RAST Inhibition Research Articles

Immunocheraical investigation of allergens from Rhizopus nigricans

The allergenic proteins of mould, Rhizopus nigricans extract (RNE) were identified and characterized by crossed immunoelectrophoresis (CIE), thin-layer isoelectrofocusing (TLIEF) and RAST inhibition. CIE revealed that the extract contained at least 31 distinct antigens. On TLIEF the extract resolved into 22 bands in pI 3.5-6.8. Two important allergens, Rhiz IIIb and VIb were purified by a combination of ammonium sulfate precipitation, anion exchange chromatography on DEAE-Sephadex column and gel filtration. Twenty and 12 micrograms of Rhiz IIIb and Rhiz VIb were sufficient to give 50% RAST inhibition as against 43 micrograms of crude RNE. Rhiz IIIb and Rhiz VIb were found to be glycoproteins with molecular weights of 12,400 daltons and 14,200 daltons, respectively. Rhiz IIIb was found to be homogeneous on polyacrylamide gel electrophoresis (PAGE) and TLIEF with a pI of 4.8, while Rhiz VIb gave a single band on PAGE and resolved into two Coomassie blue stained bands with pI 3.6 and 3.8. It was possible to separate the components of RNE on fast protein liquid chromatography (FPLC) using an anion exchanger Mono Q column. The identification and characterization of the antigenic and allergenic proteins in the extract will be useful in standardization of RNE and in preparation of an in-house reference standard.

Celery allergy associated with birch and mugwort pollinosis

Skin prick tests (SPT) with various celery, carrot and potato preparations (raw, cooked, cooking water of each vegetable and allergen extracts) as well as specific IgE determinations by RAST to celery mix, celeriac (or root celery), stick celery and heated celery extracts were performed in 70 patients with positive prick or intracutaneous tests to birch and/or mugwort pollens and celery (extract and/or raw). 94% of the patients showed positive prick tests to raw celeriac, 36% to cooked celeriac and 8/13 to cooking water. Celery-birch positive patients (n = 13) showed negative or low RASTs to heated celery extracts and to stick celery. By contrast, in the celery-mugwort sensitive patients (n = 6) the celery RASTs with heated celery extracts remained clearly positive and high RAST values to stick celery could be found. Celery-birch-mugwort-association (n = 22) favoured more positive results with relatively high values of RAST to celeriac. The results of homologous and heterologous RAST inhibition experiments with birch, mugwort, unheated and heated celery (100 degrees C) carried out in nine celery-RAST positive sera are also discussed.

Comparative safety and efficacy of short ragweed extracts differing in potency and composition in the treatment of fall hay fever

One high potency (HP) and two low potency (LP) commercial whole short ragweed (WSR) extracts were assayed for relative potency (RP) by antigen E (AgE-Amb a 1), RAST inhibition, and parallel line bioassay (PLBA). The RP of the HP extract (300 micrograms AgE) was equal to the reference WSR, but the LP extracts were only approximately 0.01 of the RP of the reference. Each extract was administered to fall hay fever patients with skin sensitivity to WSR (less than or equal to 10(-3) micrograms/ml AgE for sum of erythema = 50 mm) to the maximum tolerated dose, or 0.5 cc of the concentrate, by either a 9- or 40-dose regimen. HP-treated patients had significantly lower symptom scores than untreated controls. No significant difference was noted in the total symptom scores between HP and LP extract-treated patients. Despite approximately 100-fold differences in the RP of HP versus LP extracts, both produced similar frequencies of severe systemic and severe late phase local reactions. Furthermore, when RP of HP and LP extracts were estimated by PLBA, both reached similar peak dosages, similar dosage for IgG WSR antibody response, and comparable IgG WSR antibody levels, indicating that the WSR dose estimates based on RP were bioequivalent. The 9-dose HP regimen produced fewer late phase local and systemic reactions per patient than the 40-dose HP regimen. Most patients experiencing severe systemic reactions were among those most skin sensitive to WSR. The number of systemic reactions per injection was significantly higher at higher WSR doses. One LP extract produced a high incidence of systemic reactions in the initial three injections. With respect to HP, its RP varied approximately 10-fold depending on the skin sensitivity of the patient to heat-stable ragweed allergens, demonstrating that it was compositionally different. However, when the dose of this extract was estimated by RP based on the skin sensitivity of each patient, the peak dose of the extract as well as the doses associated with late phase and systemic reactions were found to be similar to the HP doses. This indicated that immunizing dose estimates of compositionally different WSR extracts based on RP by PLBA were also bioequivalent.

Occupational sensitivity to Tenebrio molitor linnaeus (yellow mealworm)

Tenebrio molitor is an abundant stored-grain pest in the northern United States. We evaluated an individual with work-related symptoms of rhinoconjunctivitis on exposure to this insect. Prick skin tests with extracts prepared from the larval, pupal, and adult-life stages were positive for the patient and for another individual with allergy to a closely related species of beetle, Alphitobius diaperinus. Specific IgE antibodies to the extracts were demonstrated by RAST. RAST inhibition demonstrated immunologic cross-reactivity between the life stages of T. molitor and also between T. molitor and A. diaperinus, as well as slight cross-reactivity with blowfly. The proteins in the extracts of each life stage were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 15 protein bands were detected in each of the extracts, although the patterns of separation were different for each life stage. After immunoblotting and autoradiography, six different IgE-binding proteins were identified in the larval extract, five in the pupal extract, and seven in the adult extract, with similar IgE-binding patterns noted for the larval and adult extracts. We conclude that this patient developed IgE-mediated sensitivity to T. molitor antigens as the result of occupational exposure. This study confirms the fact that beetles of the Tenebrionid family are potentially significant allergens for workers exposed to grains or grain products.

Prevalence of specific IgE to storage mites (A. siro, L. destructor and T. longior) in an urban population and crossreactivity with the house dust mite (D. pteronyssinus)

The role of pyroglyphid mites in house dust allergy is well established and the major allergens from the common house dust mites (Dermatophagoides species) have been characterized. There is, however, relatively little progress in the understanding of the human IgE response to non-pyroglyphid storage mites, allergenic crossreactivity with other mite species and extent of environmental exposure. We studied 196 individuals from an urban environment who were not occupationally exposed to storage mites and found a 24% prevalence of specific IgE antibody to Dermatophagoides pteronyssinus and a 14% prevalence of RAST positivity to at least one of three storage mites, Acarus siro, Lepidoglyphus destructor and Tyrophagus longior. All individuals with a positive RAST to storage mites had specific IgE to D. pteronyssinus. RAST inhibition studies with the eight sera with greater than 2% RAST binding to both families of mites showed considerable crossreactivity between D. pteronyssinus and the storage mites A. siro and T. longior and limited crossreactivity between D. pteronyssinus and L. destructor. This suggests that at least some of the response to storage mites observed by direct RAST is a consequence of crossreactivity with the more abundant D. pteronyssinus.

Characterization of water-soluble shrimp allergens released during boiling

Water-soluble shrimp allergens released during boiling (shrimp water) were characterized and compared to allergen extracts from boiled shrimp (shrimp meat). Both shrimp extracts contained acidic proteins (isoelectrofocusing) and demonstrated similar allergenic activity (RAST and RAST inhibition). Shrimp-water extract was analyzed further by immunoprinting with sera from 14 shrimp-sensitive, RAST-positive subjects, and six nonsensitive, RAST-negative individuals. Although none of the sera from shrimp-tolerant individuals reacted, 12 14 sera (85.7%) from shrimp-sensitive subjects reacted with shrimp-water proteins with acid isoelectric points. Shrimp-water extract was fractionated by chromatofocusing with pH and NaCl gradients. A number of eluted ultraviolet-absorbing peaks contained allergens as determined by RAST inhibition. Isoelectrofocusing demonstrated many protein bands present in these peaks, some of which bound IgE from a RAST-positive sera pool. These studies indicate that shrimp water is an excellent source of shrimp allergens, that chromatofocusing is a useful method for fractionation of shrimp allergens, and that shrimp allergens are generally protein molecules with acid isoelectric points.

Allergens in Hymenoptera venom. XXII. Comparison of venoms from two species of imported fire ants, Solenopsis invicta and richteri

Venoms were collected by electrical stimulation from the two major species of imported fire ants found in the United States, Solenopsis invicta ( Sol i) and S. richten ( Sol r). Antigens similar to three of the four known Sol i venom proteins (I, II, III, and IV) were isolated from Sol r. The N -terminal amino acid sequences for the antigens III were identical; but those for the antigens II demonstrated only nine of 20 residues to be identical. Two monoclonal antibodies raised against Sol i II did not react to Sol r. No protein IV could be detected in Sol r by molecular weight, charge, or immunologically with either polyclonal mouse antibodies or five monoclonal antibodies. Both venoms were compared with a panel of 60 sera from Sol i-allergic individuals; mean bindings were similar with an r = 0.94 for linear regression. RAST inhibition was performed with 17 individual sera representing a variety of Sol i allergen specificities. Four sera were tested from patients resident in the Sol r endemic area and five sera from patients who experienced reactions to S. xyloni stings. All sera reacted comparably to both imported fire ant venoms. The two venoms appear to be allergenically similar, although antigen IV is absent from Sol r and the antigens II have significant sequence variation. Sol i venom appears to be sufficient for diagnostic purposes.

An optimized assay of specific IgE antibodies to reactive dyes and studies of immunologic responses in exposed workers

Methods of assaying reactive dye-specific IgE antibodies were investigated with a RAST. Sera from three patients, occupationally exposed to a reactive dye, Remazol black B (Chemical Abstract registry number 17095-24-8), were used. Directly dyed disks, that is, disks without any carrier protein, resulted in poor and unreliable measures of specific IgE. In contrast, optimized preparation of conjugates between the dye and human serum albumin resulted in efficient binding of specific IgE. The patients' RAST results were strongly positive, whereas sera from 36 exposed workers but without symptoms and sera from unexposed subjects with high levels of total IgE were negative. The hapten and carrier specificity of the IgE antibodies was studied by direct RAST and RAST inhibition. In one patient, the antibodies were principally hapten specific, whereas another patient was found to have antibodies with a high degree of specificity to the carrier. The third patient's antibodies were intermediate between the other two patients' antibodies in this respect, suggesting that antibody specificity is dependent not only on the nature of the hapten but also on individual immune response factors. The study demonstrates that it is important to use an optimized preparation of dye-protein conjugates to elicit reliable results and a high degree of specific IgE binding in the RAST.

Double-blind, placebo-controlled rush immunotherapy with a standardized Alternaria extract

Specific immunotherapy is ineffective with unstandardized mold extracts. A double-blind, placebo-controlled study was performed in 24 patients (5 to 56 years of age) only allergic to Alternaria. The extract was standardized by isoelectric focusing, crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, RAST inhibition, and skin tests and contained allergen Alternaria major allergen a I and antigen B. Thirteen patients received the active treatment, and 11 received the placebo. Immunotherapy was started by a 2-day rush protocol; maintenance injections were administered for 1 year. The patient's self-evaluation of the treatment was significantly ( p < 0.001) lower in the placebo-treated group. Global symptom-medication scores, including asthma and rhinoconjunctivitis, were significantly ( p < 0.005) lower in the actively treated group. Nasal challenges with Alternaria extract were performed before immunotherapy and after 1 year of treatment. There was no difference in the placebo-treated group and a significantly ( p < 0.01) increased mean provocative dose in the actively treated group. Skin tests were significantly reduced in the actively treated group. Specific IgG increased significantly in the actively treated group and were stable in the placebo-treated group. There was a significant correlation between nasal challenges and nasal symptom-medication scores ( p < 0.03) or the patient's self-evaluation of efficacy ( p < 0.05). This study demonstrated that patients only sensitized to Alternaria benefit from specific immunotherapy with a standardized Alternaria extract.

Stinging insect allergy: sensitization to vespids in Madrid and surroundings

The study comprised 28 patients from the centre of Spain (Madrid and surroundings) who had suffered systemic reactions when stung by vespids. Specific IgE antibodies to Vespula spp. and Polistes spp. venoms were measured by RAST. All patients had positive RAST to Vespula venom and half of them also had positive RAST to Polistes venom. A patient can be sensitive to both venoms due to either a clinical sensitization to both venoms, or common antigenic determinants. To differentiate these states we used a RAST inhibition assay. We could inhibit Polistes RAST with either Polistes or Vespula venom to a similar degree. Inhibition of Vespula RAST was possible with Vespula venom, but only to a limited degree with Polistes venom. Direct RAST and RAST inhibition studies indicate that in our geographic region sensitization to Vespula venom is more common than to Polistes venom and Polistes might have cross-reactivity in our patients.

Variability of cat-allergen shedding

To study the differences in total allergen shed, nine cats were placed individually in a lucite chamber with an air sampler attached for 1 hour. With a RAST-inhibition assay that used specific cat IgE antibodies from cat-sensitive subjects, we ranked the allergen production of each cat. From these nine cats, three high producers and one low producer were studied further. Each cat's allergens were collected for two to four separate 1-hour intervals on 6 different days. The high producers' samples remained high in allergen content, and the low producer's sample remained low. An additional eight cats were selected for similar longitudinal measurements, and allergens from each of these 12 cats were collected during four 1-hour intervals on 2 different days and assayed for total allergy units (AUs) by RAST inhibition and for the major cat allergen, Fel d I, by a two-site assay with a monoclonal antibody. Shedding, particularly by high producers, varied considerably from hour to hour. We found a hundredfold difference in AUs between the mean rate of shedding of the highest and lowest producers and a sixfold difference in Fel d I units. Variation in rate of shedding of Fel d I accounted for about half the variation of shedding of AUs. Allergen shedding varies not only between cats but also in the same cat during the course of a day and between days. Male cats shed more than female cats.

Preparation of Allergen Extracts from the Green Alga &lt;i&gt;Chlorella&lt;/i&gt;

Allergen extracts were prepared from different raw materials of three strains of the unicellular green alga Chlorella (C. vulgaris, C. homosphaera and C. saccharophila). Growth variation, batch variation and composition of partially purified extracts were studied by analyzing total protein and carbohydrate content, protein and IgE-binding patterns and allergenic potency. Chlorella allergens were produced in the same proportions in early and late exponential growth phases but appeared partly degraded in the stationary phase. Uniform growth and extraction from Chlorella cells in the late exponential phase were demonstrated. Partially purified extracts from cells in late exponential phase contained 50% protein of the dry weight compared to 20% in crude extracts. The carbohydrate content was 20-30% of the dry weight of both crude and purified extracts. The allergenic potency of the purified extracts was 150-340% of the crude extracts, as assayed by RAST inhibition. The purified C. vulgaris extract showed a reduced IgE binding compared to crude extracts, while C. homosphaera and C. saccharophila extracts exhibited consistent IgE binding and are therefore suitable for in vivo and in vitro studies of allergenic properties of Chlorella.

Shared and specific allergenic and antigenic components in the two sexes of American cockroach—Periplaneta americana

The whole body extracts (WBEs) of female and male cockroaches (Periplaneta americana; Pa) were prepared separately to study the specific and/or shared allergenic and antigenic components in the two sexes. These two extracts were skin-tested on 170 respiratory allergy patients and 52 (30.6%) of them elicited a markedly positive cutaneous reaction (2+ to 4+) to any one or both the WBEs. Of these 52 patients, 32 (61.5%) produced a 2+ to 4+ response to only one, and the remaining 20 (38.5%) to both the extracts. In female and male RASTs to Pa both the extracts produced dose-related inhibition. Using rabbit anti-Pa female serum: (1) immuno-diffusion experiments resulted in lines of identity with the two extracts, and (2) the two-dimensional immunoelectrophoresis of Pa female and Pa male WBEs elicited 12 and nine precipitin peaks, respectively. In partial purification studies, only fraction Pa(F)III (approximately 50 kD) of Pa female WBE and Pa(M)I (greater than or equal to 600 kD) and Pa(M)III (approximately 50 kD) fractions of Pa male WBE revealed significant allergen activity both on skin testing and also in RAST inhibition studies. These results provide evidence for the presence of shared as well as specific allergenic and antigenic components in the two sexes of American cockroach.

Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids: III. Efficacy and safety of unfractionated and high-molecular-weight preparations in rhinoconjunctivitis and asthma

Specific immunotherapy with unmodified formalinized allergoids is effective in grass-pollen allergy, but systemic reactions have been observed. A high-molecular-weight formalinized allergoid (HMW-GOID) was fractionated by gel filtration, retaining molecules of >85,000 daltons in the expectation of improving safety without sacrificing efficacy. HMW-GOID and unfractionated allergoid (GOID) had a similar allergenic activity assessed by RAST inhibition, but the HMW-GOID preparation was 65 times less reactive when it was tested by skin prick test than the GOID preparation. A double-blind, placebo-controlled study was carried out in grass pollen-allergic patients with placebo (14 patients), GOID (15 patients), and HMW-GOID (13 patients). An additional group of 18 patients was treated by a rush schedule with a standardized orchard grass-pollen extract. A similar mean cumulative dose was administered with both allergoids. The fractionated allergoid only elicited minor systemic reactions similar to reactions elicited by placebo, whereas 20% of patients treated by GOID and 5.5% of patients receiving the standardized extract had a severe systemic reaction. For rhinitis, conjunctivitis, and asthma, the HMW-GOID and the standardized extract had a similar efficacy, significantly greater than placebo. GOID was less effective than the other two active treatments but was significantly more effective than placebo treatment for asthma and conjunctivitis.

Immunochemical characterization of Cocos nucifera pollen

The Cocos nucifera pollen, as one of the sources of allergen responsible for immediate hypersensitivity reaction, was confirmed by skin prick test, bronchial provocation test, and RAST. The whole pollen extract (WPE) of C. nucifera was fractionated by combination of gel filtration and ion-exchange columns with fast protein liquid chromatography (Pharmacia, Uppsala, Sweden). Three protein peaks designated Cocos II, Cocos VI, and Cocos VII exhibited allergenic properties, as tested by skin prick test, direct IgE ELISA, bronchial provocation test, and immunoblot analysis. In RAST inhibition, Cocos IIa (a high-molecular-weight protein) obtained by fractionation of Cocos II on Mono Q column (fast protein liquid chromatography) (Pharmacia) was found to be the most potent allergen in Cocos WPE, followed by Cocos VI and Cocos VII, which are low-molecular-weight proteins. The reference patterns of Cocos WPE on crossed immunoelectrophoresis and thin-layer isoelectric focusing were established for future standardization of Cocos WPE to be used in the diagnosis and immunotherapy of allergic patients.

Cross-reactivity of IgE antibodies to caddis fly with arthropoda and mollusca

We investigated the possibility that subjects with IgE antibodies to an inhalant insect allergen, such as caddis fly, might also have antibodies to cross-reacting carbohydrate determinants (CCDs). IgE antibodies to cross-reacting allergens in caddis flies, mussels, oysters, shrimps, crabs, honeybee, and yellow jacket venoms were determined by RAST, RAST inhibition, and immunoblot studies with sera from three different sources: (1) sera of patients with well-defined inhalant atopy to caddis fly, (2) sera with IgE anti-CCD antibodies from subjects without known exposure to caddis fly, and (3) hyperimmune antisera with IgG anti-CCD antibodies raised as a result of immunization of rabbits with grass-pollen extract, buckwheat glycoprotein, or with honeybee venom. Sera from groups 2 and 3 reacted with Sepharose-coupled caddis fly extract in a RAST-type assay and elicited virtually identical patterns on immunoblots of caddis fly extract separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the sera from group 1 atopic patients did not react with CCD-rich material. However, indications for other types of cross-reacting antibodies were detected. The IgE antibodies of one of the patients studied (who was allergic not only to caddis fly but also to shellfish) were found to detect a cross-reacting homologous protein in extracts of mussel, oyster, shrimp, crab, honeybee, and yellow jacket venom. Preliminary results suggest that this cross-reacting 13 kd protein, the most prominent caddis fly allergen, is an invertebrate hemoglobin (erythrocruorin)-like molecule. These studies suggest the possibility that patients sensitized by exposure to caddis fly antigens could develop allergic reactions during their first exposure to shellfish or to their first bee sting.

Effects of In Vitro Proteolysis on the Allergenicity of Major Whey Proteins

ABSTRACTThe effects of in vitro proteolysis on the allergenicity of major whey proteins (α‐lactalbumin and β‐lactoglobulin) by simulating the human gastrointestinal conditions were evaluated. The proteolysis of demineralized whey with pepsin (pH 2.0 for 30 min) and/or various pancreatic enzymes (pH 7.5 for 60 and 240 min) was performed by the pH‐stat technique at 37°C. The enzyme inactivation was performed by heating at 80°C for 20 min. Allergenicity of the hydrolyzates was evaluated by RAST inhibition using sera obtained from children allergic to whey proteins. Selective proteolysis of whey by pepsin and α‐chymotrypsin was the most efficient combination of enzymes to reduce the allergenicity of both α‐lactalbumin and β‐lactoglobulin. The above hydrolyzate could be used to develop an ingredient for infant milk formula with a lower allergenicity.

Species-specific shrimp allergens: RAST and RAST-inhibition studies

Two edible shrimp species are widely available in Louisiana, Penaeus setifecus (white shrimp) and Penaeus aztecus (brown shrimp). Some sensitive individuals report only occasional allergic symptoms after shrimp ingestion, suggesting that there may be species-specific allergens. To investigate this possibility, we evaluated shrimp species-specific reactivity in 31 individuals with a history of immediate hypersensitivity reactions after shrimp ingestion with skin prick tests and RASTs with white and brown shrimp extracts. On selected individuals, RAST-inhibition studies were performed with white shrimp and/or brown shrimp-coupled disks, with white and/or brown shrimp extracts as inhibiting allergen. Positive skin tests to both types of extract were observed in 77% (23130) of the subjects; one individual reacted to brown shrimp extract only. Elevated RASTs to both extracts were observed in 16131 study participants; one subject reacted only to white shrimp extracts and two subjects to brown shrimp extract alone. Sera from two individuals tested by RAST inhibition recognized qualitatively different allergens in brown and white shrimp extracts, supporting the hypothesis that there are species-specific shrimp allergens. Species specificity is important because it may explain the intermittent symptoms of some study subjects. The percentage of shrimp-sensitive subjects testing positive by skin test and RAST can be increased by use of extracts from more than one species of shrimp.

Guanine content and Dermatophagoides pteronyssinus allergens in house dust samples

The study of the relationship between the guanine content and Dermatophagoides pteronyssinus ( Dp) allergens in house dust samples is reported. Mattress and carpet dust of bedrooms from 22 different homes constituted the house dust samples. The guanine content was determined by quantitative measurements and the mite allergenicity by two immunochemical assays with a partially purified extract of Dp as internal reference: RAST inhibition and crossed and rocket line immunoelectrophoresis. A large scale range of guanine content was obtained among the 22 house dust samples studied (0.01 to 1.78 mg/0.1 gm of dust). Data of RAST inhibition, analyzed according to parallel line bioassay, demonstrated no significant difference between the slopes of the reference and the house dust sample lines, but a 100-fold variation in the relative Dp potencies was observed. By crossed and rocket line immunoelectrophoresis technique, the presence and the amounts of major Dp allergens ( Der p I and Dp 4) were established in most house dust extracts. A significant correlation was found between the guanine content of the house dust samples and their relative Dp potencies ( r = 0.86) on the one hand, and with their relative content of Der p I and Dp 4, two major Dp allergens ( r = 0.75 and r = 0.74, respectively) on the other hand; in each case, a quantitative relationship was established. These results suggest that the guanine determination could assess mite allergens in house dust and may be a useful tool in large-scale investigations of house dust.

Comparative studies of allergens from mycelia and culture media of four new strains ofAlternaria tenuis

Abstract Four new isolates, identified as Alternaria tenuis, 34–016, 34–039, 35–056 and 35–193 were grown on revised tobacco medium. Mycelial and broth extracts were prepared and their biochemical and immunological properties were examined. Compared to the mycelial extracts, the four broth extracts had lower protein contents and showed fewer protein bands in isoelectric focusing, crossed-immunoelectrophoresis and sodium dodecylsulphate polyacrylamide gel electrophoresis. However, direct radioallergosorbent and passive cutaneous anaphylaxis tests with mouse IgE revealed that mycelial and broth extracts were of similar potency and had extensive cross-reactivity. In crossed-radioimmunoelectrophoresis using human atopic sera, the mycelial extracts showed 1–2 dominant and 3–4 minor allergens while the broth extracts had one dominant and one minor allergen. In RAST inhibition tests, two isolates, 34–039 and 35–056, had 100-fold more allergen content in the broth extracts compared to the mycelial extracts, whereas the levels were similar for broth and mycelial extracts of the other two isolates. The results indicated that culture broths of 34–039 and 35–056 were excellent starting materials for purification of some of the major allergenic components of A. tenuis as they combined good yield with high allergenic activity, and lower level of other protein components. In addition, extracts containing wide spectrum of allergens and suitable for diagnostic purposes, can be prepared from mycelia and culture fluid of these isolates.