Use of Aspergillus fumigatus mycelial antigens in enzyme-linked immunosorbent assay and counter-immunoelectrophoresis.

Antigens obtained from the ruptured mycelium of Aspergillus fumigatus were separated on the basis of their attachment or non-attachment to Concanavalin A-linked Sepharose gel. They were analysed by double-diffusion and two-dimensional immunoelectrophoresis with rabbit antiserum and sera from patients with aspergillus-related disease. Their sensitivity was assessed in an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G. Sera which gave very low or high extinction readings on ELISA gave, in general, comparable results on counter-immunoelectrophoresis; but weak electrophoretic reactions were not closely paralleled by ELISA results. The unfractionated extract and the fraction bound by Concanavalin A-Sepharose had similar ability to detect specific IgG in sera from patients with aspergillosis.

Analysis of wall antigens of Aspergillus fumigatus by two-dimensional immunoelectrophoresis.

Water-soluble and surface-located antigens from Aspergillus fumigatus mycelium were analysed by two-dimensional electrophoresis (2D IEP) with selected sera. A complex pattern was seen when a water-soluble fraction reacted with hyperimmune sera. Significantly fewer precipitin peaks were seen when wall-located antigens were used in the 2D IEP system; this made possible the detection of a small number of recurring antigen-antibody interactions when sera from patients with aspergilloma or allergic bronchopulmonary aspergillosis were analysed. Many of these recurring precipitates were affected by lectins with specificity for alpha-D-glucose, alpha-D-mannose or N-acetyl-D-glucosamine. All the antigens were susceptible to hydrolysis by pronase, confirming the glycoprotein composition of many of these macromolecules.

Analysis of Aspergillus fumigatus catalases possessing antigenic activity.

Analysis of Aspergillus fumigatus water soluble fractions by electrophoresis on non-denaturing polyacrylamide gels (PAGE) showed the presence of at least three catalase bands. They were designated F, S1 and S2 in order of descending electrophoretic mobility with respect to the anode. The multiple enzyme forms appear to be distinct in their physicochemical properties. Enzyme bands S1 and S2 were simple catalases; the F band had an additional peroxidase function. All of the components were antigenic and differed in their binding to specific antibodies raised in rabbits with separate fractions of A. fumigatus mycelium. When serum from patients with aspergilloma, allergic bronchopulmonary aspergillosis, cystic fibrosis and chronic asthma were pre-incubated with A. fumigatus antigens and analysed by PAGE, 17 of 26 samples either abolished or reduced catalase activity. Enzyme F was a non-Concanavalin A (ConA)-binding antigen; the S1 and S2 enzymes were ConA-binding glycoprotein antigens. The major catalase band present in A. niger preparations represented only a minor component in A. fumigatus.

Preparation of Aspergillus fumigatus antigens and their analysis by two-dimensional immunoelectrophoresis.

The water-soluble components of Aspergillus fumigatus mycelium were partially separated by fractional precipitation with ammonium sulphate. The total protein and neutral-sugar content were determined for each of the four fractions prepared and their immunological activity was examined by double diffusion. Partial chemical characterisation of these isolates by polyacrylamide-gel electrophoresis was linked to their precipitability by allowing the separated components to diffuse from the gel into an agarose medium containing an appropriate antiserum. The distribution and reactivity of antigens was monitored by two-dimensional immunoelectrophoresis with rabbit sera raised against either mycelial or culture-filtrate antigens and human sera, obtained from patients with aspergilloma and from patients with allergic bronchopulmonary aspergillosis. This technique was also used to establish that considerable variation exists in the precipitation profile seen among patient specimens. Several antigens were found to possess sugar residues that interacted with concanavalin A, when this lectin was used in an intermediate gel in two-dimensional immunoelectrophoresis.

Surface antigens of intact Aspergillus fumigatus mycelium: their localization using radiolabelled protein A as marker.

Isotopically-labelled Protein A from Staphylococcus aureus was used as a marker to quantify binding of IgG molecules to surface antigens of Aspergillus fumigatus mycelium. The IgG class antibodies were obtained from rabbits inoculated with mycelial fractions and from patients suffering from aspergillosis and aspergillus-related diseases. The highest incorporation of label was obtained with an antiserum from rabbits inoculated with A. fumigatus wall material. Antibodies raised to other antigenic fractions of A. fumigatus and antibodies from patients infected with aspergillus gave lower levels of incorporation of Protein A. In competitive binding experiments, pre-incubation of antibodies with partially purified aspergillus antigens depressed subsequent binding to the mycelial surface by 20-30% of that of the control values. Low molecular weight disaccharides and oligosaccharides were without effect in this system. Preincubation of A. fumigatus with lectins having specificities for defined sugar residues did not reduce subsequent antigen/antibody interaction. Treatment of the mycelial surface with certain proteolytic or polysaccharolytic enzymes led to a decrease in antibody binding, while pretreatment of A. fumigatus with the hydrolytic enzyme mixture of Trichoderma harzianum culture filtrate gave increased antibody binding. Aspergillus species showed different susceptibilities to enzyme action and their surface structures could be differentiated from A. fumigatus on this basis. These differences were not obvious in direct binding experiments where antibodies raised to A. fumigatus wall bound with equal facility to antigenic sites located on the walls of other Aspergillus species.

Comparison of partially purified mycelial and culture filtrate antigens of Aspergillus fumigatus by enzyme-linked immunosorbent assay.

An aqueous extract of Aspergillus fumigatus mycelium was fractionated by precipitation with ammonium sulphate to give protein-enriched and carbohydrate-enriched preparations. The antigenic activity of the fractions so obtained was assessed in an enzyme-linked immunosorbent assay using pooled positive and negative reference sera. The best discrimination between such sera was obtained when using either the fraction insoluble in 50--75% ammonium sulphate or the material soluble in a saturate solution of the salt. This system was then used in parallel with commercial A. fumigatus extracts to test sera of patients with and without clinical aspergillosis. The implications of the results are discussed.

The preparation and chemical composition of fractions from Aspergillus fumigatus wall and protoplasts possessing antigenic activity.

A detergent-soluble fraction was prepared from the fragmented wall of Aspergillus fumigatus mycelium using the non-ionic detergent Triton X-100, and a wall-free extract was prepared from the same source in the form of protoplasts, released by a lytic enzyme system from Trichoderma harzianum. These extracts were examined by polyacrylamide gel electrophoresis and their detailed chemical composition was established. They were compared with the water-soluble fraction prepared from total mycelium, which is used routinely in this laboratory for serological tests. All fractions had immunological reactivity towards an antiserum prepared in rabbits against this water-soluble fraction of the mycelium, as shown by double diffusion. Both protein and carbohydrate moieties appear to be involved in the antigenic sites, with carbohydrate reactivity predominantly associated with the protoplast fraction. The fact that all preparations contained at least one common antigenic determinant, as judged by lines of identity to a single antiserum, is discussed in relation to antigen location.

Chemical and immunological analysis of the Aspergillus fumigatus cell wall.

Hyphal-wall preparations of Aspergillus fumigatus have been analysed by sequential treatment with KOH, nitrous acid and again with KOH. By acidification of the alkali-soluble extract, a polyglucose was precipitated which showed an X-ray diffraction pattern similar to that of (1-->3)-alpha-glucan. The remainder of the alkali-soluble fraction was precipitated with ethanol; it contained all the mannose, galactose and protein of the wall and, in addition, 6.2% of the amino sugars. This wall-associated glycoprotein, following SDS-PAGE and immunoblotting, reacted with antisera raised against several mycelial extracts of A. fumigatus. Sera from patients with aspergilloma have antibodies which recognize components of this glycoprotein. The glycoprotein nature of these antigens was shown by their ability to bind Lens culinaris lectin. In addition, the antigen/antibody binding could be disrupted by exposure of antigen to periodate oxidation, hydrolysis with dilute acid or pretreatment with a large excess of an exo-beta-D-galactofuranosidase. The alkali-insoluble fraction consisted of a covalently linked glucan-chitin complex. Nitrous acid treatment, which specifically disrupts glycosidic linkages involving glucosamine, did not solubilize much material but changed the X-ray diffraction pattern from diffuse to a pattern showing the characteristic lines of crystalline (1-->3)-beta-glucan and chitin. Most of the glucan became alkali-soluble after this treatment, and the insoluble residue appeared to contain crystalline chitin.

Effects of tunicamycin on glycoprotein antigens of Aspergillus fumigatus.

Tunicamycin, which inhibits N-glycosylation of proteins, was used as a tool to determine the type of linkage which occurs in glycoprotein antigens of Aspergillus fumigatus. When A. fumigatus extracts were electrophoretically separated and blotted then probed with anti-Aspergillus patients' sera, differences in antigenic profiles were noted when tunicamycin-treated samples were compared with controls. Tunicamycin had no detectable effect on the cellular proteinases of A. fumigatus, most of which are glycosylated. Some enzymatic components were lacking when extracellular proteinases were compared with those of control samples. The major catalase component of A. fumigatus is a concanavalin A (ConA)-binding glycoprotein. In cultures grown in the presence of tunicamycin, partially-deglycosylated catalase components were obtained which could be distinguished from the native catalase by altered mobilities in polyacrylamide gels. The effect of deglycosylation on catalase antigens was monitored using an antiserum raised to a ConA-binding fraction of A. fumigatus mycelium. These antibodies bound both to the native glycoprotein and the partially deglycosylated material. These latter two were largely unaffected when incubated with an antiserum raised to a non-ConA-binding fraction of A. fumigatus which is essentially carbohydrate free. The ability to produce partially-glycosylated antigens of A. fumigatus offers a model to study the effect of basic structural modifications on both the enzymatic and antigenic activities of these molecules.

A method to determine significant levels of immunoglobulin G to Aspergillus fumigatus antigens in an ELISA system and a comparison with counterimmunoelectrophoresis and double diffusion techniques.

A method is described which assesses results obtained from an ELISA system for the determination of human serum levels of IgG class antibodies to Aspergillus fumigatus. The method is used to discriminate positive from negative samples, and significant antibody activity may be reported to the clinician, relative to a reference positive control serum monitored simultaneously under the same test conditions. Antibody content is expressed as the absorbance of a certain dilution of serum. Duplicate samples were analysed at a single serum dilution and their absorbtion values obtained from a semi-automated ELISA microplate reader. These were entered into a computer programmed to convert the data into units on a logarithmic scale. In parallel experiments, ELISA results were compared with those obtained by the techniques of counterimmunoelectrophoresis and double diffusion which measure precipitating antibody of all classes. A relatively good degree of correlation between tests was found only among sera with a high level of antibody.

Aspergillus fumigatus antigens used in the serodiagnosis of aspergillosis.

The diagnostic antigens in Aspergillus serology are mycelial or culture filtrate concentrates. Recent work has been directed towards the separation of defined, reproducible products of greater sensitivity and specificity than crude extracts. Conventional methods of separation have been used; they include fractional precipitation, detergent extraction, chromatography, and preparative isoelectric focusing. Selective staining of the separated fractions has shown many of the more reactive components to be glycoproteins. Affinity binding to Concanavalin A has demonstrated the presence in these of alpha-D-glucopyranose, alpha-D-mannopyranose and/or terminal alpha-D-N-acetylglucosamine. Major amino acid constituents are serine and threonine, together with glutamic and aspartic acids. The more reactive and specific components, as judged by their antibody binding capacity, have molecular weights greater than 70,000; in some cases they are as high as 150,000 to 180,000. Isoelectric focusing gave products with acidic pI values which specifically bound the IgG present in sera from aspergillosis patients. Results from a number of laboratories suggest that a group of acidic glycoproteins constitute the most promising source of a diagnostic reagent for aspergillosis. However, further analysis and a comparison of these substances in an international collaborative study is needed before agreement can be reached on a "standard" antigen preparation.

Immunolocalization of Aspergillus fumigatus mycelial antigens.

Specific immunoglobulin from the sera of patients with antibodies to Aspergillus and from antisera raised in rabbits to Aspergillus fumigatus fractions bound almost exclusively to the mycelial wall, as shown by immunogold labelling of ultra-thin sections. Different layers of the wall were labelled, depending on the source of the antigen used to produce antibody. Internal, cytoplasmic components were generally not labelled, except with antisera raised to crude wall material and to a Concanavalin A (ConA)-binding fraction of a water-soluble preparation.

Structural analysis of water-soluble fractions obtained from Aspergillus fumigatus mycelium.

Fractions were prepared from the water-soluble components of Aspergillus fumigatus mycelium either by lectin-affinity chromatography or salt precipitation. While they varied considerably in their amino-acid composition, each contained a preponderance of aspartic and glutamic acids. 13C-NMR spectroscopy of these fractions, compared with that of polysaccharide obtained by alkaline extraction, indicated the presence of glycoproteins, the polysaccharide components of which contained beta-D-Galf units that are part of structures chemically different from those obtained by alkali treatment. In two of the three fractions examined, gas-liquid chromatography--mass spectrometry showed marked differences in the contents of non-reducing end-units of alpha-D-Manp and beta-D-Galf. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the preparations revealed an array of components, which stained to differing extents with silver stain and with Coomassie Blue and many of which were bound by lectins with specificity for different sugars.

The preparation and partial characterization of antigenic fractions obtained from the mycelial walls of several Aspergillus species.

Extracts with immunological activity were prepared from Aspergillus fumigatus, A. flavus, A. terreus, A. niger and A. nidulans. In each case crude mycelial wall was extracted with an aqueous solution of Triton X-100 giving detergent-soluble material. Further fractionation was achieved by removing the detergent from this solution; the resultant precipitate was removed by centrifugation, and the aqueous supernatant was used as a source of soluble antigens. The sensitivity of these preparations was compared with that of water-soluble antigenic material, prepared from whole macerated mycelium, by double diffusion and counterimmunoelectrophoresis using homologous antisera and sera from patients suffering from aspergilloma and allergic bronchopulmonary aspergillosis. The selectivity of these antigenic preparations was monitored with heterologous antisera raised in rabbits. Batch variability was analysed for one strain of A. fumigatus using chemical and immunological methods. The nature of the antigenic sites involved in these reactions was investigated by studying the susceptibility of the preparations to proteolytic hydrolysis, periodate oxidation and concanavalin A treatment. The total protein and carbohydrate content of each fraction was determined and the constituent sugars analysed in an attempt to correlate chemical composition with antigenic activity.

The preparation of protoplasts from Aspergillus fumigatus mycelium.

Protoplasts have been prepared from the mycelium of Aspergillus funigatus, using a lytic enzyme mixture from Trichoderma harzianum. A variety of experimental conditions were investigated in order to achieve optimal conditions for protoplast production. Electron micrographs showed the preparations to be free of cell-wall. Material obtained by this procedure can be used as a source of cytoplasmic antigens for further analysis.

A study of the alkaline proteases secreted by different Aspergillus species.

Strains from several species of Aspergillus were grown in the presence of soluble collagen, and the major secreted proteins present in the culture fluid were examined for proteolytic activity. The possibility of relatedness among the alkaline proteases secreted by Aspergillus was studied by probing extracts from the various species with polyclonal antisera raised to the isolated alkaline proteases of A. fumigatus and A. oryzae. The pathogenic species A. flavus, A. terreus and A. nidulans hydrolyse collagen and were found to secrete an alkaline protease related to that of Aspergillus fumigatus. In contrast, A. niger and non-pathogenic species such A. glaucus, A. versicolor and A. clavatus were unable to degrade collagen in vitro. These findings suggest a possible pathogenic role for the secreted alkaline proteases of Aspergillus species.

Evaluation of a test to detect circulating Aspergillus fumigatus antigen in a survey of immunocompromised patients with proven or suspected invasive disease.

An ELISA for the detection and measurement of Aspergillus antigenaemia has been developed and evaluated by examining sera submitted over a 12-month period from immunocompromised patients with a likelihood of invasive aspergillosis. Results from proven cases of invasive aspergillosis confirmed at post-mortem and specimens from individuals with suspected disease showed that tests on single serum samples were often negative. Multiple specimens from the same patient greatly increased the frequency of detection. Repeated monitoring of sera from a single patient showed wide fluctuations in antigen level, which was considered to be due partly to the medical regimen to which the patient was subject. Control sera from healthy laboratory personnel were consistently negative, but a number of 'at-risk' patients without other evidence of invasive aspergillosis sometimes had low amounts of antigen. Concentrations of Aspergillus antigen of 100 ng ml-1 or higher were considered to be strongly suggestive of fungal invasion.

Cellular and humoral immune responses in experimental aspergillosis in mice.

Invasive aspergillosis was established in a naive murine model. Both humoral and cellular aspects of the immunological mechanism responded to invasion. Circulating immune complexes containing IgG in the infected group of animals were detected by both the C1q and conglutinin solid phase assays. Attempts to identify these complexes as Aspergillus antigen-specific complexes were unsuccessful. Cellular responses, as measured by blastogenic activity and inhibition of migration of sensitized cells were significantly elevated in the infected group.