Variability of antigenicity/allergenicity in different strains of Alternaria alternata.

Biochemical and immunological properties were studied in crude mould extracts from 10 different strains of Alternaria alternata. The mould strains were grown on completely synthetic medium and harvested under identical conditions. The biochemical and immunological analyses (protein content, carbohydrate content, IEF, RAST-inhibition, CIE/CRIE and the content of a major allergen) showed significant variations between the individual strains of A. alternata. 32 antigens were identified in the CIE pattern of A. alternata and 19 different allergens were identified in CRIE analysis (1 major, 7 intermediate and 6 minor allergens). There was a significant variation in the content of the major A. alternata allergen (Ag-8, partially identical to Alt-I) between the different individual strains. Furthermore, a considerable variation in the content of Ag-8 was observed for a specific strain cultivated at different occasions under identical cultivation conditions during a time period of 3 years.

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.

Analysis of tobacco leaf allergens by crossed radioimmunoelectrophoresis.

The reactivity of eleven 'tobacco smoke sensitive' and eight 'non-sensitive' individuals to tobacco leaf allergens was tested by Crossed Radioimmunoelectrophoresis (CRIE). All nineteen study subjects had IgE antibodies to tobacco leaf antigens as measured by Radioallergosorbent Test (RAST) and seventeen of the nineteen individuals were atopic. Of the thirty-seven tobacco leaf precipitins detected by Cross Immunoelectrophoresis (CIE), three were identified as allergens by CRIE. All nineteen subjects reacted to at least one of the three allergens detected. However, neither the intensity nor the incidence of reactivity with any of the three allergens correlated with smoking or 'smoke sensitivity'.

Purification and partial characterization of the allergen Ag-54 from Cladosporium herbarum.

One important allergen, Ag-54, was extracted from Cladosporium herbarum and purified by a combination of diafiltration, gel filtrations and isoelectric focusing. The allergen, when examined by gel electrophoresis and high-performance liquid chromatography, was found to be essentially homogeneous with a molecular weight of about 20,000 daltons. Ag-54 is of glycoprotein nature. It contained 19.5% protein, calculated from its amino acid composition and 80.2% carbohydrate, quantified by methanolysis, trimethylsilyl derivatization and gas-liquid chromatography. The carbohydrate moiety was composed only of mannose, galactose and glucose in the ratio 1.0:0.6:1.3. Frequently occurring amino sugars like glucosamine and galactosamine could not be detected. Mass spectrometry of trimethylsilyl-derivatized methylglycosides demonstrated that sialic acid was not present.

High-performance liquid chromatography of Cladosporium herbarum. Identification of allergens with immunological techniques.

High-performance liquid chromatography (HPLC) using a gel permeation column was applied to separate components in a crude extract of the mould Cladosporium herbarum. Allergenic activity as well as individual allergens in collected fractions were analyzed by direct radioallergosorbent test (RAST) and fused rocked radioimmunoelectrophoresis (FRRIE), respectively. Thus, information of the molecular weight as well as the importance of the individual allergens were obtained simultaneously in a system showing a high degree of resolution. The study demonstrated that the crude extract of C. herbarum contained allergens mainly in the molecular weight range between 10,000 and 300,000 daltons. The results demonstrated the importance of carrying out both RAST and FRRIE to detect all allergens.

Variability in different strains of Cladosporium herbarum with special attention to carbohydrates and contents of two important allergens (Ag-32 and Ag-54).

Ten different strains of Cladosporium herbarum cultivated and extracted under identical experimental conditions were compared with respect to: 1) carbohydrates quantified by methanolysis and gas-liquid chromatography, 2) contents of two important Cladosporium allergens Ag-32 and Ag-54 estimated by rocket radioimmunoelectrophoresis, 3) protein content determined by the Lowry and Bradford methods, respectively, and 4) allergenic activity as measured by RAST. The allergen extracts contained 45-73% carbohydrate material which mainly consisted of mannose, galactose and glucose. The Ag-54 content increased with increasing mannose and decreasing glucose concentration. No linear correlation between carbohydrate content and allergenic activity was found. The strains differed extensively in immunological composition. One of the strains totally lacked Ag-32 and Ag-54. To be clinically relevant, allergen preparations of C. herbarum should be based on a representative pool of several C. herbarum strains.

Antigenic variability of different strains of Aspergillus fumigatus.

10 strains (isolates) of Aspergillus fumigatus, grown and harvested under identical conditions, were compared with respect to biochemical and immunological properties. The total protein and carbohydrate content isoelectric focusing pattern were determined for each of the individual strains. Their antigenic and allergenic activity was examined by a combination of crossed immunoelectrophoresis (CIE)/crossed radioimmunoelectrophoresis (CRIE) and RAST inhibition. Biochemical and immunochemical data indicated that considerable variation exists between the various isolates of A. fumigatus. 44 antigens were demonstrated in CIE of A. fumigatus, and 18 different allergens were identified in CRIE (2 major, 10 intermediate and 6 minor allergens).

Studies on the antigenic composition of aqueous cotton dust extracts.

An immune response to cotton dust components has been proposed as a possible mechanism of byssinosis. To evaluate this possibility, specific precipitating antibodies against aqueous cotton dust extracts (ACDE) were raised in rabbits, and a reference pattern for crossed immunoelectrophoresis (CIE) was established using this extract. A significant portion of the immune response was directed against Alternaria tenuis, Aspergillus niger, and Fusarium solani, all documented contaminants of cotton dust. Preliminary crossed radio-immunoelectrophoretic studies demonstrated that some of these antigens are allergenic in man, suggesting that an IgE-mediated hypersensitivity against cotton dust, or a fungal component of the dust, may be important in byssinosis pathogenesis.

Crossed radioimmunoelectrophoretic studies of bee venom allergens.

Crossed immunoelectrophoresis (CIE) glass slides of honey bee venom showing 18 immunoprecipitates were used in crossed radioimmunoelectrophoresis (CRIE) experiments with sera from 25 patients allergic to honey bee venoms. Phospholipase A, acid phosphatase, hyaluronidase and melittin were demonstrated in the immunoprecipitates using zymographic techniques and direct hemolysis of sheep erythrocytes. The CRIE experiments verified the allergenicity of these proteins. In addition, another antigen (Ag-1) with allergenic activity was identified. This allergen is probably identical to the previously described allergen C in honey bee venom. The presence and complexity of multiple forms of the known allergenic proteins in honey bee venom, was well illustrated in the CIE/CRIE system employed.

Severe hypersensitivity or intolerance reactions to measles vaccine in six children. Clinical and immunological studies.

Six reported cases of anaphylactic reactions due to measles vaccinations of children were investigated. The reactions appeared to be immediate hypersensitivity reactions most probably due to allergy. Trace amounts of calf serum proteins were demonstrated in the measles vaccine by means of crossed immunoelectrophoresis whereas egg antigens could not be detected by this method. The reason for the untoward reactions was not identified. Atopic traits were not reported in any of the patients but were found in first degree family members of three of them. Skin prick testing with measles vaccine was positive in one of the patients only, whereas skin prick testing with egg yolk, egg white, cow's milk and calf serum were negative in all cases. RAST was performed with allergosorbents containing different material used during the vaccine production, but was negative in all cases. The results with control individuals were in accord with previous reports that patients with allergy to hen's egg tolerate the injection of measles vaccine. Immediate hypersensitivity reactions to measles vaccine may occur independent of allergy to egg and even in children without any known allergy or atopic heredity.

Immediate type hypersensitivity to common moulds. Comparison of different diagnostic materials.

Extracts of five common atmospheric moulds (Alternaria, Aspergillus, Cladosporium, Mucor and Penicillium) obtained from different manufacturers were compared by means of skin prick testing and, partly, with the radioallergosorbent test. Considerable differences were found between the extracts with respect to positive tests found in mould-allergic individuals and controls. A purified allergen preparation of Cladosporium herbarum was superior to the crude allergen extracts. The results suggest that the allergen contents of the extract differed both with respect to total allergenic potency and to allergenic composition.