Optimized preparation of Aspergillus fumigatus extracts for allergy diagnosis.

Extracts of Aspergillus fumigatus are required for the measurement of specific antibodies that are important indices in the diagnosis of allergic bronchopulmonary aspergillosis (ABPA). This study investigated the effect of different culture conditions on the production and release of antigenic and allergenic proteins of A. fumigatus. Increasing the incubation temperature from 25 degrees C to 37 degrees C altered the production of proteins by the mycelium which resulted in the release of a greater number of proteins that reacted with precipitating antibodies. Static sporulating cultures produced a much wider antigenic spectrum than shake cultures although the number of precipitating proteins (5 and 3 respectively) and major IgE binding proteins (5 and 3 respectively) was not greatly altered. The widest range of proteins bound by precipitating antibody or IgE from ABPA serum were released into the culture filtrate during 28 day static incubation at 37 degrees C. The resultant extract proved useful for screening patients for specific IgE and will provide a starting material for the identification of individual antigens or allergens.

Aspergillus fumigatus: identification of 16, 18, and 45 kd antigens recognized by human IgG and IgE antibodies and murine monoclonal antibodies.

The immunochemical properties of antigens produced by Aspergillus fumigatus were investigated with biochemical purification techniques in conjunction with the production of murine monoclonal antibodies (MAbs) and binding studies with human IgG and IgE antibodies. A. fumigatus antigens were partially purified by gel filtration and hydrophobic interaction chromatography on phenyl-Sepharose. Two fractions that eluted with either 2 mol/L or 0.15 mol/L of NaCl demonstrated strong binding to human IgG and IgE antibodies. Immunoprecipitation analysis with IgG antibodies from six patients with different Aspergillus-related diseases demonstrated that the 2M and 0.15M fractions contained major antigens of molecular weight 18 kd (Asp f I) and 45 kd, respectively. The 125I-labeled 2M fraction was used to compare IgG antibodies to A. fumigatus in sera from 25 patients with Aspergillus-related diseases. IgG antibodies were significantly higher in patients with allergic bronchopulmonary aspergillosis (geometric mean, 437 U/ml) than in patients with asthma (geometric mean, 14 U/ml; p less than 0.001), but undetectable (less than 5 U/ml) in 43/48 control subjects. A good correlation was found between levels of IgG antibodies to the 125I-labeled 0.15M fraction and the 125I-labeled 2M fraction in sera from 106 patients with cystic fibrosis (r = 0.77; p less than 0.001). Five murine IgG MAbs and two IgM MAbs were raised against the 2M fraction, and immunoprecipitation with the IgG MAb demonstrated two distinct antigens within the 2M fraction, Asp f I, and a 16 kd antigen. The results of a solid-phase RIA with IgG MAb 4A6 demonstrated that approximately 85% of A. fumigatus-allergic patients with allergic bonchopulmonary aspergillosis had IgE antibodies to Asp f I. The three protein antigens defined in these studies are useful probes for investigating the immunopathogenesis of diseases associated with colonization by A. fumigatus.

Allergy to rabbits. III. Further identification and characterisation of rabbit allergens.

Crossed immunoelectrophoresis and crossed radio-immunoelectrophoresis techniques have been employed for analysis of extracts of various rabbit source materials to identify rabbit allergens in addition to the already described Antigen R1 (AgR1). Urine and dander extracts were found to contain only low levels of AgR1 and its presence in urine was as a contaminant due to mode of collection--it was not present in urine collected directly from the bladder. Other allergens were only recognised by highly rabbit-sensitive individuals, one in particular (Ag2) being present in several source materials. Serum albumin proved to be of minor allergenic importance and except for dander its presence was only in minimal amounts. As both AgR1 and Ag2 are significant components in extracts of fur and dust these extracts are therefore most recommended for use in investigations of individuals sensitive to rabbits.

Measurement of airborne mite antigen in homes of asthmatic children.

The airborne concentration of major house dust mite antigen Der p1 was measured by low volume sampling (2 litres/min) in the homes of 68 allergic, asthmatic children. The presence of detectable airborne antigen was strongly associated with sensitivity to the mite, whereas there was no significant relation between sensitivity and the previously recommended threshold level of 2 micrograms Der p1 per g carpet dust. There was a significant association with lower threshold levels in carpet dust (0.5 microgram/g) but at no level was the association as strong as that with air measurements. Concentrations of airborne antigen were higher in rooms with wool carpets than in those with synthetic carpets or hard floors, but there was no significant difference between the dust levels of Der p1 in the two carpet types. Air sampling is a more appropriate method of assessing antigen exposure than dust sampling for asthmatic patients.

Controlled trial of natamycin in the treatment of allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis often requires treatment with oral corticosteroids to control the host response to Aspergillus fumigatus. In a double blind study 25 patients with allergic bronchopulmonary aspergillosis taking maintenance oral corticosteroids were randomly allocated to receive 5 mg natamycin or placebo by nebuliser twice daily for one year. The primary aim of the study was to assess the steroid sparing potential of natamycin. Standardised reductions in corticosteroid dosage were therefore undertaken every five weeks, unless clinically contraindicated. Five patients were withdrawn in the first four months: two (1 natamycin, 1 placebo) died, two (1 natamycin, 1 placebo) had suspected drug reactions, and one (natamycin) was non-compliant. The pretreatment characteristics of the 20 patients (10 in each group) who completed the study were similar, 17 (9 natamycin, 8 placebo) having evidence of recent disease activity. At the end of the study prednisolone dose had been reduced by a similar amount in each group (median natamycin 2.25 mg, placebo 2.5 mg). Evidence of disease activity during the study year (transient shadowing on the chest radiograph, blood eosinophilia, or increases in antibodies to A fumigatus, or any combination of these) was observed in similar numbers of patients in each group (5 natamycin, 7 placebo). There was no evidence that natamycin conferred benefit on these patients with allergic bronchopulmonary aspergillosis.

Allergy to mice. II. Further characterization of two major mouse allergens (AG 1 and AG 3) and immunohistochemical investigations of their sources.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and quantitative immunoelectrophoretic techniques have been used to characterize further the two major mouse allergens, antigen 1 (Ag 1) and antigen 3 (Ag 3). Gel filtration using Sephacryl S-200 showed Ag 1 to have a molecular weight of 18 kD and Ag 3 of 21 kD. SDS-PAGE followed by Western blotting onto nitrocellulose then incubation with individual antisera directed against each of the two major allergens, and an alkaline phosphatase enzyme system, was used to distinguish between the two allergens and indicated a molecular weight of 17 kD for Ag 1 and 16 kD for Ag 3. Ag 3 but not Ag 1 was shown to contain polysaccharide residues. Immunohistochemical staining of mouse skin sections demonstrated that antigens detected in whole dust extracts were present in the hair follicles, on the hair shafts and on the stratum corneum. Staining of similar sections using the rabbit anti-Ag 3 showed the presence of this major allergen in the hair follicles coating the hairs and extending along the skin surface. Serum from a pool of mouse-allergic subjects also demonstrated staining in the same areas when detected using a fluorescein-labelled anti-human IgE as second antibody. As both major allergens were present in extracts of fur this would appear to be most appropriate for use in diagnosis (i.e. skin test and RAST) and also possibly desensitization. However, dust from isolators (available in greater amounts) would be equally suitable.

Pigeon breeders' disease: quantitative immunoelectrophoretic studies of pigeon bloom antigen.

This study employed quantitative immunoelectrophoretic techniques, on sera from confirmed cases of pigeon breeders' disease (PBD), to investigate the antigenicity of a pigeon bloom extract, implicated as a sensitizing agent in this disease. On crossed immunoelectrophoresis the maximum number of antigenic components identified was 29 for the bloom compared to 10 for pigeon serum. A major component was shown to be closely related to pigeon IgA, and demonstrated partial crossreactivity to the pigeon IgG. This component also showed identity with the major component of a pigeon droppings extract, considered to be derived from intestinal IgA. Only trace amounts of serum albumin were detected and most other bloom components were not serum-related. Although greater overall antigenic similarity was found to pigeon droppings extract, at least three of the bloom components appeared to be specific. The bloom extract also contained a low amount of an alpha-techoic acid-like component, causing some non-specific reactivity. Pigeon feather dust or 'bloom', like pigeon droppings, is therefore a potent source of antigens associated with PDB--pigeon IgA being a major component of both antigens.

IgG antibodies in relation to exposure to laboratory-animal allergens.

An ELISA technique has been used to measure levels of specific IgG in individuals exposed to laboratory animals in comparison to levels in unexposed individuals. The technique was to determine whether these levels could be useful as a monitor of exposure. Both symptomatic (with and without specific IgE) and nonsymptomatic individuals were studied. Several antigenic source materials were investigated for each animal. The IgG response was extremely complex and elicited a varied pattern of response between animals (mouse, rat, and rabbit). No evidence was found for raised IgG levels being directly related to the degree of exposure, more to the presence of clinical symptoms and specific IgE. Long-term heavy exposure to rats in nonsymptomatic individuals appeared to produce a suppression of the IgG response in relation to control levels. These findings suggest that IgG is not an indicator of exposure but demonstrate a varied response related to the presence of specific allergy to an animal, the particle size on which antigens are carried, and the physiologic state of the mucosa onto which the particles impact.

Task-related variation in airborne concentrations of laboratory animal allergens: studies with Rat n I.

To define airborne allergen exposure during various tasks with rats in a laboratory, concentrations of allergen Rat n I were measured by radioimmunoassay in extracts from filters in personal air sampling devices that were worn by laboratory workers while they were performing these tasks. The tasks included feeding, cage cleaning, handling, injection, surgery, and sacrifice. Median concentrations encountered during feeding or cleaning (21 ng/m3) and injection or handling (13 ng/m3) were higher than those associated with surgery or sacrifice (3.1 ng/m3; p less than 0.01). Area samples in animal-holding rooms contained 3.4 ng/m3 during animal handling and 2.3 ng/m3 at other times. Very low concentrations were found in air outside the handling room, in unused laboratories, or outside air. We concluded that certain tasks incur a higher risk of allergen exposure but that exposure may occur anywhere within an animal laboratory environment.

Lymphocyte sensitization to Aspergillus fumigatus in allergic bronchopulmonary aspergillosis.

Peripheral blood mononuclear cell (PBMC) proliferation induced by an extract of Aspergillus fumigatus (AF) was examined in patients with allergic bronchopulmonary aspergillosis (ABPA), all of whom had an immediate skin prick test reaction (SPT) and increased RAST binding to AF, and, for comparison, in individuals without immediate SPT reactivity or increased RAST binding to AF. The proliferative responses of PBMC from the ABPA patients were greater than those from the comparison donors. A substantial proportion of the comparison group, however, showed evidence of a specific immune response to AF, with AF-specific IgG measured by ELISA and specific lymphoproliferative responses. AF-responsive T cell lines and T cell clones were established from both ABPA patients and IgE-negative individuals. These clones, of helper/inducer (CD4+) phenotype, showed antigenic specificity and MHC restriction. The stimulating antigen was determined for four of six clones derived from a skin-prick-test-negative individual, and found to be of Mr 18 kD, possibly the major allergen, 'Ag 3'. ABPA patients showed a marked diminution of the proliferative response during disease exacerbation.

Inhalant allergy in asthmatic children: skin prick test, radioallergosorbent test and chemiluminescent assay compared with allergen levels in their mattress dusts.

Mattress dusts from the beds of 51 asthmatic children with positive skin tests to house dust mite were assayed for Der p I, Fel d I and certain viable fungi. The results of skin tests to grass, cat, dog, Alternaria, Aspergillus, Cladosporium and Penicillium, radioallergosorbent tests to cat, mite, dog and grass and the chemiluminescent assay were compared and correlations made with allergen concentrations in the dust. The sensitivity of the three tests appeared comparable (percentage agreement = 54.5-94), however, an increased level of allergen in the mattress dust was not a good indicator of the degree of sensitization of the child (p greater than 0.05).

Characterization of immunologically important antigens and allergens of Aspergillus fumigatus.

Using a variety of immunochemical methods, including quantitative immuno-electrophoretic techniques, combined with gel filtration and iso-electric focusing, and production of monospecific antisera for identification and affinity purification, 4 major components of Aspergillus fumigatus have now been partially characterized. Numbering of these was derived from a reference allergic bronchopulmonary aspergillosis (ABPA) self-crossed radio-immuno-electrophoresis pattern of reactivity. Two major intracellular/cytoplasmic, concanavalin A (Con A)-binding antigens, Ag 7 and Ag 13, of molecular weights 150-200 and 70 kilodaltons (kD), respectively, were confirmed to be of importance for both ABPA and aspergilloma in specific sandwich enzyme-linked immunosorbent assays. A rapidly released component, Ag 5, of molecular weight 35 kD, proved both antigenic and allergenic, with aspergilloma patients having especially high-titre IgG antibodies. The major allergenic component Ag 3, of molecular weight 24 kD by gel filtration and 18 kD by SDS-PAGE was, like Ag 5, relatively heat-labile and non-Con-A-binding. Interestingly, T cell clones have been identified which respond primarily to an 18-kD fraction.

Smoking, atopy, and laboratory animal allergy.

This study examined data from three cross sectional surveys of 296 laboratory workers exposed to small mammals. Four indices of laboratory animal allergy were studied: symptoms suggestive of occupational asthma, symptoms suggestive of any occupational allergy, skin weals to animal urine extracts, and serum binding in radioallergosorbent tests with urine extracts. Pooled data from the three surveys showed an association between smoking and all indices except radioallergosorbent tests; the association was significant for symptoms of occupational asthma. One of the three surveys consistently showed a stronger association of allergy indices with smoking than with atopy (defined on skin tests with non-animal aeroallergens). Associations with smoking persisted after stratifying by atopic status, suggesting that smoking may be a risk factor for laboratory animal allergy.

Partial characterization of a rapidly released antigenic/allergenic component (Ag 5) of Aspergillus fumigatus.

An antigenic component of Aspergillus fumigatus, which was responsible for inducing an initial early antibody response in rabbits immunized with A. fumigatus germlings, has been identified and partially characterized. By immunofluorescent antibody and ELISA techniques, this antigen was demonstrated to be present on the germling surface, although it was also detected in supernatants of conidia/germlings within 1 hour and was present in all shake and stationary culture-filtrate extracts. By incorporation of a monospecific antiserum to this component in an intermediate gel of the reference self-crossed radioimmunoelectrophoresis pattern for allergic bronchopulmonary aspergillosis sera, it was recognized as Ag 5 and was also demonstrated to bind specific IgE. Further immunochemical analyses have revealed that Ag 5 is relatively heat labile, does not bind to concanavalin A, and has a molecular weight of approximately 35 kd. This rapidly released antigenic/allergenic component may play an important role in the initiation of immunologic responses in patients with allergic bronchopulmonary aspergillosis.

ELISA method for measurement of airborne levels of major laboratory animal allergens.

Three types of air-sampling apparatus (the Air-Sentinel, Burkard Personal Sampler and Casella Personal Sampler) were compared for their use in sampling the air in situations where individuals are exposed to laboratory animals, and a cascade impactor was used to determine the sizes of particles carrying the allergens under investigation. An ELISA, using monospecific antisera raised to selected major allergens of the mouse, rat and rabbit, i.e. mouse Ag 1 (mouse urinary prealbumin), rat Ag 4 (rat urinary prealbumin) and rabbit Ag R1, was developed to assay the extracts obtained from the samplers. This ELISA system was able to detect greater than 5 ng/m3 of allergen in sampled air. The rat and mouse allergens were shown to be carried mainly on particles of 6-18 micron, whereas the rabbit allergen was also found on particles of 2 micron and smaller. Allergen levels correlated well with the number of animals present in the room and the degree of activity during sampling. A protective filtered-air hood, when worn during surgical operation procedures, was shown to reduce effectively the level of rat allergen breathed by an individual.

Allergy to rabbits. II. Identification and characterization of a major rabbit allergen.

Quantitative immunoelectrophoretic techniques have been used to study the antigenic components found in extracts of dust collected from rabbit housing areas. To determine the possible source of these antigens, comparisons have been made to rabbit saliva, urine, fur and dander. Specific antisera for the rabbit extracts were raised in guinea pigs. One major component of the dust (Ag Rl) was also found in large amounts in saliva, slightly less in fur and in only minimal amounts in urine and dander. Crossed radioimmunoelectrophoresis (XRIE) of the dust, performed with sera from 14 rabbit allergic individuals who were RAST positive to rabbit saliva, urine and dust identified four IgE-binding constituents. Individual responses varied but all sera reacted with Ag Rl, identifying this as a major rabbit allergen. Dust RAST inhibition studies with rabbit dust, saliva and urine indicated saliva to be closely related to the dust. Ag Rl is a glycoprotein which appears to be very heterogeneous in nature. It produced a broad biphasic precipitin peak on immunoelectrophoresis and eluted from Sephacryl S-200 gel filtration over the molecular weight range 30-50 Kd, although a molecular weight of 17 Kd was indicated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gradient gel electrophoresis. The RAST inhibition results and the antigenic similarity of saliva to the dust suggest this to be the most likely source of the major rabbit allergen, Ag Rl.

Characterization of a second major antigen Ag 13 (antigen C) of Aspergillus fumigatus and investigation of its immunological reactivity.

A second major antigen of Aspergillus fumigatus (Ag 13) has been identified and characterized as a 70 kD, heat-labile component which is able to bind to the lectin, concanavalin A. Ag 13 proved to be identical to the previously recognized C antigen, known to possess chymotryptic activity. A monospecific antiserum to Ag 13 has been affinity purified and used to develop a sandwich ELISA for measuring levels of Ag 13 specific IgG antibodies in patients' sera. Although all ABPA sera (25/25) had significantly raised levels, some aspergilloma sera (2/5) had only very low levels; of the control sera, 10/10 fungal atopics were negative, but 5/12 farmer's lung sera had low but positive values of Ag 13 specific IgG, i.e. sensitivity 100%, specificity 80%. Ag 13 is therefore a diagnostically important component of A. fumigatus and the antigen-specific ELISA may be of importance for the improved detection of disease-specific antibodies.

Allergy to rats: quantitative immunoelectrophoretic studies of rat dust as a source of inhalant allergen.

The antigenic composition of an extract of rat dust, as a source of aeroallergens for rat-sensitive individuals, has been investigated and compared to the antigenic composition of rat saliva and urine. Of four main antigenic peaks identified by crossed immunoelectrophoresis, one antigenic peak (Ag 4) was demonstrated to be antigenically closely related to and with similar molecular weight (approximately 22 kd) and isoelectric point values as urinary prealbumin, already recognized as a major rat allergen. Ag 4 was present in all dusts studied and was also identified as a minor component of saliva. However, no component with the same electrophoretic mobility or physicochemical characteristics of the alpha 2-euglobulin of male rat urine that shares partial identity with the prealbumin was detected, even in dust collected from a male rat room. A second high molecular weight (greater than 200 kd) component, Ag 1, present in most of the dust extracts, could not be detected in either urine or saliva. Crossed immunoelectrophoresis and skin prick tests confirmed the allergenicity of both these antigens. Analysis of an air filter sample taken within a male rat room revealed significant amounts of the "prealbumin" component, and a monospecific antiserum to this component was used to quantitate levels in dusts collected from various locations. These findings suggest that a major inhalant allergen present in rat dust is closely related to urinary prealbumin but that this and other allergenic components may not be derived predominantly from rat urine or saliva but possibly from secretions originating from the skin of the animals and present in the fur.

Release of antigens and allergens during shake-culture of Aspergillus fumigatus.

The appearance of IgG and IgE binding components in the medium of shake cultures of Aspergillus fumigatus has been studied. Cultures were grown in synthetic asparagine medium at 35 degrees C and flasks harvested in duplicate 1, 2, 3, 4, 7 and 14 days after inoculation. The pH of the medium dropped from its initial value of 5.5 to pH 3, and then after 4 days gradually increased up to pH 7.5 in the 14-day medium. The weight of mycelium, after an initial peak followed by a slight decline, increased as the pH of the medium increased. Components able to bind IgG and IgE from pooled ABPA sera were detected by crossed immunoelectrophoresis/self-crossed radioimmunoelectrophoresis within 24 h of growth, but maximal release of both antigens and allergens coincided with the increase in pH of the medium and was seen in the 14-day culture filtrate. Two recognised "major" antigens, Ag 7 and Ag 13, detected using the relevant monospecific antisera, were present in the culture medium after 14 days of growth and similarly for Ag 3, the major allergen, although another allergen, Ag 1, was identified in the 1-day extract. None of the culture filtrates was found to contain the "C-substance" polysaccharide.