IgE-binding components of baker's yeast (Saccharomyces cerevisiae) recognized by immunoblotting analysis. Simultaneous IgE binding to mannan and 46–48 kD allergens of Saccharomyces cerevisiae and Candida albicans

The Saccharomyces cerevisiae allergens were characterized by IgE-immunoblotting with serum samples of 83 patients; 63 represented patients with atopic dermatitis with previous positive skin prick test or RAST for S. cerevisiae, seven patients with AD but negative test results and 13 were non-atopic controls. Disrupted whole body extract of S. cerevisiae was used in the assays. From the patients tested 41 patients with atopic dermatitis appeared positive in IgE immunoblotting revealing 22 IgE stained bands. From these bands 10 represented intermediate allergens, and 12 minor allergens. The most frequent staining was obtained with the 48 kD band (39%). When the staining pattern of 45 kD and 48 kD bands and mannan was compared with Candida albicans allergens or purified baker's yeast enolase a simultaneous binding was seen with the 48 kD band of S. cerevisiae and the 46 kD band of C. albicans and enolase whereas the 45 kD band was neither associated with the 46 kD band of C. albicans nor purified enolase. High molecular weight staining was found in five samples. The staining pattern was associated with the mannose containing structures in parallel with C. albicans.     

Candida albicans and atopic dermatitis

The role of sensitization and exposure to Candida albicans in atopic dermatitis (AD) was studied with skin-prick tests, yeast cultures and immunoblotting in 156 young adults with AD attending the Department of Dermatology, University of Turku, during 1983–89. Eighteen patients with allergic rhinitis without eczema and 39 non-atopics were included as controls. Parameters associated with severe AD were simultaneous anti-C. albicans IgE and saprophytic C. albicans growth. A statistically significant correlation between C. albicans sensitization (specific IgE antibodies) and AD symptoms was observed only in patients with saprophytic C. albicans exposure. No correlation between C. albicans-specific IgE and AD severity was shown in patients without gastrointestinal growth. Furthermore, severe eczema was seldom seen in patients without saprophytic C. albicans growth. The most important IgE-binding components of C. albicans in immunoblotting were 27 and 46 kD proteins and mannan, a polysaccharide. IgG and IgA antibodies to C. albicans, mainly towards C. albicans mannan, were found in practically all 70 sera studied. These results suggest a continuous exposure and induction of IgE antibodies by C. albicans in AD patients. Severe phases of AD in colonized patients are associated with IgE synthesis against C. albicans. These findings suggest a role for C. albicans in the exacerbations of AD but the clarification of this subject needs double-blind placebo-controlled treatment trials.     

IgE-binding components of wheat, rye, barley and oats recognized by immunoblotting analysis with sera from adult atopic dermatitis patients

Abstract. The allergen extracts of wheat, rye, barley and oats flours were characterized by IgE-immunoblotting with serum samples from 40 adult patients; 35 patients with atopic dermatitis, one with rhinitis and four with urticaria. All these patients had been positive when skin-prick testing was carried out with one or more of the four flour extracts or displayed one or more positive cereal RAST results. Four non-atopic sera were used as negative controls. Acidic and neutral protein extracts of wheat, rye, barley and oats flours were processed for the immunoblotting experiments and 35 patients appeared positive in IgE immunoblotting with wheat and rye. 32 with barley and 33 with oats. The IgE immunoblots showed polyspecific binding patterns; wheat exhibited 36 IgE stained bands, rye 35, barley 33 and oats 10. Eighteen of the IgE stained bands could be classified as intermediate allergens for wheat, 23 for rye and 15 for barley. The 66 kDa protein in oats was visualized by 28 out of 33 sera (84%). however, there was evident non-specific binding to this region and thus it may also represent lectin-like binding. The most frequent staining with wheat extract was seen in the 26 kDa protein region (1 5 35. 43%), with rye in the 40 kDa (16/35,46%) and with barley in the 26 and 46 kDa protein bands (14/32, 44%). Simultaneous staining with wheat, rye and barley extracts were observed with 16 bands suggesting crossreactivity between these cereals.     

Immediate hypersensitivity to bakery, brewery and wine products in yeast-sensitive atopic dermatitis patients

Ultrafiltered (> 1000 Da) samples of beer, aged red wine, young white wine, sparkling wine and extracts of fresh wheat bread and dried rye bread were analysed by skin-prick test (SPT), radioallergosorbent test (RAST) inhibition, sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to find out if they contain Saccharomyces cerevisiae(S. cerevisiae, baker's yeast) allergens. Serum pool consisting of S. cerevisiae positive sera was used in the assays. The results were compared with freeze-dried reference. V. cerevisiae and cereal antigens. The beer, bread, red wine and sparkling wine extracts elicited immediate reactions. However, no evident correlation with suspected symptoms was observed. White wine extract caused reactions in four out of six atopic dermatitis (AD) patients with symptoms, and in five out of seven symptom-free AD patients and in two of the 24 controls. In SDS-PAGE, protein bands were found in wheat and rye bread extracts and beer. In IgE immunoblotting, however, no staining was seen with the S. cerevisiae positive sera suggesting that they were of cereal origin. In white wine and champagne extracts a nonspecific staining was seen in the region 20kDa representing, e.g. lectin-like activity. No bakers yeast antigen could be detected in brewery and bakery products with IgE-immunoblotting even in the excessively concentrated extracts. The IgE mediated allergy to baker's yeast alone should thus not lead to denial of bakery, brewery and wine products.     

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.     

Antigliadin IgE--indicator of wheat allergy in atopic dermatitis

BACKGROUND: Cereal grains are recognized as the cause of adverse reactions in some patients exposed to grain or flour by either inhalation or ingestion. Cereal-related diseases, such as celiac disease and baker's asthma, have been well studied and the causative cereal proteins have been characterized. Although cereals form an essential part of daily nutrition, the allergenic proteins causing symptoms on ingestion in atopic dermatitis (AD) have remained obscure. In this study, we have investigated the allergenic fraction of wheat in AD. METHODS: Skin prick tests (SPT) with a NaCl wheat suspension and the ethanol-soluble wheat gliadin were performed on 18 wheat-challenge-positive or -negative children with AD, six adult AD patients with suspected cereal allergy, and one adult with wheat-dependent exercise-induced urticaria/anaphylaxis. Serum total IgE and specific IgE-antibody levels to wheat and gluten were measured with the radioallergosorbent test (RAST) simultaneously. In addition serum samples of all 25 patients were analyzed by IgE immunoblotting with the ethanol-soluble wheat-protein extract. RESULTS: Thirteen of the AD children were wheat-challenge-positive, 11/12 of them appeared to be positive with gliadin SPT, and all had an elevated gluten RAST value. Those challenge-negative were negative with both gliadin SPT and gluten RAST. Positive wheat SPT and RAST alone were not associated with positive challenges. Four of the adult patients responded to a cereal-free diet, although only two of them appeared to be positive with gliadin SPT and gluten RAST. A broad and intensive staining of gliadin peptides in IgE-immunoblotting studies was seen in challenge-positive children with positive gliadin SPT and/ or gluten RAST. Besides staining of peptides in the main gliadin area of 30-46 kDa, a characteristic finding was the staining of small, <14-kDa proteins with sera of challenge- and gliadin-SPT-positive patients. CONCLUSIONS: We found that wheat-allergic AD patients have IgE antibodies against gliadin that can be detected by both SPT and the sensitive immunoblotting method. This suggests that gliadin peptides are important allergens, and ingestion of wheat causes symptoms of AD. A broad and intensive IgE staining was seen of gliadin peptides against both the previously characterized peptides in the main gliadin area and small, previously uncharacterized peptides of less than 14 kDa. The gliadin SPT and gluten RAST are good screening methods. Further characterization of the IgE-stained gliadin proteins is needed.     

Candida albicans mannan- and protein-induced humoral, cellular and cytokine responses in atopic dermatitis patients

BACKGROUND: The cytokine observed most often in atopic dermatitis (AD) is IL-4, but a role for IL-5 and IFN-gamma in the late and delayed phase reactions has been suggested. In AD with head, neck and shoulder distribution, hypersensitivity to saprophytic yeasts is an important pathogenetic factor. The yeast allergens include both the mannan polysaccharides and the proteins. Mannans are major cross-reacting allergens likely to be involved in the pathogenesis of AD. OBJECTIVE: To characterize the humoral, lymphoproliferative and cytokine (IL-2, 4, 5 and IFN-gamma) responses of peripheral blood mononuclear cells (PBMCs) induced by Candida albicans mannan and protein antigens in AD. METHODS: Fifteen AD patients and seven healthy controls were included. Ficoll-isolated PBMCs were stimulated by PHA and laboratory-generated mannan and protein extracts of C. albicans. Lymphocyte proliferation was measured and cytokine production was studied by ELISA. The antigen-specific IgG and IgE antibodies were analysed by ELISA and nitrocellulose RAST. RESULTS: In AD mannan (P < 0.005) and protein (P < 0.002), specific IgE levels were higher than in healthy controls. Both mannan and protein-specific lymphoproliferations (both: P < 0.02) were higher in AD than in healthy controls. Mannan, but not protein, induced long lasting IL-2 and IL-4 productions from 24 h lasting up to 66-96 h and IL-5 and IFN-gamma productions with elevated levels at 66 and 96 h. The mannan-induced IL-2 (P = 0.015) and IFN-gamma (P < 0.005) were increased in AD as compared with healthy controls. Significant correlations were seen between the protein-induced proliferation responses and both serum total IgE (r = 0.59, P < 0.01) and protein-specific IgE (r = 0.65, P < 0.005). The mannan-induced IL-2 responses correlated with the specific IgE (r = 0.62, P < 0.01) and proliferation (r = 0.51, P < 0.02) and S-IgE level (r = 0.71, P < 0. 002). Mannan-induced IL-4 and IFN-gamma productions also correlated (r = 0.43, P < 0.05). CONCLUSIONS: C. albicans mannan induced elevated IL-2 and IFN-gamma responses in AD patients. The correlations of the cytokine responses with mannan-induced IgE and proliferation responses suggest that C. albicans mannan induced TH1 type cytokine responses are involved in AD.     

Enhanced IgE response to Candida albicans in postoperative invasive candidiasis

Background Invasive candidiasis is a life-threatening complication problem in postoperative and immunocompromized patients, e.g. those treated by intensive care. Candida is frequently cultured from the mucous membranes of hospital patients and fungal cultures offer httle diagnostic help. Other diagnostic methods, such as blood cultures, serology and diagnostic imaging techniques produce results too late and, if positive, low sensitivity. Objective To study the value of Candida-specific antibodies, especially those of IgE class, in diagnosing invasive Candida infection. Methods The immunoglobulins IgE, IgG and IgM responses to antigens of Candida albicans in the sera of 14 patients with culture, biopsy and/or autopsy proven postoperative invasive candidiasis and of 11 colonized and 19 non-colonized operated patients were studied by mannan radioallergosorbent test (RAST), mannan enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results Detection of IgE antibodies to C. albicans polysaccharide (mannan) and protein antigens proved specific and sensitive in diagnostics of invasive candidiasis after major abdominal surgery. IgE rose early in the course of the infection and the method made a clear distinction between invasive infection and mucous colonization. Immunoblotting for protein antibodies was most sensitive while nitrocellulose-RAST for mannan antibodies was most specific. The combined use of immunoblotting and RAST increased the sensitivity and the specificity. Determinations of anti-Candida IgG and IgM antibodies had low sensitivity and specificity. Conclusion Critically ill patients with invasive candidiasis develop IgE antibodies to Candida antigens probably because of disturbed TH₁/TH₂ responses. Determination of specific IgE antibodies can be used as a diagnostic aid in the early stage of invasive Candida infection.     

Cross-reacting IgE and IgG antibodies to Pityrosporum ovale mannan and other yeasts in atopic dermatitis.

Atopic dermatitis (AD) patients often demonstrate positive skin prick test results and serum IgE antibodies to a range of different yeasts. This has been thought to be due to cross-reactivity. In this study, the cross-reactivity of IgE and IgG antibodies between mannan and crude antigens of Pityrosporum ovale, Candida albicans, and Saccharomyces cerevisiae and crude antigens of Cryptococcus albidus and Rhodotorula rubra was examined by RAST and ELISA inhibition with two serum pools of AD patients. We found cross-reacting IgE and IgG antibodies. In the IgE response, the main cross-reacting pattern was the mannan region, although inhibition could be achieved also with crude antigens of C. albicans, S. cerevisiae, and, to some extent, C. albidus. P. ovale was the most potent inhibitor of IgE-binding components, and against it the highest IgE antibody levels were detected in AD serum pools. In contrast, C. albicans was found to be the most important inducer of IgG antibodies, since the IgG level against P. ovale mannan in both AD serum pools was very low. Cross-reacting antibodies were also seen in ELISA inhibition with both crude and mannan antigens, but since the IgG antibody level of P. ovale mannan in AD serum pools was low, further studies are needed to confirm the IgG results.     

IgE, IgA and IgG antibodies and delayed skin response towards Candida albicans antigens in atopies with and without saprophytic growth

Immunoblotting and RAST were used to analyse IgE, IgA and IgG responses to antigens of Candida albicans. These were compared with the delayed skin response and C. albicans carriage in 40 atopic subjects. The majority of the atopic patients showed a strong IgG and IgA antibody response towards mannan, a carbohydrate, but only occasionally to proteins. Altogether 22 of the 40 patients showed specific IgE towards C. albicans by immunoblotting. The IgE response was mainly towards proteins, particularly to ones with molecular weights of 29 kD and 46 kD, and only in eight out of 22 IgE-positive subjects towards mannan. The IgG and IgA responses to mannan and the total IgE response towards C. albicans assessed by RAST showed an association with C. albicans carriage, whereas the delayed skin response showed an inverse relationship. The immunological parameters characteristic of C. albicans carriage were found to be C. albicans-specific depressed delayed skin response and elevated IgE, IgA and IgG responses. This situation in the atopics presenting such parameters may favour simultaneous sensitization and exposure by colonization. The degree of sensitization may be sufficiently high to produce symptomatic allergy, such as asthma, in some individuals during occasional overgrowth of C. albicans, e.g. due to antibiotic therapy.     

Predominance of the major allergen (Alt a I) in Alternaria sensitized patients

Thirty-nine patients sensitized to Alternaria were evaluated using titrated skin-prick test (SPT), histamine release studies (HR), inhibition of RAST and immunoblotting studies. To determine the relevance of the major allergen, Alt a I, specific rabbit antibodies against Alt a I and Alt a B were used. The antibodies were preincubated at different concentrations: (i) with the Alternaria allergen dose required for maximum response in the HR assay (10 BU/ml) and (ii) with the Alternaria antigen coupled to RAST paper discs (1000 BU/disc). Dose dependent inhibition of histamine release (n= 30, x?= 80%± 4%, IC30 = 0.69 μg/ml) and of RAST (n= 7, IC30 = 4.4 μg/ml) was found in all patients sensitized to Alternaria as indicated by allergen induced HR. The greater the response to Alternaria in HR, the higher the antibody concentrations necessary for inhibition (P<0.05). Immunoblot experiments (n=25) using SDS-PAGE showed in all cases IgE- and IgG binding at approximately 28 kD, which is the size reported for the major allergen. All a I. In two cases, slight IgE binding at 45 and 66 kD was also found, while in two other patients, only IgE binding at 66 kD was seen. Our findings emphasize the major importance of Alt a I in patients sensitized to Alternaria.     

Crossreacting IgE antibodies to Pityrosporum ovale and Candida albicans in atopic children

Sera from 13 patients with positive Pityrosporum ovale skin prick test were analysed with IgE immunoblotting. Both P. ovale and Candida albicans antigens were used to reveal possible crossreactivity of these yeasts. Of the 13 sera, eight sera showed IgE binding to P. ovale and six sera to C. albicans. Simultaneous IgE-binding to both C. albicans and P. ovale was observed in five of these sera. The most common IgE-binding band pairs were, a 23 kD band of P. ovale and a 27 kD band of C. albicans, as one pair, and a 26 kD band and a 13 kD band, respectively, as another pair. In addition to these protein bands, simultaneous reactivity was observed to C. albicans mannan, a polysaccharide, and to a diffuse high molecular weight component of P. ovale, possibly also a polysaccharide. The five sera with simultaneous IgE-binding were analysed with RAST-inhibition, which revealed presence of crossreacting IgE-antibodies to P. ovale and C. albicans in two of these sera. Crossreacting epitopes were suggested to be located in the mannan polysaccharide of C. albicans and high molecular weight diffuse stain of P. ovale, based on the immunoblotting and RAST-inhibition patterns of the studied sera. Crossreacting sera were pooled and used as an IgE probe in CRIE and Tandem-CRIE. These experiments revealed a fused precipitin line indicating presence of a common structure of P. ovale and C. albicans. This suggested crossreactivity may imply an interesting phenomenon in atopics who are sensitized by C. albicans growth in the gastrointestinal tract and get exposed by P. ovale growing on the skin.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgE crossreactivity between mugwort pollen (Artemisia vulgaris) and hazelnut (Abellana nux) in sera from patients with sensitivity to both extracts

Background An association between sensitization to Compositae pollens and hypersensitivity to hazelnut has been previously described. There is no previous in vitro study about crossreactivity between mugwort pollen and hazelnut. Objectives To study mugwort pollen and hazelnut allergens and to assess if there is IgE crossreactivity between mugwort pollen and hazelnut. Methods A serum pool formed by 28 individual sera with specific IgE to mugwort pollen and hazelnut was used to investigate IgE crossreactivity. RAST-inhibition, SDS-PAGE/IEF immunoblotting inhibition assays were performed by preineubation of the sera with mugwort pollen and hazelnut. Results RAST to hazelnut was inhibited up to 63% by mugwort pollen, but the mugwort pollen RAST was only inhibited up to 36% by hazelnut. In SDS-PAGE immunoblotting mugwort pollen showed nine allergens ranging from < 16 to 65kDa and hazelnut had four main allergens: 42kDa, 17kDa and < 16kDa (two bands). In the SDS-PAGE immunoblotting inhibition hazelnut partially inhibited all the mugwort pollen bands, except that with 19kDa, whereas mugwort pollen produced a nearly total inhibition of all the hazelnut allergens. In isoeleetrofocusing immunoblotting mugwort pollen had two groups of allergens: pI 7.5–8.5 and pi 3.5–5.2 and hazelnut one group of allergens: pI 5.2–5.8. In the isoeleetrofocusing immunoblotting inhibition hazelnut produced a partial inhibition of all the bands of mugwort pollen and mugwort pollen partially inhibited all the allergenic bands of hazelnut. Conclusions The RAST and SDS-PAGE/IEF immunoblotting inhibition results provide evidence of IgE cross reactivity between mugwort pollen and hazelnut allergens. The inhibition of hazelnut by mugwort pollen is higher than the inhibition of mugwort pollen by hazelnut in both RAST inhibition and SDS-PAGE immunoblotting inhibition. These results suggest that mugwort pollen allergens would behave as primary immunogens in the association between sensitivity to mugwort pollen and hazelnut.     

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.     

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.     

Detection of specific IgE antibodies towards cat flea (Ctenocephalides felis felis) in patients with suspected cat allergy

Cat flea sensitivity is considered one of the most important skin diseases in cats and dogs. Cat fleas, however, are also a growing allergen problem for humans. Cat flea-specific IgE antibodies were studied in serum samples from 70 patients with suspected cat allergy, using RAST-based techniques and the nitrocellulose immunoblotting method. Results from RAST studies, using cat and cat flea as allergosorbents, showed that 46% of the patients were RAST positive against both cat and cat flea. 9% of the patients were RAST positive only against the cat flea. The nitrocellulose immunoblotting experiments were in agreement with the RAST results showing specific IgE to cat flea. The results indicate that some cat-allergic patients have specific IgE both towards cat and cat flea but also that some of the patients with suspected cat allergy might have specific IgE towards the cat flea and not the cat. RAST-inhibition and immunoblotting experiments also indicate that the allergen composition of cat flea extract differs from that of cat extract, even if common allergens have been detected, leading to cross-reactivity in some sera.     

Codfish allergy in adults. Specific tests for IgE and histamine release vs double-blind, placebo-controlled challenges

Background At present, several in vitro tests for immunoglobulin E (lgE)-mediated food allergy are available. An estimation of the diagnostic accuracy of the various tests used in predicting clinical sensitivity to codfish in a well-characterized allergic material is necessary. Objectives To compare the diagnostic value of four specific IgE tests, and histamine release from basophils (HR) in identifying clinical type I allergy to codfish. As a true diagnosis, double-blind, placebo-controlled food challenges (DBPCFC) were employed. Methods Eight clinically codfish-allergic adult patients were investigated together with 30 codfish-tolerant control subjects for evidence of codfish-specific reactivity by Phadebas RAST® (PHA). Pharmacia CAP System RAST® (CAP), Magic® Lite (ML) and HR. To characterize the diagnostic properties of a freshly prepared raw codfish extract, experiments were conducted employing an in-house radioallergosorbent test (RAST). the Maxisorp RAST (MAXI) and HR. Finally, protein profile and IgE-reacting allergens were detected by means of sodium dodecyl sulfate-polyacrylamide gel elcctrophoresis (SDS-PAGE) and immunoblotting. Results The sensitivities of HR with commercial extract and the three commercially available specific IgE analyses were 0.83 and 1.00 respectively. Specificities were 1.00 (H R) and 0.87-1.00 (specific IgE tests). Ereshly prepared codfish extracts improved the sensitivity of HR. SDS-PAGE revealed ～29 bands (< 14.3-200 kDa) including a band of 12-13 kDa. and in immunoblotting 18 sera identified 17 IgE-binding bands. The protein migrating at 12-13 kDa was identified in the fresh codfish extract tested with gen from all clinical codfish allergies, while no significant reaction was seen in the control subjects. Conclusion Based on the small number of adult patients included in our study, the in vitro assays with commercial and fresh extracts have high sensitivity and are acceptable for screening for codfish allergy. Specificity of Phadebas. CAP. and our in-house RAST was less than unity, whereas ML and strong binding of IgE to a 12-13kDa protein completely matches DBPCFC results, and thus seems sufficient for establishing the diagnosis.     

Rice pollen allergy in Taiwan

A panel of tests including intracutaneous skin testing (ST), radioallergosorbent test (RAST), immunoblotting and allergen-induced lymphoproliferation was done to study rice pollen allergy in asthmatic children and to characterize the allergens. Of the 312 asthmatic patients skin tested, 29 (9.3%) had positive reactions (wheal greater than or equal to 6 mm) to rice pollen extract at a concentration of 10(-5) g/mL and the remaining 283 (90.7%) were negative. While eight (34.8%) of the 23 ST-positive patients were also RAST-positive, RAST was negative in all 34 ST-negative patients and 20 normals. Immunoblotting revealed three major allergens, with molecular weights of 16 kD, 26 kD, and 32 kD, respectively. Interestingly, RAST-positive patients showed IgE responses to most allergens but only a few of them had IgG antibodies, while normal controls had stronger IgG responses to the same allergens, particularly to 32 kD, but none had IgE antibody. The preliminary results of rice pollen protein induced-lymphoproliferation were not informative; thus, rice pollen proteins do elicit a specific response in asthmatic children and normals, but its pathogenic role in bronchial asthma needs further study.     

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.