander were highly significant independent risk factors associated with the development of asthma (whether defined as recurrent typical respiratory symptoms, increased airway responsiveness, or the concurrent presence of both), whereas grass sensitivity was not a significant independent risk factor for asthma.     

Skin-prick test and RAST responses to cereals in children with atopic dermatitis. Characterization of IgE-binding components in wheat and oats by an immunoblotting method

Background Hypersensitiviiy to cereals may occur via inhalation or ingestion. Although cereals are essential in the daily nutrition, only little information is available of the allergens causing symptoms in patients with atopic dermatitis (AD). Objective The purpose of the present study was to analyse the IgE immune-response to various cereals and specific cereal fractions of wheat and oats in children with severe AD and correlate the results with challenge studies. Methods Skin-Prick tests (SPT) with a NaCL suspension of wheat. oats, rice, corn. millet and buckwheat and the ethenol soluble sliadin fraction of wheat were performed to 34 wheat/oats challange positive or negative children with AD Simultaneously serum total IgE and specific IgE antibody radioallergosorbent test (RAST), levels to wheat, oats and gluten were determined, In addition serum samples of these 34 AD patients and five age matched controls were analysed with IgE immunoblotting using neutral and acidic protien extracts of wheat and oats. Results From the 34 AD children 33 were SPT positive with wheat and 18 with oats. Positive RAST to wheat and oats could be detected in 32 and 30 samples respectively. From the oral Wheat challange positive children 12/14 appeared positive with gliadin SPT and revealed positive RAST to gluten, but each of the wheat challenge negative were negtive in SPT with gliadin. In immunoblotting using neutral and acidic fractions of cereals the IgE binding with sera of challenge positive children showed the most intensive staining, but no correlation was found between differrent staining patterns and the clinical wheat sensitivity. The 26,38 and 69 KDa bands in wheat and the 46 and 66 KDa in oats could be classified as major IgE binding proteins of these cereals (>50% of the sera were positive). SPT with rice, corn, miller or buckwheat and oats was positive in 16/34 patients. Conclusion Intensive IgE staining to natural acidic soluble proteins in wheat and oats was seen with major IgE binding to 26.38 and 69 KDa protiens in wheat and 46 and 66 KDa in oats, but no specific IgE staining patterns correlating with clinical cereal sensitivity were found. The strong association between the positive SPT with the ethnol soluble gliadin suggest that also gliadin is an important allergen in wheat-allergic children with AD. The allergens in rice, corn, millet and buckwheat should be better studied before they can be recommended as alternatives for cereal allergic children.