Identification of allergens involved in occupational asthma due to carmine dye.

BACKGROUND: Carmine has been implicated as an etiologic agent of occupational asthma, but the allergens involved have not been yet identified. OBJECTIVE: To identify the allergens involved in occupational asthma due to carmine dye. METHODS: An in vitro study based in SDS-PAGE and IgE immunoblotting with carmine and cochineal extracts was performed. Sera from three carmine dye workers diagnosed with occupational asthma induced by carmine dye and from seven nonatopic subjects were used. RESULTS: Three proteins of around 30, 28, and 17 kD in raw cochineal extract and another protein of 50 kD in the boiled one were demonstrated by SDS-PAGE. Two proteins of around 50 and 28 kD were observed in the carmine extract by the same technique. Specific IgE binding bands at 17 kD in cochineal raw extract, at 50 kD in the boiled one, and at 28 kD in carmine extract were demonstrated by IgE immunoblotting. CONCLUSIONS: We have identified three allergens of around 17, 28, and 50 kD implicated in occupational asthma of three carmine workers.     

Occupational asthma and food allergy due to carmine

Carmine (E120), a natural red dye extracted from the dried females of the insect Dactylopius coccus var. Costa (eochineal), has been reported to cause hypersensitivity reactions. We report a case of occupational asthma and food allergy due to carmine in a worker not engaged in dye manufacturing. A 35-year-old nonatopic man, who had worked for 4 years in a spice warehouse, reported asthma and rhinoeonjunctivitis for 5 months, related to carmine handling in his work. Two weeks before the visit, he reported one similar episode after the ingestion of a red-colored sweet containing carmine. Peak flow showed drops higher than 25% related to carmine exposure. Prick tests with the cochineal insect and carmine were positive, but negative to common aeroallergens, several mites, foods, and spices. The methacholine test was positive. Specific bronchial challenge test with a cochineal extract was positive with a dual pattern (20% and 24% fall in FEV_i). Double-blind oral challenge with E120 was positive. The patient's sera contained specific IgE for various high-molecular-weight proteins from the cochineal extract, as shown by immunoblotting. Carmine proteins can induce IgE-mediated food allergy and occupational asthma in workers using products where its presence could be easily overlooked, as well as in dye manufacture workers.     

Occupational asthma to carmine in a butcher

Hypersensitivity to carmine (E120) has been identified as a cause of food intolerance and occupational asthma. We present a case of occupational asthma following exposure to carmine in a manufacturer of sausages and review the literature. CASE REPORT: A 42-year-old non-atopic male presented with a 5-year history of rhinoconjunctivitis and asthma on occupational exposure to food additive dusts. Symptoms increased after work. The patient had been exposed for more than 20 years. METHODS: Skin prick tests were performed with a battery of common inhalant allergens and spices. Cochineal, carmine lake and additive mixes used by the patient were extracted and subsequently used for skin prick test, bronchial provocation and in vitro measurements (specific IgE, Western blot and chromatographic fractionation). RESULTS: Prick tests were positive to carmine and carmine-containing additives; carmine-specific IgE and bronchial challenge tests were also positive (PC20 = 0.0004 mg/ml and 1.6 kU/l). Western blot showed IgE binding to bands of about 30 kDa on cochineal extract and a diffuse pattern at 40-97 kDa on carmine. This result was confirmed by gel filtration chromatography and dot blot. Carmine completely inhibited IgE binding to cochineal extract. DISCUSSION: Carmine is a potential sensitizer in an occupational setting: 18 cases of occupational asthma have been described to date. Carmine allergens are poorly defined; in general, proteins from cochineal not removed by the extraction process are considered as the main allergens in carmine. Our results are consistent with this, but show that these proteins may be subject to chemical modification.     

Systemic allergic reaction to coconut (Cocos nucifera) in 2 subjects with hypersensitivity to tree nut and demonstration of cross-reactivity to legumin-like seed storage proteins: New coconut and walnut food allergens

BACKGROUND: Two patients with tree nut allergy manifested by life-threatening systemic reactions reported the subsequent onset of systemic reactions after the consumption of coconut. OBJECTIVE: Herein, the IgE-binding proteins from coconut are described, and in vitro cross-reactivity with other nuts is investigated. METHODS: The IgE-binding profile of coconut endosperm tissue extract was analyzed by SDS-PAGE followed by immunoblotting. Immunoblot inhibition studies with walnut, almond, peanut, and coconut were performed. RESULTS: Sera IgE from both patients recognized reduced coconut allergens with molecular weights of 35 and 36.5 kd. IgE from 1 patient also bound a 55-kd antigen. Preabsorption of sera with nut extracts suppressed IgE binding to coconut proteins. Preabsorption of sera with coconut caused the disappearance of IgE binding to protein bands at 35 and 36 kd on a reduced immunoblot of walnut protein extract in 1 patient and suppression of IgE binding to a protein at 36 kd in the other patient. CONCLUSION: The reduced coconut protein at 35 kd was previously shown to be immunologically similar to soy glycinin (legumin group of seed storage proteins). The clinical reactivity in these 2 patients is likely due to cross-reacting IgE antibodies primarily directed against walnut, the original clinical allergy reported, and most likely to a walnut legumin-like protein. Coconut allergy in patients with tree nut allergy is rare; these are the first 2 patients ever reported, and therefore there is no general indication to advise patients with tree nut allergy to avoid coconut.     

O-glycans as a source of cross-reactivity in determinations of human serum antibodies to Anisakis simplex antigens

BACKGROUND: Anisakis simplex is a seafood-borne parasite that may both infect humans and cause allergy. Serodiagnosis of anisakiasis and allergy caused by this nematode is difficult since most Anisakis antigens show cross-reactivity problems. OBJECTIVE: To analyse the possible role of sugar epitopes contained in Anisakis simplex antigens as causes of false-positive results in serodiagnostic assays. METHODS: The antigens UA2R and UA3R recognized by two anti-Anisakis monoclonal antibodies were used in this study. Capture ELISA techniques were used to compare the reactivities with native or O-deglycosylated antigens of sera from Anisakis-free children (most of them infected by several other parasites) and from Anisakis allergy patients. O-deglycosylation was done by mild alkali treatment with NaOH. SDS-PAGE and immunoblotting were used to characterize the effects of NaOH or N-glycanase F treatment on UA3R. RESULTS: Native UA2R was recognized by IgG1 and IgM antibodies in the sera of both Anisakis-free subjects and allergy patients. Native UA3R was recognized by most sera from allergy patients (92% considering immunoglobulin (Ig) G1, 100% considering IgE), but also by a significant proportion of sera from Anisakis-free subjects (36% considering IgG1, 14% considering IgE). O-deglycosylation of UA3R greatly improved specificity: none of the sera from Anisakis-free patients showed either IgG1 or IgE reactivity with O-deglycosylated UA3R, while the proportion of sera from allergy patients showing IgE reactivity with this antigen was practically unaffected. O-deglycosylation of UA2R did not improve the specificity of assays using this antigen. Our results also show that the protein core of glycoproteins may be altered by even very mild alkali treatment, depending on the nature of the protein. CONCLUSION: Native glycoproteins of A. simplex should not be used for diagnostic purposes. O-deglycosylated UA3R seems to be an excellent candidate for use as target antigen in the serodiagnosis of anisakiasis and A. simplex allergy.     

Comparison of ammoniated and nonammoniated extracts in children with latex allergy

BACKGROUND: The use of ammoniated or nonammoniated latex extracts for the diagnosis of latex allergy is still a matter of debate. The aim of our study was to compare the characteristics of the two types of extracts by immunoblotting and RAST techniques in children with ascertained latex allergy. METHODS: Ammoniated (AL) and nonammoniated latex (NAL) extracts were prepared and blotted on SDS-PAGE to resolve their components. Also a solid phase for RAST assays was prepared with the two extracts. The sera from 18 children (mean age 11.4 years, range 6-15 years), with ascertained latex allergy (clinical history, skin test, CAP-RAST and provocation) were used for the experiments. RESULTS: The NAL extract is resolved in many bands (5-100 kDa), whereas AL showed only few components, likely Hev b 4, 6 and 7. IgE reactivity against AL was observed only in 5/18 patients, whereas 12/18 were positive with NAL. The blotting profile against NAL was complex and the IgE recognition pattern involved different bands. CONCLUSION: The extract obtained from NAL is able to detect specific IgE against a greater number of allergenic determinants, and therefore a greater diagnostic accuracy can be expected.     

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.     

Anaphylactic reactions to ingested carmine (E120)

We report five cases of anaphylactic reaction to carmine (cochineal. E120) after patients drank an alcoholic beverage. By means of positive skin prick tests (SPT) and positive RAST to carmine, IgE-mediated sensitization could be established. One nonalopic patient showed also a great amount of serum IgE antibodies to the carmine acid-albumin conjugate. Due to its widespread use in the food and cosmetic industry, carmine should be tested in the allergy work-up in case of allergic reactions after a drink or a meal.     

Oral allergy syndrome to chicory associated with birch pollen allergy

BACKGROUND: A few cases of IgE-mediated chicory allergy with oral, cutaneous, and/or respiratory symptoms are reported. We present 4 patients with inhalant birch pollen allergy and oral allergy syndrome to chicory. IgE-binding proteins in chicory and cross-reactivity with birch pollen were studied. METHODS: Chicory extract was prepared and immunoblotting was used to study IgE reactivity and cross-reactions with birch pollen. RESULTS: The pattern of IgE binding to chicory was variable among the patients, with protein bands recognized at 18, 21, 40, 52 and 71 kD. Bet v 1-like proteins were detected in chicory by monoclonal antibody binding. Chicory-birch pollen cross-reactivity, as studied in 2 patients from whom enough serum was available, could be demonstrated but did not involve the Bet v 1 protein family. In one of these cases, a 51-kD protein of birch pollen was found to be responsible for cross-reactivity. CONCLUSIONS: Chicory should be added to the list of foods that can cross-react with birch pollen and cause the birch pollen-associated oral allergy syndrome.     

Identification of Epicoccum purpurascens allergens by two-dimensional immunoblotting and mass spectrometry

Sensitization to allergens of Epicoccum purpurascens is reported from many places world over. Previously, the IgE binding proteins of Epicoccum were studied by one-dimensional immunoblotting (1-DE). However, the allergens recognized by 1-DE may be a mixture of proteins appearing at the same molecular weight. In the present study, IgE-binding components of E. purpurascens were detected using two-dimensional immunoblotting and mass spectrometry. Approximately 250 protein spots were identified by Coomassie staining in the range of 14-110kDa and pI 3.5-9.5. Of these, 147 showed specific IgE reactivity with pooled patients' sera whereas 16 protein components reacted with more than 50% of the individual patients' sera. The frequency of IgE reactivity was highest (87.5%) for a 25-kDa protein (pI-7.85). Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of 16 protein components led to identification of 6 important allergens as phospho-glycerate kinase (PGK), RNA-dependent RNA polymerase, pyrroline-5-carboxylate dehydrogenase, 40S ribosomal protein and two proteins of unknown functions. The major allergens identified may be assessed for cross reactivity with other fungi. In conclusion, 2-DE followed by immunoblotting allowed characterization of E purpurascens allergens. Six new allergens have been identified using this technique and mass spectrometry. The availability of such relevant allergens would help in component-based diagnosis and therapy of fungal allergy.     

Multiplicity of mitochondrial inner membrane antigens from beef heart reacting with antimitochondrial antibodies in sera of patients with primary biliary cirrhosis

Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular weights of 70 kDa, 54 kDa, 51 kDa and 45 kDa, as defined by their RF in SDS-PAGE gel. There was no correlation between the number of specificities and the titers of AMA as determined by immunofluorescence analysis. The 70-kDa protein was dissociated into a 36-kDa protein by trypsin digestion which still reacted with AMA. The reactivity to AMA of the 54-, 51- and 45-kDa proteins was abolished by trypsin digestion.     

Identification and characterization of the allergens in the tomato fruit by immunoblotting

BACKGROUND: Although the tomato fruit (Lycopersicon esculentum) has been widely investigated for breeding purposes, there have been few studies on tomato allergenicity. We attempted to identify the tomato fruit allergens and to compare the concentrations of IgE-binding proteins among the different growth stages with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. METHODS: An immunoblot experiment on tomato fruit extracts was performed using sera from 11 patients with oral allergy syndrome (OAS) to tomatoes. Bands reacting with IgE from more than half of the OAS patients' sera were excised and subjected to determination of N-terminal amino acid sequences using the automated Edman degradation method. Moreover, we compared the concentrations of these proteins at each growth stage of the tomato fruit with SDS-PAGE and immunoblotting. RESULTS: Four proteins binding with IgE from more than half of the OAS patients' sera were determined to be polygalacturonase 2A (PG2A), beta-fructofuranosidase, superoxide dismutase (SOD) and pectinesterase (PE). The concentrations of PG2A, beta-fructofuranosidase and PE were highest in the red ripening stage with both SDS-PAGE and immunoblotting. CONCLUSION: The concentrations of 3 of 4 tomato allergens increased during ripening.     

Identification of casein as the major allergenic and antigenic protein of cow's milk

The objective of this study was to analyze both the allergenicity and immunogenicity of cow's milk proteins. To this end, 80 milk-atopic patients were selected on the basis of the presence of cow's milk-specific IgE antibodies in serum and compatible clinical history. Fifteen patients allergic to other allergens and 10 nonatopic subjects were studied as controls. The specificity of serum IgG and IgE antibodies was determined by immunoblotting, employing both cow's milk and milk components, i.e., α- and β-casein, β-lactoglobulin, and α-lactalbumin separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The experiments showed that casein-specific IgE antibodies were present in all (80/80) sera examined; 10/80 showed reactivity to α-lactoglobulin, and 5/80 showed reactivity to α-lactalbumin. None of the 25 negative control sera analyzed showed the presence of specific IgE antibodies against milk proteins. These results were similar to those corresponding to the detection, by the radioallergosorbent test, of IgE antibodies against the milk components coupled to paper disks. All sera from milk-atopic patients also showed IgE reactivity against a high-molecular-mass fraction that hardly enters the gel. This fraction, after separation by gel filtration and treatment with β-mercaptoethanol and urea, was shown by SDS-PAGE analysis to be formed by casein monomers. All sera analyzed by immunoblotting reacted against the components corresponding to casein monomers. Inhibition of immunoblotting by adsorption with different milk components confirmed that those high-molecular-mass aggregates are formed by casein components. The results presented here strongly suggest that casein is the major allergenic component of cow's milk.     

Antigenicity of the proteins in soy lecithin and soy oil in soybean allergy

BACKGROUND: Soy lecithin and soy oil are usually produced from the hexane extract of soybean. Some of the soybean proteins are included in the extract and are therefore present in small amounts in both soy lecithin and soy oil. The antigenicity of the proteins present in defatted soybean has been studied with respect to soybean allergy, but the antigenicity of those found in the extract is yet to be investigated. OBJECTIVE: The antigenicity of soy lecithin and soy oil proteins with regard to soybean allergy were investigated. METHODS: The proteins present in soy lecithin and soy oil were determined according to already established method and analysed by SDS-PAGE. The IgE- and IgG4-binding abilities of the soy lecithin proteins were investigated by immunoblotting with sera from 30 soybean-sensitive patients, including seven with a positive challenge test. Immunoblotting of soy oil proteins was performed with the sera from some of these patients. RESULTS: In 100 g of sample, the soy lecithin and soy oil contained 2.8 mg and 1.4-4.0 microg of proteins, respectively. The results of SDS-PAGE demonstrated the presence of only three proteins, with molecular weights of about 58-67 kDa in soy oil, and suggested that soy lecithin also contains these proteins. The soy lecithin also contained many proteins besides these. In the soy lecithin, the detection rate of only one protein, with a molecular weight of 31 kDa, by the serum IgE of patients was significantly different compared with controls (detection rate: 40%). The proteins with molecular weights of 58-67 kDa rarely bound to serum IgE. Only one of the patients who presented a positive challenge test had IgE antibodies to soy lecithin proteins. IgG4-binding proteins were found only rarely in soy lecithin. Neither the IgE nor the IgG4 present in the patients' sera reacted to any soy oil protein. CONCLUSION: Proteins present in soy lecithin and soy oil have little antigenicity with regard to soybean allergy.     

Antigen characterization of major cork moulds in Suberosis (cork worker's pneumonitis) by immunoblotting

BACKGROUND: We characterized by immunoblotting the antigenicity of the most frequent fungi colonizing cork during its industrial processing, Penicillium glabrum and Chrysonilia sitophila. Penicillium glabrum is the main causative agent of Suberosis, a hypersensitivity pneumonitis of cork workers. Chrysonilia sitophila induces both IgE sensitization and occupational asthma in the wood processing industry. METHODS: Serum-specific IgG, IgG4 and IgE to P. glabrum and C. sitophila from nine cork workers with hypersensitivity pneumonitis (HP) and seven with asthma (four with occupational asthma) were analysed by immunoblotting. RESULTS: Both HP and asthmatic patients' sera showed immunoreactivity to several proteins resolved in the specific immunoblot strips. The frequency of specific IgG recognition to 12-13.5 and 33 kDa proteins of P. glabrum was significantly higher in HP patients. The sera of HP patients had significantly higher specific IgG recognition to 16 and 51-55 kDa proteins of C. sitophila. There was no specific IgE recognition in the sera of HP or asthmatic patients to both fungi. CONCLUSIONS: Different patterns of antibody reactivity to P. glabrum and C. sitophila are seen in cork workers with hypersensitivity pneumonitis or asthma. The 12-13.5 and 33 kDa proteins of P. glabrum and the 16 and 51-55 kDa proteins of C. sitophila may be major antigens in Suberosis.     

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.     

Clinical role of a lipid transfer protein that acts as a new apple-specific allergen.

BACKGROUND: Allergy to apple is commonly associated with birch pollinosis because the two share homologous allergens. However, some patients have apple allergy but no birch pollinosis, suggesting that there are allergens that do not cross-react with birch. OBJECTIVE: The aim of the study was to evaluate the IgE reactivity pattern to an apple extract in subjects with allergic reactions to apple, with and without birch hay fever. METHODS: Forty-three patients with oral allergy syndrome for apple and positive open food challenge, skin prick test, and serum specific IgE antibodies to apple were admitted to the study. Thirty-two had birch pollinosis (documented by specific IgE for birch) and 11 were not allergic to birch. The IgE reactivity pattern to apple extract was identified by SDS-PAGE and immunoblotting. The consistent allergen, a 9-kd protein, was then purified by HPLC and characterized by periodic acid-Schiff staining, isoelectric point, and N-terminal amino acid sequencing. RESULTS: The sera from 28% of patients allergic to apple with birch pollinosis, but from all patients allergic only to apple, recognized the 9-kd protein. This protein has an isoelectric point of 7.5 and is not glycosylated. Determination of its partial amino acid sequence showed that it belongs to the family of lipid transfer proteins, which act as major allergens in Prunoideae fruits. CONCLUSIONS: These results indicate that a lipid transfer protein is an important allergen in patients allergic to apple but not to birch pollen. The prevalent IgE reactivity to this allergen in subjects with no birch pollinosis and the physicochemical characteristics of this protein suggest that sensitization may occur through the oral route.     

IgE binding to soluble and insoluble wheat flour proteins in atopic and non-atopic patients suffering from gastrointestinal symptoms after wheat ingestion

BACKGROUND: The involvement of IgE-mediated hypersensitivity reactions in the genesis of gastrointestinal symptoms after ingestion of foods containing wheat has been rarely reported. OBJECTIVE: To detect IgE specifically binding to wheat proteins in the sera of atopic and non-atopic patients suffering from gastrointestinal symptoms after ingestion of wheat and to evaluate the reliability of skin prick test and CAP in the diagnosis of food allergy to wheat. METHODS: The sera of patients (10 atopic and 10 non-atopic) previously diagnosed as suffering from irritable bowel syndrome and complaining of symptoms after wheat ingestion were analysed by immunoblotting for IgE binding to water/salt-soluble and insoluble wheat flour proteins. RESULTS: All the atopic patients and only one of the non-atopic patients were positive to wheat CAP. For the patients tested, skin prick test was positive for all the atopic patients and for only one of the non-atopic patients. However, immunoblotting experiments showed the presence of specific IgE to wheat proteins in all the patients. Ten out of 11 of the wheat CAP-positive patients had IgE binding to a soluble 16-kDa band, but the same band was recognized, in a slighter way, by only two out of nine of the wheat CAP-negative patients. Moreover, although almost all of the patients were negative in CAP testing with gluten, 19 out of 20 recognized protein bands belonging to the prolamin fraction. CONCLUSIONS: For the atopic patients the positivity to skin prick test and CAP to wheat was in accordance with the immunoblotting results and a food allergy to wheat could be diagnosed. In these patients a major allergen was a 16-kDa band corresponding to members of the cereal alpha-amylase/trypsin inhibitors protein family, the major allergens involved in baker's asthma. In the non-atopic patients the positive immunoblotting results contrasted with the responses of the allergologic tests, indicating that the allergenic wheat protein preparations currently used are of limited value in detecting specific IgE to wheat and that the fraction of irritable bowel syndrome (IBS) patients with food allergy may be larger than believed.     

Esterase from ammoniated latex: Biochemical characterization and antigenicity

Latex allergy is a recently increasing hazard to people who are repeatedly in contact with Latex products. Notably, with this allergy, cross‐reactivity to vegetable foods and pollen is frequently observed. It is postulated that pathogenesis‐ or, rather, defence‐related proteins induced in rubber trees are responsible for the Latex allergy and the cross‐reactivity. To evaluate this hypothesis, an esterase was selected as one of the probable defence‐related proteins in rubber Latex and its involvement in Latex allergy was investigated. The biochemical properties of a chromatographically purified esterase from ammoniated Latex were compared with those of esterases (hevains) previously purified from rubber Latex. The antigenicity of the esterase was examined by immunoblotting using sera from Latex‐allergic patients. The purified esterase (of molecular weight 80 kDa) dissociated into subunits under denaturing conditions and shared biochemical properties with hevain 1 from lutoids. The esterase was recognized by IgE in patients’ sera. This suggests the relevance of the purified esterase to Latex allergy.     

Culture filtrate antigens and allergens of<Emphasis Type="Italic"> Epicoccum nigrum</Emphasis> cultivated in modified semi-synthetic medium

Epicoccum nigrum (EN) is an important fungal allergen for nasobronchial allergy. Fungal extracts should contain all the relevant allergen components from spores, mycelium and culture medium for the purpose of allergy diagnosis and therapy. EN extract from spore-mycelial mass has been standardized, but the culture filtrate (CF) allergens of EN have not been studied as EN grows poorly in synthetic medium. The objective of the present study was to obtain a standard CF extract of EN by cultivating the source material in a modified semi-synthetic medium and to compare this with the EN cellular extract. Sabouraud's medium containing yeast extract (50 mg/l) was filtered using 10-kDa cut-off membrane and the lower molecular mass media components were used to cultivate EN. The CF obtained after removing the spore-mycelia was dialyzed to remove media components. The CF extract was characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot. It was compared with EN spore-mycelial extract by enzyme-linked immunosorbent assay (ELISA), ELISA inhibition and by intradermal testing on allergy patients. The CF extract of EN resolved into 30 protein bands on SDS-PAGE. About 27 IgG bands were detected using anti-EN rabbit antibodies and 12 IgE bands by EN-sensitive pooled patients' sera. Periodate modification of CF proteins showed that the carbohydrate moieties are not important for IgE binding. Protein components of 26, 34 and 43 kDa were recognized as the major CF allergens. Three different batches of CF extract required 7.5-9 ng of self protein for 50% inhibition of binding to anti-EN rabbit antibodies in ELISA. Intradermal testing with CF extract showed comparable allergenic potency to standardized EN spore-mycelial extract, although it contained some allergenic proteins in higher amounts as compared to the spore-mycelial extract. In summary, the semi-synthetic medium has been suitably modified for obtaining EN CF antigens. This medium can be an important substitute for producing potent CF allergens of fungi that grow poorly in synthetic medium. The EN CF extract elicited good allergenic reactivity and may be used for allergy diagnosis along with spore-mycelial extract.