Shared allergenic and antigenic determinants in Alternaria and Stemphylium extracts

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p < 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibition using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibition, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.     

Immunochemical partial identity between two independently identified and isolated major allergens from Alternaria alternata (Alt-I and Ag 1)

Partially purified preparations of Alt-I, the main allergenic fraction of Alternaria alternata isolated by Yunginger, and of Ag 1, shown in crossed radioimmunoelectrophoresis (CRIE) to be the dominating major allergen of A. alternata (Løwenstein, Nyholm), were compared by tandem crossed immunoelectrophoresis (CIE), RAST inhibition, and the CRIE-related technique, single radial radioimmunodiffusion (SRRID). The two allergen preparations showed reaction of identity in tandem-CIE and indistinguishable specific IgE binding in CRIE and SRRID, regardless of antibodies and serum pools used. In RAST inhibition, the relative potencies of the allergen preparations and of the crude extracts correlated well with their Alt-I/Ag 1 content as estimated by rocket immunoelectrophoresis. Moreover, all inhibition curves were parallel, confirming identical IgE binding by Alt-I and Ag 1 with the serum pools used. A second preparation of Alt-I, isolated from another strain of Alternaria, showed reaction of partial identity with Ag 1 in tandem-CIE, indicating that different variants of Alt-I (Ag 1) may exist in different strains of A. alternata.     

The international collaborative study establishing the first international standard for timothy (Phleum pratense) grass pollen allergenic extract

A selected candidate international reference preparation of timothy grass (Phleum pratense)-pollen extract was studied together with two other freeze-dried timothy pollen allergenic extracts in a multinational study. The collaborators used RAST inhibition, histamine release, quantitative immunoelectrophoresis (crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis and rockets), isoelectric focusing, and other methods. The total allergenic potencies measured in RAST inhibition were evaluated for validity of linearity and parallel-line response. The relative concentrations of some important individual allergenic components were measured. The relative potencies for the total allergenic activity and the timothy components studied in each preparation were expressed relative to the selected candidate. This preparation was established in 1983 by the World Health Organization expert committee on biologic standardization as the international standard for timothy grass-pollen extracts with assigned units of 100,000 IU per ampule.     

Production of an international reference standard alternaria extract. I. Testing of candidate extracts

As part of a program to establish international standards of selected allergens, 6 coded extracts of Alternaria were assessed in 6 laboratories by immunochemical, biochemical and physicochemical procedures. Direct RAST, RAST inhibition, quantitative skin tests and leukocyte histamine release were used to assign relative orders of potency to the 6 extracts. The composition and major allergen content was tested by thin-layer isoelectric focusing and quantitative immunoelectrophoresis (crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis). Three laboratories determined the quantity of purified allergens in each of the preparations. In addition, source materials were sent to an expert Alternaria taxonomist for independent identification. The results showed considerable variation with respect to total allergenic potency and content of individual allergens. Source materials could not be confirmed as Alternaria in some instances. Based on fulfillment of written specifications and assay results, extract No. 6 was recommended by the Alternaria Working Group as the candidate international standard to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies.     

Crossed radioimmunoelectrophoretic studies of distinct allergens in two extracts of Cladosporium herbarum

Cladosporium herbarum was supplied from two sources and extracted identically. Antisera against the extracts were produced in rabbits and two reference patterns were established using crossed immunoelectrophoresis (CIE). Both patterns showed more than 60 precipitates but less than 50% of the detectable antigens appeared to be identical in the two extracts. The allergens were identified by means of crossed radioimmunoelectrophoresis (CRIE) using sera from 35 individuals with proven or suspected allergy to C. herbarum. Four important and 10-20 less important allergens were demonstrated. Among the allergens present, there were none reacting with all patient sera. Only 1 out of 3 rabbits immunized with a suspension of broken cells of C. herbarum showed precipitating antibodies to the statistically most important allergen, while 9 rabbits immunized with aqueous extracts of the mold did not. The composition of the two extracts with respect to allergens differed. Allergens present in one extract were not always detectable in the other. The experiments also showed how CIE/CRIE with various combinations of antigens and antisera may be combined with CRIE inhibition, radioallergosorbent test (RAST) and RAST inhibition for comparing complex allergen extracts at the molecular level.     

Standardization of Dermatophagoides pteronyssinus: assessment of potency and allergen content in ten coded extracts

As part of the studies to establish an international reference preparation of Dermatophagoides pteronyssinus allergens, ten coded extracts of this mite were assessed in four laboratories. The extracts were compared for total potency using direct RAST, RAST inhibition and quantitative skin tests, and also for composition and major allergen content using crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, rocket immunoelectrophoresis and radioimmunoassay. In addition, the source materials were examined by light microscopy, and the extracts were examined for the presence of proteins/allergens derived from the culture media. To help with standardization, a new reference pool of sera from patients allergic to D. pteronyssinus was also established (National Institute of Biological Standards and Control, NIBSC, 82/528). The results showed that techniques are available for measurements of potency and allergen content. In several of the extracts, culture medium derived allergens and antigens were demonstrated. It also became clear that extracts varied not only in their total potency but also in the distribution of the identifiable allergens. In particular, extracts derived from isolated mites contained more AgX and/or Ag 23 relative to their content of antigen P1 (= Ag42). These studies lead to the choice of an extract for an international reference preparation (NIBSC 82/518) and helped to establish the methods used subsequently in the international collaborative study.     

Investigation of the allergenic activity of isolated fractions of a standardized partly purified whole mite body (Dermatophagoides pteronyssinus) extract

Twelve fractions of a molecular weight range of 1.35-670.00 kilodaltons (kD) were isolated from a biologically standardized partly purified whole mite body extract (Dermatophagoides pteronyssinus) by preparative size exclusion high-performance liquid chromatography. The allergenic activity and the antigen and allergen patterns of the isolated fractions were investigated by RAST, RAST inhibition and crossed (radio)immunoelectrophoresis (CIE/CRIE). By CRIE, each of the fractions showed allergen patterns, mostly of different compositions. Each fraction showed allergenic activity of different degrees by RAST inhibition. The highest allergenic activity could be measured by RAST inhibition with fractions which contained predominantly the major allergens Der pI and PY as detected by CRIE. Also proteins of higher molecular weights (greater than 158 kD) showed IgE-binding capacities. Nearly all antigens detected by CIE could be identified as allergens using CRIE.     

Antigens of alternaria. I. Isolation and partial characterization of a basic peptide allergen

A basic peptide allergen has been isolated from Alternaria extracts. Flat-bed gel preparative isoelectric focusing followed by dialysis and lyophilization allowed for concentration of substantial quantities of this heretofore unrecognized allergen. Rocket immunoelectrophoresis and autoradiography (using a 2 X concentrated, IgE serum fraction from Alternaria patient sera and 125I-anti human IgE), a RAST inhibition assay, and skin testing of Alternaria-sensitive patients were used to demonstrate the allergenicity of this fraction. The allergenicity of this protein was sufficient to give 50% RAST inhibition at a concentration of 100 micrograms/ml using standard RAST procedures. The immunoelectrophoresis autoradiography also indicated allergenicity. Skin tests indicated that over 90% of patients hypersensitive to Alternaria crude extracts were also hypersensitive to the basic allergen fraction.     

Immunochemical quantitation of airborne short ragweed, Alternaria, antigen E, and Alt-I allergens: a two-year prospective study

We conducted a 2 yr prospective study to measure atmospheric short ragweed and Alternaria allergens by RAST inhibition analysis of eluates from filter sheets exposed in air samplers. In both years ragweed pollen and Alternaria spore counts, obtained with a rotoslide sampler, correlated significantly with immunochemically measured airborne ragweed and Alternaria allergenic activity. Airborne levels of the purified allergens AgE and Alt-I were successfully quantitated; these levels correlated closely with total airborne ragweed and Alternaria allergenic activities, respectively, and also with ragweed pollen and Alternaria spore counts. Eluates from filter sheets exposed during late summer and fall produced positive wheal-and-flare skin tests in patients with fall hay fever. In both years immunochemical measurements of allergenic activity due to airborne short ragweed correlated closely with mean symptom score indices in groups of short ragweed-sensitive individuals. Measurable levels of atmospheric ragweed allergenic activity were noted before and after the ragweed pollination season, and at these times we noted small increases in mean symptom score indices in the short ragweed-sensitive groups. Thus immunochemical analyses provide important information concerning levels of environmental allergens.     

Fusarium solani: Evidence for common antigenic/allergenic determinants with other Fungi Imperfecti

Aqueous extracts of Fusarium solani and other members of the Fungi Imperfecti were evaluated for the presence of common antigenic/allergenic determinants using skin-prick testing, radio-allergo-sorbent test (RAST) inhibition, and immunoelectrophoretic methods. Prevalence of skin reactivity in forty-four atopic individuals, tested with commercially available fungal extracts, ranged from 27.3% for Alternaria tenuis to 6.8% for Penicillium notatum. No specific patterns of reactivity emerged from statistical analyses of skin test data. In contrast, RAST inhibition demonstrated common allergenic determinants. P. notatum and Aspergillus glaucus inhibited F. solani RAST by 79% and 84%, respectively. This was supported by crossed line immunoelectrophoresis; both P. notatum and A. glaucus had antigenic determinants in common with F. solani. Collectively, these studies suggest that F. solani, P. notatum , and A. glaucus have several common antigenic/allergenic determinants.     

Comparative studies on tree pollen allergens. XVII. Immunochemical analysis of the international standardization extracts of birch (Betula verrucosa) pollen as compared with a local partially purified extract

Six different birch pollen extracts were analyzed by 20 laboratories for the standardization of birch (Betula verrucosa) pollen extracts used for diagnosis and specific therapy of patients with birch pollen allergy. The extracts were collected and delivered by the International Union of Immunological Societies, Allergen Standardization Subcommittee. One of the extracts, designated M, was proposed as an international standard (IS)-candidate of birch pollen extracts. The protein content of the IS candidate M was found to be 1.12 mg/mL, more than 2-fold higher than any of the other extracts analyzed. This preparation was among the extracts containing the highest number of protein components, as shown by isoelectric focusing, 28 lines, and by 11 precipitates in crossed immunoelectrophoresis. The allergenic reactivities were tested by crossed radioimmunoelectrophoresis (CRIE) and by radioallergosorbent test (RAST)-inhibition. In CRIE, the proposed IS (M) showed similar affinity for binding patients' IgE as the other extracts, as judged by the autoradiographic illustrations. Except for extract L, the values of RAST-inhibition for the rest were very similar. An IS extract should qualify for the criteria suggested for an optimal allergen preparation, containing minimal amounts of non-allergenic antigens and providing quantitatively and qualitatively all the allergenic proteins. The appropriateness of this selection seems unjustified in view of the above criteria.     

Allergens of mammalian origin. V.

Eight commercial cat dander extracts and two pelt extracts derived from mongrel and Siamese cats were compared. Cat allergen 1 and cat albumin were measured by radial immunodiffusion. Allergenic activity was evaluated by prick test and a modified radioallergosorbent test. In the latter, the dilution of each extract that produced 50% inhibition of binding of IgE antibodies to insolubilized cat allergen 1 (RAST 1) and insolubilized cat serum (RAST 2) was determined. The total non-dialysable solid content of the extracts did not correlate with any other parameter. Cat allergen 1 content determined by radial immunodiffusion correlated with average prick test results in ten cat-sensitive subjects and with RAST 1 activity. Cat albumin content correlated weakly with RAST 2 activity but not with any other measure of allergenic activity. Absorption of each extract with the γ-globulin fraction of rabbit antiserum to cat allergen 1 significantly reduced prick test reactivity and RAST 1 activity, but not RAST 2 activity. These results indicate that cat allergen 1 is an important allergen in cat dander extracts and its measurement may be used to standardize the allergenic activity of such extracts.     

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 less than 0.001) lower in the placebo-treated group. Global symptom-medication scores, including asthma and rhinoconjunctivitis, were significantly (p less than 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 less than 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 less than 0.03) or the patient's self-evaluation of efficacy (p less than 0.05). This study demonstrated that patients only sensitized to Alternaria benefit from specific immunotherapy with a standardized Alternaria extract.     

Antigenic/allergenic analysis of basidiomycete cap, mycelia, and spore extracts

Since airborne basidiospores may be important inducers of respiratory allergy, extracts of spores, caps and mycelia from Pleurotus ostreatus were studied by immunologic techniques. Crossed immunoelectrophoresis of the Pleurotus spore extract showed it to be a complex mixture containing at least 27 precipitating antigens. Crossed-line immunoelectrophoresis comparing Pleurotus spore extract with extracts of Pleurotus cap or mycelia demonstrated both common antigens and antigens unique to the spore extract. Inhibition of Pleurotus spore RAST by extracts of Pleurotus spore, cap, and mycelia suggested that these extracts contained common allergenic components. However, wheal and flare skin reactivity of allergic patients to extracts of P. ostreatus or Cantharellus cibarius demonstrated little correlation between reactivity to cap and spores. These results demonstrate that basidiomycete spore extracts are the best diagnostic reagents to use in clinical studies, although cap and mycelia extracts may provide useful material for further allergen analysis.     

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.     

Standardization of allergen extracts by inhibition of RAST, skin test, and chemical composition

Five allergen extracts of Dermatophagoides pteronyssinus, Lolium perenne, Alternaria tenuis, Aspergillus fumigatus and Cladosporium herbarum, obtained from four different manufacturers, were examined by inhibition of RAST, content of protein and carbohydrate, contents of phosphorylcholine (Pc) and tridacnin reactive components, and by skin test. Inhibition of RAST was used as a primary method for establishing allergenic potency and demonstrated wide variations for each preparation supplied by the different manufacturers. The extracts also varied widely in protein and carbohydrate content and in the ratio of these parameters, indicating internal heterogeneity. Pc content was significantly related to RAST potency for extracts of A. fumigatus and A. tenuis, suggesting that Pc content may be used as a primary standarization procedure for these extracts. Skin test reactions undertaken at a single concentration did not show any significant variation in weal size between preparations of a given allergen extract. However, of particular importance to practising clinicians is the finding that varying numbers of patients showed negative skin reactions to one preparation of a particular allergen yet were positive to the corresponding preparations supplied by the other companies.     

Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA. Correlation with biologic activity

A solid-phase, monoclonal antibody-based ELISA was set up to quantitale group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1–100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleumpratense extracts, and a very good quantitative correlation was found ( r =0.98; P <0.0001). A highly significant correlation ( r >0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.     

Evaluation of Basidiomycete and Deuteromycete (Fungi Imperfecti) extracts for shared allergenic determinants

Aqueous extracts of select members of the Basidiomycetes and Deuteromycetes (Fungi Imperfecti) were evaluated for the presence of shared allergenic determinants using skin prick and radio-allergosorbent test (RAST) inhibition. Twenty adults with perennial symptoms of rhinitis, with or without asthma, were skin-prick tested with six species of Deuteromycetes and seven species of Basidomycetes. Positive weal-and-flare reactivity to Pleurotus ostreatus was associated with Alternaria alternata, Fusarium solani and Epicoccum purpurescens. Positive skin reactivity to Calvatia cyathiformis was also associated with A. alternata and F. solani. Coprinus quadrifidus was associated only with F. solani, and Psilocybe cubensis was only associated with Aspergillus fumigatus. No other skin test associations were demonstrated. For every allergen tested by RAST inhibition, significant dose-dependent homologous inhibition was demonstrated. Although the ability of an individual heterologous extract to inhibit the direct RAST varied, inhibition was generally minimal. In the most extreme example, no heterologous allergen inhibited the A. alternata RAST. However, the Armillaria tabescens RAST was inhibited 52.6%, 38.1% and 25.1% by A. fumigatus, E. purpurescens, and Penicillium notatum, respectively, suggesting significant cross-reactivity. These results suggest that, although shared allergenic determinants exist between select species of Basidiomycetes and Deuteromycetes, crossreactivity is minimal and its clinical significance is not clear. These data confirm that for reliable diagnosis of fungal allergy, representatives of both major groups must be used.     

Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test

The potencies of 11 commercial extracts of short ragweed pollen were analyzed by skin test end point titration and compared to potency as measured in vitro: (1) by the radioallergosorbent test (RAST), (2) by AgE concentrations, and (3) by protein nitrogen determinations (PNU). RAST potency was determined by the capacity of the extract to inhibit the binding of IgE antibody to solid-phase allergen in the first step of the RAST, and it was expressed as the quantity of extract needed for 50 per cent inhibition of binding. Potencies determined by skin testing in 7 patients were strongly related among the various patients in spite of a 1,000-fold difference in the patients' sensitivity to the extracts. Similarly, potency as measured by RAST inhibition showed about a 1,000-fold difference among the extracts, and the RAST potencies were strongly related to potency as measured by skin testing. Purified antigens derived from short ragweed inhibited binding of IgE antibodies, but the slopes of the inhibition curves were significantly less than those produced by crude or partially purified short ragweed. AgE concentration and PNU concentrations also were correlated with skin test potency, but the range of potencies was less than that found by RAST inhibition. Measurement of the potency of extracts by RAST inhibition should prove useful as a general procedure for the standardization of allergy extracts.     

Basidiospore extracts: evidence for common antigenic/allergenic determinants

Spore extracts, prepared from Armillariella tabescens, Pleurotus ostreatus, Coprinus quadrifidus, Amanita muscaria, Ganoderma lucidum, Psilocybe cubensis, Pisolithus tinctorius, Scleroderma sp. and Calvatia cyathiformis, were examined for antigenic/allergenic relationships by Ouchterlony and radioallergosorbent testing (RAST) inhibition, respectively. Ouchterlony, using hyperimmunized rabbit sera, demonstrated a high degree of cross-antigenicity among the extracts tested; however, some unique antigens were also present. RAST inhibition, evaluated by comparing extract concentrations which inhibited the RAST by 50% (IC-50), varied with the allergen tested. P. cubensis was the most potent inhibitor (IC-50 ranged from 0.034 mg/ml for A. tabescens RAST to 0.29 mg/ml for G. lucidum RAST). P. tinctorius was the least potent inhibitor, failing to reach IC-50 at 10 mg/ml for any basidiospore extract. Evaluation of slopes and intercepts of the dose-response lines demonstrated qualitative and quantitative differences among allergens in these extracts. These results indicate the presence of shared allergenic epitopes, and suggest that representative extract panels could be developed for future use in diagnosis and treatment of basidiospore-sensitive individuals.